Open AccessBacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC)
Author(s) -
Manuel Pazos,
Christian Otten,
Waldemar Vollmer
Publication year - 2018
Publication title -
bio-protocol
Language(s) - English
Resource type - Journals
ISSN - 2331-8325
DOI - 10.21769/bioprotoc.2761
Subject(s) - peptidoglycan , lipid ii , chromatography , lysis , chemistry , high performance liquid chromatography , bacterial cell structure , biochemistry , thin layer chromatography , size exclusion chromatography , pyrophosphate , cell wall , enzyme , biology , bacteria , genetics
Peptidoglycan encases the bacterial cytoplasmic membrane to protect the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis require a membrane-anchored substrate called lipid II, in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid II is the target of glycopeptide antibiotics and several antimicrobial peptides, and is degraded by ‘attacking’ enzymes involved in bacterial competition to induce lysis. Here we describe two protocols using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC), respectively, to assay the digestion of lipid II by phosphatases such as Colicin M or the LXG toxin protein TelC from Streptococcus intermedius . The TLC method can also monitor the digestion of undecaprenyl (pyro)phosphate, whereas the HPLC method allows to separate the di-, mono- or unphosphorylated disaccharide pentapeptide products of lipid II.