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Immunology and Infection

Detecting Cortex Fragments During Bacterial Spore Germination

Published: June 25th, 2016



1Department of Biology, Texas A&M University

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

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.

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 filament3,4. An asymmetr....

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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 previously7,9,10.
  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 so.......

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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.......

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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.......

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The Project described is supported by Award Number 5R01AI116895 from the National Institute of Allergy and Infectious Diseases. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health.


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Name Company Catalog Number Comments
2.0 mL screw cap tube USA scientific 1420-3700
p1000 eppendorf pippet Eppendorf
p200 eppendorf pippet Eppendorf
p20 eppendorf pippet Eppendorf
100-1250uL pipet tips VWR 89079-486
1-200uL pipet tips VWR 89079-458
N-acetyl-D-glucosamine Sigma A3286-25G
Hydrochloric Acid - 10N 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
Heat Blocks
Vortex Machine

  1. Al-Hinai, M. A., Jones, S. W., Papoutsakis, E. T. The Clostridium sporulation programs: diversity and preservation of endospore differentiation. Microbiol Mol Biol Rev. 79, 19-37 (2015).
  2. Setlow, P. Germination of spores of Bacillus species: what we know and do not know. J Bacteriol. 196, 1297-1305 (2014).
  3. Tan, I. S., Ramamurthi, K. S. Spore formation in Bacillus subtilis. Environ Microbiol Rep. 6, 212-225 (2014).
  4. Fimlaid, K. A., Shen, A. Diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes. Curr Opin Microbiol. 24, 88-95 (2015).
  5. Popham, D. L., Helin, J., Costello, C. E., Setlow, P. Analysis of the peptidoglycan structure of Bacillus subtilis endospores. J Bacteriol. 178, 6451-6458 (1996).
  6. Atrih, A., Zollner, P., Allmaier, G., Foster, S. J. Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation. J Bacteriol. 178, 6173-6183 (1996).
  7. Sorg, J. A., Sonenshein, A. L. Bile salts and glycine as cogerminants for Clostridium difficile spores. J. Bacteriol. 190, 2505-2512 (2008).
  8. Paredes-Sabja, D., Shen, A., Sorg, J. A. Clostridium difficile spore biology: sporulation, germination, and spore structural proteins. Trends Microbiol. 22 (7), (2014).
  9. Francis, M. B., Allen, C. A., Sorg, J. A. Spore cortex hydrolysis precedes DPA release during Clostridium difficile spore germination. J Bacteriol. 197 (14), (2015).
  10. Francis, M. B., Allen, C. A., Shrestha, R., Sorg, J. A. Bile acid recognition by the Clostridium difficile Germinant Receptor, CspC, is important for establishing infection. PLoS Pathog. 9, e1003356 (2013).
  11. Moir, A., Smith, D. A. The Genetics of bacterial spore germination. Annu. Rev. Microbiol. 44, 531-553 (1990).
  12. Zhang, P., Kong, L., Wang, G., Setlow, P., Li, Y. Q. Combination of Raman tweezers and quantitative differential interference contrast microscopy for measurement of dynamics and heterogeneity during the germination of individual bacterial spores. Journal of biomedical optics. 15, 056010 (2010).
  13. Ghuysen, J. -. M., Tipper, D. J., Strominger, J. L. Enzymes that degrade bacterial cell walls. Methods Enzymol. 8, 685-699 (1966).
  14. Park, J. T., Johnson, M. J. A submicrodetermination of glucose. J Biol Chem. 181, 149-151 (1949).
  15. Thompson, J. S., Shockman, G. D. A modification of the Park and Johnson reducing sugar determination suitable for the assay of insoluble materials: its application to bacterial cell walls. Anal Biochem. 22, 260-268 (1968).
  16. Paredes-Sabja, D., Setlow, P., Sarker, M. R. SleC is essential for cortex peptidoglycan hydrolysis during germination of spores of the pathogenic bacterium Clostridium perfringens. J Bacteriol. 191, 2711-2720 (2009).
  17. Paredes-Sabja, D., Sarker, M. R. Effect of the cortex-lytic enzyme SleC from non-food-borne Clostridium perfringens on the germination properties of SleC-lacking spores of a food poisoning isolate. Can J Microbiol. 56, 952-958 (2010).
  18. Hindle, A. A., Hall, E. A. Dipicolinic acid (DPA) assay revisited and appraised for spore detection. The Analyst. 124, 1599-1604 (1999).
  19. Kort, R., et al. Assessment of heat resistance of bacterial spores from food product isolates by fluorescence monitoring of dipicolinic acid release. Appl Environ Microbiol. 71, 3556-3564 (2005).
  20. Elson, L. A., Morgan, W. T. A colorimetric method for the determination of glucosamine and chondrosamine. The Biochemical journal. 27, 1824-1828 (1933).
  21. Ramirez, N., Abel-Santos, E. Requirements for germination of Clostridium sordellii spores in vitro. J. Bacteriol. 192, 418-425 (2010).
  22. Akoachere, M., et al. Indentification of an in vivo inhibitor of Bacillus anthracis spore germination. J. Biol. Chem. 282, 12112-12118 (2007).
  23. Duncan, C. L., Strong, D. H. Improved medium for sporulation of Clostridium perfringens. Appl Microbiol. 16, 82-89 (1968).
  24. Heffron, J. D., Lambert, E. A., Sherry, N., Popham, D. L. Contributions of four cortex lytic enzymes to germination of Bacillus anthracis spores. J Bacteriol. 192, 763-770 (2010).
  25. Meador-Parton, J., Popham, D. L. Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation. J Bacteriol. 182, 4491-4499 (2000).
  26. Lambert, E. A., Popham, D. L. The Bacillus anthracis SleL (YaaH) protein is an N-acetylglucosaminidase involved in spore cortex depolymerization. J Bacteriol. 190, 7601-7607 (2008).
  27. Wang, S., Shen, A., Setlow, P., Li, Y. Q. Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy. J Bacteriol. 197, 2361-2373 (2015).

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