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. Issue 2 (30th September 2018)

• Record Type: Journal Article
• Authors: Nilsson, Inga; Prathapam, Ramadevi; Grove, Kerri; Lapointe, Guillaume; Six, David A.
• Is Part Of: Molecular microbiology. Volume 110:Issue 2(2018)
• Page Start: 204
• Page End: 218
• Publication Date: 2018-09-30
• Subjects: Molecular microbiology -- Periodicals; 572.829
• DOI: 10.1111/mmi.14098
• Languages: English
• ISSNs: 0950-382X

Abstract

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‐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 Δ nanT were not labeled with Kdo‐N3, while expression of NanT in the Δ nanT 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. Abstract : Lipopolysaccharides (LPS) form the outside of the Gram‐negative bacterial outer membrane. The uptake and incorporation of a Kdo sugar analog, Kdo‐N3, into Escherichia coli LPS is inefficient. Our work identifies the sialic acid sugar transporter NanT as the uptake mechanism of exogenous Kdo and Kdo‐N3 . This insight will help to enhance Kdo‐N3 incorporation, to understand which bacterial strains can be labeled with Kdo‐N3, and to enable LPS labeling in bacteria which lack the transporter.