O-GlcNAcylation had been shown to be required for infection by phage serogroups A,B and F(ref,parkk74), though other functions were unclear. Brown and colleagues identified TarM as the glycosyltransferase responsible for a-O-GlcNAcylation(xia10) through observations that a RN4220 strain with a transposon interruption in this gene lacked a-GlcNAcylated WTAs. This was a significant advance in their continued efforts to elucidate the steps of WTA biosynthetic pathway and was the first S.aureus glycosyltransferase whose function was confirmed in vitro. Conversely, the gene responsible for B-O-GlcNAcylation remained elusive, providing scope for their 2012 …show more content…
This required knowledge of the genes involved in WTA modification, so they first aimed to identify the gene responsible for B-O-GlcNAcylation. This allowed subsequent use an array of genetic and biochemical techniques to explore the correlations between loss of specific tailoring modifications and the observed phenotypes of WTA null mutants, allowing them to demonstrate B-O-GlcNAcylation is required for B-lactam resistance and therefore hypothesise that this modification could act as an effective target for new antimicrobials.
Identification of B-O-GlcNAc Transferase in S.aureus
Genetic screening of the WTA operons of S.aureus strain NCTC8325 identified two putative glycosyltransferase genes SAOUHSC_00228 and SAOUHSC_0064. An in vitro reconstitution approach was used to test the function of these genes, which were expressed as N-terminal 10-His fusion proteins and incubated with UDP-[14C]-GlcNAc and poly(RboP). PAGE phophoimaging revealed that the SAOUHSC_00228 encoded protein produced radiolabelled products of similar mobility to [14C]-WTA polymers, confirming the gene encoded a WTA-glycosyltransferase, redesignated TarS.
UDP-GlcNAc is the preferred donor of TarS and is specific for