Fig. 4: gspD is an essential gene in Myxococcus xanthus.
A Immunoblotting reveals that removal of copper from the medium of a cell line expressing gspD under a copper-inducible promoter leads to a gradual decrease of GspD, which eventually stabilizes at a low but detectable level. GspD is visualized as a ~100 kDa band, with occasionally a second band at ~47 kDa or lower molecular weight. These lower-weight bands represent proteolytically degraded GspD (Table S2). Size markers (first lane) indicate molecular weight in kDa. Specific time-points post-copper withdrawal were performed once, and reliance on copper for GspD was assessed by immunoblot twice independently for each of the two mutants. B Immunofluorescence microscopy reveals low-level expression of GspD signal in cells grown without inducer. Cells were grown in the indicated concentration of CuSO4 for 24 h prior to fixation and imaging. Top panel, anti-GspD signal; bottom, merged image with anti-GspD (red), DAPI (blue), and phase contrast. Images are representative of the field of cells from each growth condition, and the effects of copper depletion on GspD expression and localization were assessed by IFM in three independent experiments. Scale bar, 1 μm. C Plating of wild type (wt) cells and cells containing a copper-inducible version of GspD reveal that gspD is an essential gene. Cells pre-cultured in 0.2 mM CuSO4 were shifted to culture lacking CuSO4 for 48 h. Cells were then concentrated, and 4-fold serial dilutions were spotted on agar media containing the indicated concentration of CuSO4. In the absence of copper (left panel) the mutant strain fails to grow, while with increasing amounts of copper (intermediate concentration, middle panel; high concentrations, right panel) the cells grow at rates indistinguishable from the wild type. Source data are provided as a Source Data file.
