Abstract
High-performance liquid chromatography (HPLC) analysis has been critical for determining the structural and chemical complexity of the cell wall. However this method is very time consuming in terms of sample preparation and chromatographic separation. Here we describe (1) optimized methods for peptidoglycan isolation from both Gram-negative and Gram-positive bacteria that dramatically reduce the sample preparation time, and (2) the application of the fast and highly efficient ultra-performance liquid chromatography (UPLC) technology to muropeptide separation and quantification. The advances in both analytical instrumentation and stationary-phase chemistry have allowed for evolved protocols which cut run time from hours (2–3 h) to minutes (10–20 min), and sample demands by at least one order of magnitude. Furthermore, development of methods based on organic solvents permits in-line mass spectrometry (MS) of the UPLC-resolved muropeptides. Application of these technologies to high-throughput analysis will expedite the better understanding of the cell wall biology.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Vollmer W, Blanot D, de Pedro MA (2008) Peptidoglycan structure and architecture. FEMS Microbiol Rev 32(2):149–167. doi:10.1111/j.1574-6976.2007.00094.x, FMR094 [pii]
Alvarez L, Espaillat A, Hermoso JA, de Pedro MA, Cava F (2014) Peptidoglycan remodeling by the coordinated action of multispecific enzymes. Microb Drug Resist 20(3):190–198. doi:10.1089/mdr.2014.0047
Cava F, de Pedro MA (2014) Peptidoglycan plasticity in bacteria: emerging variability of the murein sacculus and their associated biological functions. Curr Opin Microbiol 18:46–53. doi:10.1016/j.mib.2014.01.004
Glauner B, Holtje JV, Schwarz U (1988) The composition of the murein of Escherichia coli. J Biol Chem 263(21):10088–10095
Desmarais SM, De Pedro MA, Cava F, Huang KC (2013) Peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell wall structure and assembly. Mol Microbiol 89(1):1–13. doi:10.1111/mmi.12266
Ikeda S, Hanaki H, Yanagisawa C, Ikeda-Dantsuji Y, Matsui H, Iwatsuki M, Shiomi K, Nakae T, Sunakawa K, Omura S (2010) Identification of the active component that induces vancomycin resistance in MRSA. J Antibiot 63(9):533–538. doi:10.1038/ja.2010.75
Filipe SR, Tomasz A (2000) Inhibition of the expression of penicillin resistance in Streptococcus pneumoniae by inactivation of cell wall muropeptide branching genes. Proc Natl Acad Sci U S A 97(9):4891–4896. doi:10.1073/pnas.080067697
Lortal S, Van Heijenoort J, Gruber K, Sleytr UB (1992) S-layer of Lactobacillus helveticus ATCC 12046: isolation, chemical characterization and re-formation after extraction with lithium chloride. Microbiology 138(3):611–618. doi:10.1099/00221287-138-3-611
Liang OD, Flock JI, Wadstrom T (1995) Isolation and characterisation of a vitronectin-binding surface protein from Staphylococcus aureus. Biochim Biophys Acta 1250(1): 110–116
Regulski K, Courtin P, Meyrand M, Claes IJ, Lebeer S, Vanderleyden J, Hols P, Guillot A, Chapot-Chartier MP (2012) Analysis of the peptidoglycan hydrolase complement of Lactobacillus casei and characterization of the major gamma-D-glutamyl-L-lysyl-endopeptidase. PLoS One 7(2):e32301. doi:10.1371/journal.pone.0032301
Hill SA, Judd RC (1989) Identification and characterization of peptidoglycan-associated proteins in Neisseria gonorrhoeae. Infect Immun 57(11):3612–3618
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Alvarez, L., Hernandez, S.B., de Pedro, M.A., Cava, F. (2016). Ultra-Sensitive, High-Resolution Liquid Chromatography Methods for the High-Throughput Quantitative Analysis of Bacterial Cell Wall Chemistry and Structure. In: Hong, HJ. (eds) Bacterial Cell Wall Homeostasis. Methods in Molecular Biology, vol 1440. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3676-2_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3676-2_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3674-8
Online ISBN: 978-1-4939-3676-2
eBook Packages: Springer Protocols