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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

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‐N3) has been reported but is inefficient. For optimization, it is important to understand how exogenous Kdo‐N3 enters the cytoplasm. Based on similarities between Kdo and sialic acids, we proposed and verified that the sialic acid transporter NanT imports exogenous Kdo‐N3 into E. coli. We demonstrated that E. coli ΔnanT were not labeled with Kdo‐N3, while expression of NanT in the ΔnanT mutant restored Kdo‐N3 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‐N3 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‐N3 incorporation and labeling, though this led to abnormal morphology and growth arrest. NanT seems to define which bacteria can be labeled with Kdo‐N3, provides opportunities to enhance Kdo‐N3 labeling efficiency and spectrum, and raises the possibility of Kdo biosynthetic bypass where exogenous Kdo is present, perhaps even in vivo.