Abstract
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.
Highlights
The peptidoglycan (PG) sacculus is an essential bacterial macromolecule that protects the cell from bursting due to its turgor and maintains the shape of the cell (Vollmer and Bertsche, 2008; Typas et al, 2012)
Whilst the group of actinomycetes are known for their capability to secrete a repertoire of small metabolites that often show antibacterial activity, many Gram-negative bacteria utilize sophisticated type VI secretion systems to target adjacent bacterial cells by antimicrobial enzymes (Russell et al, 2011; 2012 and 2014)
We provide a detailed description of the thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC) methods that established the degradation of lipid II and C55-PP by TelC (Whitney et al, 2017)
Summary
The peptidoglycan (PG) sacculus is an essential bacterial macromolecule that protects the cell from bursting due to its turgor and maintains the shape of the cell (Vollmer and Bertsche, 2008; Typas et al, 2012). PG glycosyltransferases (GTases) polymerize lipid II at the outer leaflet of the membrane to glycan chains This reaction releases C55-PP which is dephosphorylated for new rounds of precursor synthesis and transport. Whilst the group of actinomycetes are known for their capability to secrete a repertoire of small metabolites that often show antibacterial activity, many Gram-negative bacteria utilize sophisticated type VI secretion systems to target adjacent bacterial cells by antimicrobial enzymes (Russell et al, 2011; 2012 and 2014). We provide a detailed description of the TLC and HPLC methods that established the degradation of lipid II and C55-PP by TelC (Whitney et al, 2017) These methods can be generally used to assess the activity and specificity of phosphatases against membrane-bound bacterial cell wall precursors. Brown glass vials (Fisher Scientific, catalog number: 11531474) Part I: Thin-layer chromatography assay
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