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. 2022 Apr 14;13(3):e00514-22. doi: 10.1128/mbio.00514-22

FIG 4.

FIG 4

Localization of DnaK1 and DnaJ3 and interaction of DnaK1 with the T4P motor complex. (A) Fluorescence micrographs of the dnaK1-gfp and dnaJ3-gfp strains at 0 and 24 h post hormogonium induction. Depicted are merged images of fluorescence micrographs acquired using a 63× lens objective from cellular autofluorescence (red) and GFP fluorescence (cyan) from immobilized filaments. (B) Dynamic localization of DnaK1-GFP in response to light in a motile filament. Depicted are fluorescence micrographs of GFP fluorescence from a motile filament of the dnaK1-gfp strain preincubated in white light for 1 min (light), followed by darkness for 1 min (dark), and subsequently incubated in white light for 1 min (1 m light). Arrow indicates the direction of movement for the filament. White bar = 5 μm. The lower panel depicts the quantification of positional fluorescence intensity. Using ImageJ, a line was drawn along the length of the filament and the pixel intensity was measured at the indicated time points in the light regimen. (C) Localization of DnaK1-GFP and DnaJ3-GFP in response to treatment with CCCP or DMSO alone in a wild-type genetic background or DnaK1-GFP in a ΔhmpF background. Depicted are fluorescence micrographs of GFP fluorescence (cyan). White bar = 5 μm. (D) Quantification of the fraction of DnaK1-GFP or DnaJ3-GFP localized to the poles and cytoplasm. *, P < 0.05 as determined by two-tailed Student’s t test between the CCCP and DMSO treatment for each strain and position measured, n = 3. (E) BACTH analysis between DnaK1, and various cytoplasmic facing proteins of the T4P in N. punctiforme or (F) DnaK1 and DnaJ3 or T4P proteins from Synechocystis sp. strain PCC 6803. Proteins are fused to the C (25) and N (n25) terminus of the T25 fragment or C (18c) and N (18) terminus of the T18 fragment of B. pertussis adenylate cyclase. Depicted are the results from assays on MacConkey agar. The positive-control strain (+) harbors plasmids pKT25-zip and pUT18c-zip, while the negative-control strain (−) harbors the empty vectors pKT25 and pUT18c.