Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Summary

Germinant receptor proteins cluster in ‘germinosomes’ in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

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 co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Introduction

Spores of the orders Bacillales and Clostridiales are metabolically dormant and extraordinarily resistant to harsh decontamination regimes, but unless they germinate, cannot cause deleterious effects in humans¹. In nutrient germinant triggered germination of Bacillus subtilis (B. subtilis) spores, the initiation event is germinant binding to germinant receptors (GRs) located in the spore’s inner membrane (IM). Subsequently, the GRs transduce signals to the SpoVA channel protein also located in the IM. This results in the onset of the exchange of spore core pyridine-2,6-dicarboxylic acid (dipicolinic acid; DPA; comprising....

Protocol

1. B. subtillis Sporulation (Timing: 7 Days Before Microscopic Observation)

1. Day 1
   1. Streak a bacterial culture on a Luria-Bertani Broth (LB) agar plate (1% tryptone, 0.5% yeast extract, 1% NaCl, 1% agar)¹³ and incubate overnight at 37 °C to obtain single colonies. Use the B. subtilis KGB80 (PS4150 gerKA gerKC gerKB-mCherry cat, gerD-gfp kan) strain and its parent background strain B. subtilis PS4150 (PS832 ΔgerE::spc, ΔcotE::tet) as described previously⁷.
      NOTE: The use of spores with the &#....

Results

The current protocol presents a SIM microscope imaging procedure for bacterial spores. The sporulation and slide preparation procedures were carried out as shown in Figure 1 before imaging. Later, the imaging and analysis procedures were applied both for dim (fluorescent protein labeled germination proteins) and bright (lipophilic probe stained IM) spore samples as shown in the following text.

Discussion

 The protocol presented contains a standard 3D-SIM procedure for analysis of FM4-64 stained B. subtilis spores that includes sporulation, slide preparation and imaging processes. In addition, the protocol describes a modified SIMcheck (ImageJ)-assisted 3D imaging process for SIM microscopy of B. subtilis spore germinosomes labeled with fluorescent reporters. The latter procedure allowed us to observe this dim substructure with enhanced contrast. By coupling two imaging procedures, it is possible to.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Air dried glass slides	Menzel Gläser	630-2870
APO TIRF N20R8 100× oil objective (NA=1.49)
B. subtilis KGB80 (PS4150 gerKA gerKC gerKB-mCherry cat, gerD-gfp kan)
B. subtilis PS4150 (PS832 ΔgerE::spc, ΔcotE::tet)
Erlenmeyer flasks 1 L	Sigma-Aldrich	Z567868
Erlenmeyer flasks 250 mL	Sigma-Aldrich	Z723088
FluoSpheres carboxylate-modified microspheres	Invitrogen, 0.1 μm	F8803
FM4-64	Thermo Fisher Scientific	F34653
Histodenz nonionic density gradient medium	Sigma-Aldrich	D2158
ImageJ
iXON3 DU-897 X-6515 CCD camera	Andor Technology		https://imagej.net/Welcome
LB Agar	Sigma-Aldrich	L2897
Microfuge tubes 1.5 mL	Thermo Fisher Scientific	3451PK
Microscope imaging software	Nikon, Japan	NIS-Element AR 4.51.01
MilliQ Ultrapure Deminerilzed Water	Millipore	Milli-Q IQ 7003
Nikon Eclipse Ti microscope
Polypropylene Screw Cap Bottle 180 mL	Thermo Fisher Scientific	75003800
Precision Coverslips	Paul Marienfeld	117650
Round Bottom tubes 15 mL	Thermo Fisher Scientific		Nunc TM
Screw cap tubes 50 mL	Thermo Fisher Scientific		Nunc TM