Bacterial strains and plasmids

Escherichia coli CLM24 (O polysaccharide ligase WaaL negative derivative of W3110, [3]) was used as host strain for all in vivo glycosylation experiments. For construction of plasmid libraries by cassette mutagenesis, E. coli XL10 Gold cells were used (Agilent-Stratagene, La Jolla, CA, USA).

Plasmids used in this study are listed in Table 1. Expression vector pACT3Kan was constructed by insertion of a Kanamycin resistance cassette into the cat chloramphenicol resistance gene of pACT3 [25] by homologous recombination. Plasmids pGVXN112Kan and pGVXN113Kan were constructed by PCR-sub cloning of the sequence coding for wt PglB with C terminal HA tag and inactive PglB-HA W458A-D459A from pGVXN114 and pGVXN115, respectively, into pACT3Kan using restriction sites KpnI and BamHI. Construction of plasmid pGVXN345 for expression of the CP5 polysaccharide biosynthesis cluster of S. aureus will be described elsewhere (GlycoVaxyn and Jean Lee, manuscript in preparation).