The sialic acid transporter NanT is necessary and sufficient for uptake of 3‐deoxy‐d‐manno‐oct‐2‐ulosonic acid (Kdo) and its azido analog in Escherichia coli

Summary 3‐Deoxy‐ d ‐ manno ‐oct‐2‐ulosonic acid (Kdo) is an essential component of lipopolysaccharides (LPS) in the Gram‐negative bacterial outer membrane. Metabolic labeling of Escherichia coli LPS with 8‐azido‐3,8‐dideoxy‐ d ‐ manno ‐oct‐2‐ulosonic acid (Kdo‐N 3 ) has been reported but is inefficient. For optimization, it is important to understand how exogenous Kdo‐N 3 enters the cytoplasm. Based on similarities between Kdo and sialic acids, we proposed and verified that the sialic acid transporter NanT imports exogenous Kdo‐N 3 into E. coli . We demonstrated that E. coli Δ nanT were not labeled with Kdo‐N 3 , while expression of NanT in the Δ nanT mutant restored Kdo‐N 3 incorporation. Induced NanT expression in a strain lacking Kdo biosynthesis led to higher exogenous Kdo incorporation and restoration of full‐length core‐LPS, suggesting that NanT also transports Kdo. While Kdo‐N 3 incorporation was observed in strains having NanT, it was not detected in Pseudomonas aeruginosa and Acinetobacter baumannii , which lack nanT . However, heterologous expression of E. coli NanT in P. aeruginosa enabled Kdo‐N 3 incorporation and labeling, though this led to abnormal morphology and growth arrest. NanT seems to define which bacteria can be labeled with Kdo‐N 3 , provides opportunities to enhance Kdo‐N 3 labeling efficiency and spectrum, and raises the possibility of Kdo biosynthetic bypass where exogenous Kdo is present, perhaps even in vivo.