Extraction and Purification of Wall-Bound Polymers of Gram-Positive Bacteria
The envelope of gram-positive bacteria encompasses the cell wall, a rigid exoskeleton comprised of peptidoglycan that provides protection against lysis and governs bacterial cell shapes. Peptidoglycan also serves as the site of attachment for proteins and nonproteinaceous polymers that interact with the bacterial environment. Nonproteinaceous molecules include teichoic acids, capsular polysaccharides, and secondary cell wall polysaccharides (SCWP). Treatment of gram-positive bacterial cells with proteases, nucleases, and detergents results in the isolation of “murein sacculi” (i.e., peptidoglycan with bound carbohydrate polymers). Incubation of sacculi with acid or base releases carbohydrate polymers that can be purified for further biochemical characterization. This protocol describes the hydrofluoric acid extraction and purification of the secondary cell wall polymer of Bacillus anthracis that is also found in the envelope of the other members of the Bacillus cereus sensu lato group of bacteria.
Key wordsSecondary cell wall polysaccharide Bacillus cereus Murein sacculus Peptidoglycan Hydrofluoric acid Phosphodiester bond
We thank current and past members of the laboratory who helped develop and refine this protocol. Research in our laboratory is supported by grant number AI069227 from the National Institute of Allergy and Infectious Diseases, Infectious Diseases Branch.
- 20.Forsberg LS, Abshire TG, Friedlander A et al (2012) Localization and structural analysis of a conserved pyruvylated epitope in Bacillus anthracis secondary cell wall polysaccharides and characterization of the galactose deficient wall polysaccharide from avirulent B. anthracis CDC 684. Glycobiology 22:1103–1117CrossRefGoogle Scholar
- 21.Chateau A, Lunderberg JM, Oh SY et al (2018) Galactosylation of the secondary cell wall polysaccharide of Bacillus anthracis and its contribution to anthrax pathogenesis. J Bacteriol 200(5). https://doi.org/10.1128/JB.00562-17