Detecting Cortex Fragments During Bacterial Spore Germination

Summary

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

Abstract

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Introduction

Endospores are metabolically dormant forms of bacteria that allow bacteria to persist in unfavorable environments. In many spore-forming bacteria, spore formation is induced by nutrient deprivation but this process can be controlled by changes to pH, exposure to oxygen or other stresses¹. While in their metabolically dormant spore form, bacteria resist UV radiation, desiccation, higher temperatures, and freezing². Most of the knowledge on the sporulation process comes from studies in the model organism, Bacillus subtilis. The sporulation process begins with DNA replication and the formation of an axial filament^3,4. An asymmetr....

Protocol

1. Generating Samples 

1. Heat activate C. difficile spores at 65 °C for 30 min and store on ice.
   Note: C. difficile spores can be produced and purified as described previously^7,9,10.
2. Prepare the germination solution at X + 1 ml where X is the number of samples to be taken during the assay.
   Note: The germination solution is specific to the species of bacteria spore being studied. For assaying C. difficile spore germination, we used a solution of 10 mM Tris (pH 7.5), 150 mM NaCl, 100 mM glycine and 10 mM taurocholic acid in deionized water.
3. Before beginning the assay, draw a....

Results

Table 1 shows typical results using NAG standards. Data are used to generate a standard curve. Table 2 shows typical results from a germination assay in germination buffer supplemented with 100 mM glycine and 10 mM TA (germination-promoting conditions) or 100 mM glycine only (non-germinating conditions). In the absence of TA, C. difficile spores do not germinate and there is little change in the presence of cortex fragments in the solution. Howev.......

Discussion

Upon stimulation, spores undergoing the process of germination lose their resistance properties without the need for macromolecular synthesis. When spore germination is triggered, the spore core releases DPA in exchange for water². Due to the high DPA content of the dormant spore, the spore core is under intense osmotic pressure and the specialized peptidoglycan, cortex, helps prevent the core from swelling by acting, presumably, as a barrier to expansion².

The method des.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2.0 ml screw cap tube	USA scientific	1420-3700
p1000 Eppendorf pipet	Eppendorf
p200 Eppendorf pipet	Eppendorf
p20 Eppendorf pipet	Eppendorf
100 - 1,250 μl pipet tips	VWR	89079-486
1 - 200 μll pipet tips	VWR	89079-458
N-acetyl-D-glucosamine	Sigma	A3286-25G
Hydrochloric Acid - 10 N	BDH-Aristar	BDH3032-3.8LP
Acetic Anhydride	Alfa Aesar	L04295
Sodium Bicarbonate	BDH	BDH0280-500G
Potassium Tetraborate tetrahydrate	Alfa Aesar	39435
Sodium Hydroxide	BDH	BDH8019-500G
4-(Dimethylamino)benzaldehyde	Sigma	156477-100G
Saturated Phenol	Fisher	BP1750-400
2-Mercaptoethanol	Aldrich	M6250-100ML
SpectraMax M3	Molecular Devices
Acetic Acid, Glacial	BDH	BDH3098-3.8LP
Heated, Circulating Water Bath	VWR Scientific	Model 1136
Microtest 96	Falcon	353072	96 well clear tissue culture plates
Culture tubes	VWR	89000-506
Lyophilizer
Heat Blocks
Vortex Machine