Figure 5. The N‐terminal intracellular globular domain of YqgP binds divalent cations.

1. Detection (left panel) and quantification (right panel) of steady‐state conversion of endogenous MgtE by YqgP (strain BS72, Table EV2) grown in minimal M9 medium containing 10 μM MgSO₄ and 1 μM each of MnCl₂, ZnCl₂, CoCl₂, NiCl₂ and CaCl₂ (reference conditions) with the additions of 10 μM or 100 μM of a given divalent cation salt solution (MnCl₂, ZnCl₂, CoCl₂ or NiCl₂), or 50 μM and 500 μM for CaCl₂. Western blots were quantified by near‐infrared fluorescence detection (left), quantified by densitometry and displayed as relative specific activity (graph on the right), which is substrate conversion normalised to enzyme expression level. Black arrow denotes full‐length substrate (MgtE), and red arrow denotes the N‐terminal cleavage product(s) generated by YqgP. All bands originate from the same Western blot and identical treatment series. Irrelevant lanes have been cropped out for clarity, and the source blot is available online as Source Data.
2. Isothermal titration calorimetry of purified recombinant YqgP_NTD and selected divalent cations.
3. Cation toxicity assays and their relationship to YqgP activity. Wild‐type Bacillus subtilis (BTM2, Table EV2), its variant lacking YqgP (ΔyqgP, BTM78, Table EV2) and the rescue strain ectopically expressing YqgP (BTM501, Table EV2) were cultivated in minimal M9 medium containing 10 μM MgSO₄ and 1 μM each of MnCl₂, ZnCl₂, CoCl₂, NiCl₂ and CaCl₂ (i.e. reference conditions), and in mid‐exponential phase were stressed by the addition of either 70 μM MnCl₂, 500 μM ZnCl₂, 25 μM CoSO₄ or 400 μM NiCl₂ (pale blue area). YqgP activity specifically improved cell fitness during manganese and zinc stress, while it had no effect on growth of cells cultivated in the presence of high cobalt and nickel concentrations. Representative experiments of 2–3 independent replicates are shown.

Source data are available online for this figure.