Computational protocol: Cell wall remodeling drives engulfment during Bacillus subtilis sporulation

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Protocol publication

[…] MreB tracking experiments were performed using the strain JLG2411, which produced GFP-MreB in the forespore after polar septation from spoIIQ promoter. Sporulation and agarose pads were done as described in Timelapse fluorescent microscopy, except that FM 4–64 was only added to the agarose pads and not to the sporulating cultures. A static membrane picture was taken at the beginning of the experiment, and was used as a reference to determine the position of the GFP-MreB foci. GFP-MreB motion at the cell surface was determined by TIRF microscopy (; ), taking pictures every 4 s for 100 s. Imaging was performed at 30°C. We used two different microscopes to perform TIRF microscopy: (i) An Applied Precision Spectris optical sectioning microscope system equipped with an Olympus IX70 microscope, a Photometrics CoolSNAP HQ digital camera and a 488 nm argon laser. To perform TIRF in this microscope, we used an Olympus 1003 1.65 Apo objective, immersion oil n = 1.78 (Cargille Laboratories), and sapphire coverslips (Olympus). Laser power was set to 15%, and exposure time was 200 ms. (ii) An Applied Precision OMX Structured Illumination microscopy equipped with a Ring-TIRF system and a UApoN 1.49NA objective, immersion oil n = 1.518. Exposure time was 150 ms.Images were analyzed using the ImageJ-based FIJI package. Sporangia were aligned vertically using the rotation function in FIJI. GFP-MreB foci were tracked using the TrackMate pluging (), using the LoG detector, estimated blob diameter of 300 nm, simple LAP tracked and linking max distance of 300 nm. Only tracks that contained more than four points were used to determine the MreB foci speed. […]

Pipeline specifications

Software tools ImageJ, TrackMate
Application Microscopic phenotype analysis
Organisms Bacillus subtilis
Chemicals Penicillins