Figure 5. DnaK has growth phase dependent patterns of subcellular localization.

(A) DnaK-mCitrine localizes to peripheral foci during growth. Cells expressing DnaK fused at the C-terminus to mCitrine (MGM6003) at A₆₀₀ 0.2 stained with FM 4-64 and Hoechst prior to imaging. (B). DnaK-mCitrine foci are dynamic. Time-lapse microscopy of DnaK-mCitrine expressing strain (MGM6003) at 37°C. YFP images were acquired every 15 minutes as indicated in bottom left corner (Also see Movie S1 and Figure S12). (C). DnaK-mCitrine localizes to peripheral foci in Mycobacterium bovis BCG. BCG expressing DnaK fused at the C-terminus to mCitrine (MGM6023) at A₆₀₀ 0.2. Overlay is of YFP and DIC images. (D) DnaK-mCitrine localizes to 1 or 2 large foci in stationary phase cells. Cells of DnaK-mCitrine strain (MGM6003) from late stationary phase culture. Overlay is of YFP and DIC images. (E) Catalytically inactive DnaK fails to localize to peripheral foci. Cells expressing DnaK(K70A)-mCitrine (MGM6024) at A₆₀₀ 0.2. Overlay is of YFP and DIC images. (F) GrpE overexpression disperses DnaK-mCitrine foci. Time-lapse microscopy of Tet-GrpE DnaK-mCitrine expressing strain (MGM6016). Time at lower left of panel indicates minutes after addition of ATc to induce GrpE overexpression. Overlay is of YFP and DIC images. For all images: White bars indicate 2 µm. Exposure times were YFP 250 ms, FM 4-64 500 ms, Hoechst 500 ms.