Visualization Of Germinosomes And The Inner Membrane In Bacillus Subtilis Spores
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS(2019)
Abstract
The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores' germinosomes, cluster(s) of germination proteins. We also present a (standard) 3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that > 80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores' 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess colocalization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.
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Key words
Bioengineering,Issue 146,Gram-Positive Bacteria,Bacillales,Microscopy,microbiology,Organisms,Bacteria,Analytical,Diagnostic and Therapeutic Techniques and Equipment,Investigative Techniques,Life sciences,Life Sciences (General),Bacillus,Spores,germination proteins,Germinosomes,Spore Inner Membrane,super resolution microscopy,Fluorescence Microscopy
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