<i>kdpF’</i>::GFP functions as a specific reporter of Mtb response to environmental K⁺ levels.

(A) Mtb response to K+ is concentration-dependent. CDC1551(kdpF’::GFP, smyc’::mCherry) was grown in standard 7H9 broth, pH 7.0, or in K+-free 7H9 broth, pH 7.0, supplemented with indicated amounts of K+. Samples were taken at indicated time points, fixed, and GFP induction analyzed by FACS. Data are shown as means ± SD from 3 independent experiments. (B-C) kdpF’::GFP induction is specific. CDC1551(kdpF’::GFP, smyc’::mCherry) was grown in 7H9 broth, pH 7.0, supplemented with 250 mM NaCl (green triangles) or 300 mM sucrose (blue inverted triangles), in 7H9 broth, pH 5.7 (red squares), or in K+-free 7H9 broth, pH 7.0 (dotted black line, circles) (B). In (C), CDC1551(kdpF’::GFP, smyc’::mCherry) was grown in media in stirred, aerated, cultures, before exposure to 0.5% O2 for 6 days. The hypoxia responsive strain, CDC1551(hspX’::GFP, smyc’::mCherry), was used as a control. All samples were analyzed as in (A), and data are shown as means ± SD from 3 independent experiments. (D) KdpDE inactivation abolishes kdpF’::GFP induction in low [K+]. WT, ΔkdpDE, or kdpDE* (complemented mutant) Mtb expressing the kdpF’::GFP reporter were grown in standard 7H9 broth, pH 7.0, or in K+-free 7H9 broth, pH 7.0, supplemented with 0.05 mM KCl. All samples were analyzed as in (A), and data are shown as means ± SD from 3 independent experiments.