Figure 1: Radioactive labelling and detection of the PG sugars GlcNAc and MurNAc by TLC.

(a) Principle of the radioactive kinase assay: polymeric PG is first partly digested by mutanolysin into disaccharides (stem peptides are still linked to MurNAc). The GlcNAc/MurNAc kinase MurK is not able to transfer phosphate (P) from [γ-³²P]-ATP to polymers (1) and disaccharides (2) and thus, no signal is detected in TLC. Subsequently, the disaccharides are further digested into monomers and stem peptides (3). MurK is able to transfer the [γ-³²P] from [γ-³²P]-ATP to the sugar monomers GlcNAc and MurNAc and thus, a radioactive signal can be detected by TLC. (b) Radioactive detection of GlcNAc and MurNAc in either undigested, partly digested (treatment with mutanolysin) or fully digested (treatment with mutanolysin, AmiD and BsNagZ) preparations of P. limnophilus, R. baltica and G. obscuriglobus cells on incubation with [γ-³²P]-ATP and MurNAc/GlcNAc kinase MurK and subsequent TLC separation. Shown are time 0 and 1 h of incubation with [γ-³²P]-ATP. C−: water negative control; C+: GlcNAc and MurNAc standards as positive controls.