FIGURE 6.

O-antigen modification is dependent on a functional ABC transporter. The samples were proteinase K-treated whole cell lysates of E. coli CWG1217 (Δwzx-wbbK) and CWG1219 (Δwzx-wbbK ΔgtrA) transformed with pWQ677 (carrying wbbL-wzm-wbbB) and pWQ114 (encoding FLAG-Wzt). Gene expression was induced with 2.5 ng ml⁻¹ anhydrotetracycline to give a constant level of the biosynthesis enzymes. The amount of FLAG-Wzt (NBD) was varied by repression with glucose or induction with different concentrations of l-arabinose. The presence of the O12 antigen in LPS and Und-PP-linked glycans was assessed using silver stain (top panel) and immunoblotting (middle panel) of proteinase K-treated whole cell lysates. The silver stain profile detects only lipid-A linked O-antigen, whereas the immunoblot detects O-antigen linked to both lipid-A and UndPP. The Western immunoblot of FLAG-tagged Wzt confirmed the presence of the NBD (bottom panel). The markers indicated on the left are protein standards to allow comparison of LPS migrations in different backgrounds.