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. 2019 Mar 12;10(2):e02716-18. doi: 10.1128/mBio.02716-18

FIG 6.

FIG 6

Photokinetic analysis of MamK filament dynamics upon popZMgr absence and effects on cell morphology upon overexpression of popZMgr. (A) Photobleaching of mCherry-MamK was used to follow the recovery of the fluorescence corresponding to the MamK filament during 10 min in the ΔpopZMgr strain. The mCherry-MamK translational fusion was expressed from a replicative plasmid under the control of the native PmamAB promoter. The upper panel shows representative cells for this assay, indicating the selected bleached areas (white dashed circle) and fluorescence recovery progression over time. The prebleaching image is a composite of the bright-field and fluorescence channels to display subcellular localization. The lower panel shows the quantification of the MamK filament fluorescence recovery over time. Time point zero was measured immediately after the laser pulse. The half-time fluorescence recovery is presented as t1/2 in the plot. Scale bar = 1 μm. (B) MamK filament pole-to-midcell treadmilling growth behavior analysis by FRAP in the ΔpopZMgr strain. Kymographs display the fluorescence signal intensity (x axis) of bleached mCherry-MamK filaments over time (y axis). The corresponding duplicated kymograph indicates bleaching time/area (red box) and filament fluorescent signal progression (red dashed line). The bleach-marked filaments were followed for 10 min (imaging every 30 s). Scale bars = 500 nm. (C) Prolonged overexpression (23 h postinduction) of popZMgr-gfp results in polar regions devoid of chromosomal DNA (stained with DAPI) and PHB granules (Nile red stain). The inset shows an additional cell with multiple PopZ foci distributed across the cell body. Scale bars = 5 µm. (D) Upon overproduction of PopZMgr (25 h postinduction), a polar exclusion zone devoid of ribosomes, polyhydroxybutyrate (PHB), and polyphosphate (PP) granules is formed. Formation of a large PopZ exclusion zone does not prevent formation of flagella (F) at the same pole. In some cells, the magnetosome chains (M) were found to be enclosed in the PopZ-rich zone. Scale bars = 1 µm.