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 asymmetric septum then divides the cell into two unequally sized compartments. The larger compartment, the mother cell, engulfs the smaller compartment, the forespore. Both the mother cell and forespore coordinate gene expression to mature the spore and the mother cell eventually lyses, releasing the dormant spore into the surrounding environment¹.

The spore structure and composition is conserved across many bacterial species. The spore core has a lower water content than the vegetative cell and is rich with dipicolinic acid (DPA)^1,2. During spore formation, DPA is pumped into the spore core in exchange for water. Surrounding the core is an inner spore membrane where, in most spore forming bacteria, the receptors which recognize the small molecules that stimulate germination (germinants) are located². Located just outside the spore's inner membrane is a layer of cell wall peptidoglycan. A specialized peptidoglycan layer (cortex) surrounds the cell wall peptidoglycan and is composed of many of the same components as cell wall peptidoglycan [alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM)]. However, approximately 50% of the NAM residues in the cortex have been converted to muramic-δ-lactam^5,6. During spore germination, these muramic-δ-lactam residues are recognized by the spore cortex lytic enzymes (SCLEs), thus allowing the cortex to be degraded (a process essential to complete germination) but not the cell wall. Surrounding the cortex is an outer membrane and several layers of coat proteins ².

Controlling the process of germination is critically important for spore-forming bacteria. Germination is initiated when germinant receptors interact with their respective germinants². In many spore-forming bacteria, these germinants are amino acids, sugars, nucleotides, ions or combinations thereof². C. difficile spore germination is initiated by combinations of certain bile acids, [e.g., taurocholic acid (TA)] and amino acids (e.g., glycine)^7-10. Though there are differences between the C. difficile germination pathway and the pathways studied in other spore-forming bacteria, such as B. subtilis, common to all is the absolute requirement to degrade the spore cortex to allow a vegetative cell to grow from the germinated spore^2,8. Cortex degradation can be accomplished by the SCLEs CwlJ/SleB (as found in B. subtilis) or SleC (as found in many Clostridia). Cortex hydrolysis reduces the constraint on the cell wall and the spore. This allows for full core rehydration, a necessary step in reactivating many of the proteins necessary for cellular metabolism ².

When spores germinate, the dormant spore changes from a phase-bright state to a phase-dark state and this process can be measured by a change in optical density (OD) at 600 nm¹¹. A previous report suggests that much of this change in OD is due to the release of DPA from the spore¹². During our recent study, we sought to compare the timing of C. difficile spore germination and monitored the release of DPA and cortex fragments⁹. For this study it was critical to monitor the release of cortex fragments as they began to be released by germinating spores.

The colorimetric assay used here was based upon a method for detecting sugars with reducing ends developed Ghuysen et al.¹³. Because others have described protocols for the detection of reducing sugars¹⁴ or have modified those protocols¹⁵, the literature on this subject can be confusing. Here, we detail a step-by-step method for the colorimetric detection of reducing sugars liberated from germinating C. difficile spores. Though this study uses C. difficile spores, the reducing sugars released during the germination of spores from other spore-forming bacteria are able to be detected with this protocol^9,16,17.

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 1.0 ml sample of the germination solution without spores to serve as a blank for cortex fragment detection.
4. Add spores to an OD₆₀₀ of ~3.0 to the germination solution and vortex quickly.
   Note: For our germination studies in B. subtilis, we used the same OD₆₀₀ spores as for C. difficile.
   Note: This amount of spores worked well for monitoring cortex fragment release during C. difficile spore germination. The amount of spores used in this assay should be determined experimentally for each bacterium or strain.
5. Remove a 1.2 ml sample immediately after adding the spores and centrifuge at room temperature for 1 min at 14,000 × g. This is the zero time point.
6. Transfer 1.0 ml of the supernatant from the centrifuged sample to a fresh tube for subsequent cortex fragment analysis.
   Note: Should DPA need to be monitored, remove a separate 100 μl sample for the detection of DPA in the solution using an assay based on terbium fluorescence^9,18,19.
7. Freeze the collected sample at -80 °C.
8. Repeat steps 1.4 and 1.5 at regular intervals until all time points completed.
   Note: Because of time required for centrifugation and sampling, time points less than 2 min apart were not possible during our procedure, so our sampling intervals were once every 2 min for 14 min.
9. As a positive control for reducing sugar detection and quantification, include the following amounts of N-acetylglucosamine (nmol): 0, 12.5, 25, 50, 100, 250, 500, and 5,000. Prepare these samples at the same time as cortex samples and run along with the cortex samples so that the standard curve is relevant to the data generated by germinating spores.
10. After all time point samples are collected, lyophilize the samples.
    Note: We lyophilized our samples in 2 ml screw-cap tubes.

2. Measuring Cortex Fragments

1. Resuspend lyophilized samples in 120 µl of 3 N HCl supplemented with 1% phenol and 0.5% β-mercaptoethanol.
2. Transfer the resuspended samples to 2-ml screw-cap tubes.
   Note: For reproducibility, ensure all the lyophilized sample is resuspended and transferred.
3. Incubate the samples in a recirculating water bath at 95 °C for 4 hr.
4. After incubation, place the samples on ice until they are cool.
5. Neutralize the samples with 120 µl of 3 M NaOH.
6. Add 80 µl of a saturated sodium bicarbonate solution and 80 µl of a 5% acetic anhydride solution to each sample and vortex.
7. Incubate the samples at room temperature for 10 min.
8. Transfer the samples back to the 95 °C water bath for EXACTLY 3 min.
   Note: This step is time sensitive and longer times can affect downstream signal development.
9. Remove samples from the water bath and cool on ice.
10. Add 400 µl of 6.54% K₂B₄O₇·4H₂O to each tube and vortex.
11. Incubate the samples in a recirculating water bath at 95 °C for EXACTLY 7 min.
    Note: This step is time sensitive and longer times can affect downstream signal development.
12. Remove the samples and place on ice for 5 min.

3. Color Reagent

1. While samples are on ice (Step 2.12), prepare the color reagent. Dissolve 0.320 g of p-dimethylaminobenzaldehyde (DMAB; Ehrlich's reagent) in 1.9 ml of glacial acetic acid.
   Note: The reagent is time, temperature and light sensitive and should not be made in advance.
2. After the DMAB completely dissolves, add 100 µl of 10 N HCl and vortex.
3. Add 5 ml of glacial acetic acid and vortex.

4. Colorimetric Reaction

1. Transfer 100 µl of each cooled cortex sample to a new 1.5 ml microcentrifuge tube.
2. Add 700 µl of the freshly-prepared color solution to each sample.
3. Immediately transfer the samples to a 37 °C water bath and incubate for EXACTLY 20 min.
4. Transfer 200 µl of each sample to a clear 96-well plate.
5. Quantify the samples at 585 nm on a plate reader. Samples should start at a yellow color and a positive reaction will shift to purple. The more cortex present, the deeper the purple color.

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

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