Journal: The Journal of Biological Chemistry

Article Title: Noncanonical lipooligosaccharide assembly in Acinetobacter baumannii is mediated by the glycosyltransferases KdoT and GnaT

doi: 10.1016/j.jbc.2025.111103

Figure Lengend Snippet: Cellular localization and purification of KdoT and GnaT proteins. A , Top : SDS-PAGE with Coomassie staining of cell-free extract (CFE), soluble, and membrane fractions of Acinetobacter baumannii Δ kdoT with empty vector (EV) or expressing KdoT-His 8 (∼35 kDa) to identify subcellular localization. Bottom: Western blot of KdoT-His 8 . B , Top : SDS-PAGE with Coomassie staining of cell-free extract (CFE), soluble, and membrane fractions of A. baumannii Δ gnaT with empty vector or expressing GnaT-His 8 (∼42 kDa) to identify subcellular localization. Bottom : Western blot of GnaT-His 8 . Data in ( A ) and ( B ) are representative of three biological replicates. C , SDS-PAGE with Coomassie staining of cell-free extract (CFE), membrane fractions, and purified proteins isolated from Escherichia coli BLR (DE3) pLysS containing either pET21a:: kdoT-His 8 or:: gnaT - His 8 .

Article Snippet: The amino acid sequences of KdoT and GnaT from A. baumannii ATCC 17978 were submitted to ChaiDiscovery ( ) for structure prediction.

Techniques: Purification, SDS Page, Staining, Membrane, Plasmid Preparation, Expressing, Western Blot, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Noncanonical lipooligosaccharide assembly in Acinetobacter baumannii is mediated by the glycosyltransferases KdoT and GnaT

doi: 10.1016/j.jbc.2025.111103

Figure Lengend Snippet: Early core oligosaccharide assembly in Acinetobacter baumannii . A , pathway overview of early lipid A and core OS synthesis. Early lipid A synthesis from UDP-GlcNAc yields a bis -phosphorylated lipid IV A precursor. The Kdo transferase WaaA (also known as KdtA) begins core OS synthesis via the transfer of Kdo residues using CMP-Kdo as the donor to form Kdo 2 -lipid IV A . Late acylation steps complete Kdo 2 -lipid A assembly using acyl-ACPs as donor substrates. The addition of the acyloxyacyl-linked fatty acid at the 2-position, however, is partial ( dashed bond) resulting in both hexa- and hepta-acylated Kdo 2 -lipid A species in A. baumannii . Core OS is extended via transfer of CMP-Kdo and UDP-GlcNAcA by two proteins, KdoT and GnaT, characterized here . The KdoIII residue added by KdoT diverges from inner core assembly in other bacteria, as WaaA is normally responsible for the transfer of all Kdo sugars. The core OS is then extended by a series of enzymes not shown in the schematic, completing LOS assembly. B , diagram of the complete LOS structure of A. baumannii ATCC 17978. Dashed lines indicate proteins predicted to transfer their corresponding residues based on knockout mutation analysis and prior literature ( , ). Note that deletion of either kdoT or gnaT results in an identical Kdo 2 –lipid A chemotype and expression of both proteins is required for KdoIII and GlcNAcA addition ( red box ) in whole cells. For most strains characterized to date, the inner core structure of A. baumannii LOS is conserved. Sugar residues are depicted using the official Symbol Nomenclature for Glycans (SNFG) .

Article Snippet: The amino acid sequences of KdoT and GnaT from A. baumannii ATCC 17978 were submitted to ChaiDiscovery ( ) for structure prediction.

Techniques: Residue, Bacteria, Knock-Out, Mutagenesis, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Noncanonical lipooligosaccharide assembly in Acinetobacter baumannii is mediated by the glycosyltransferases KdoT and GnaT

doi: 10.1016/j.jbc.2025.111103

Figure Lengend Snippet: Cellular localization and purification of KdoT and GnaT proteins. A , Top : SDS-PAGE with Coomassie staining of cell-free extract (CFE), soluble, and membrane fractions of Acinetobacter baumannii Δ kdoT with empty vector (EV) or expressing KdoT-His 8 (∼35 kDa) to identify subcellular localization. Bottom: Western blot of KdoT-His 8 . B , Top : SDS-PAGE with Coomassie staining of cell-free extract (CFE), soluble, and membrane fractions of A. baumannii Δ gnaT with empty vector or expressing GnaT-His 8 (∼42 kDa) to identify subcellular localization. Bottom : Western blot of GnaT-His 8 . Data in ( A ) and ( B ) are representative of three biological replicates. C , SDS-PAGE with Coomassie staining of cell-free extract (CFE), membrane fractions, and purified proteins isolated from Escherichia coli BLR (DE3) pLysS containing either pET21a:: kdoT-His 8 or:: gnaT - His 8 .

Article Snippet: The A. baumannii ATCC 17978 KdoT protein sequence was queried against the NCBI nonredundant database using BLASTp and all available and complete homologs returned by the search were collected.

Techniques: Purification, SDS Page, Staining, Membrane, Plasmid Preparation, Expressing, Western Blot, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Noncanonical lipooligosaccharide assembly in Acinetobacter baumannii is mediated by the glycosyltransferases KdoT and GnaT

doi: 10.1016/j.jbc.2025.111103

Figure Lengend Snippet: Early core oligosaccharide assembly in Acinetobacter baumannii . A , pathway overview of early lipid A and core OS synthesis. Early lipid A synthesis from UDP-GlcNAc yields a bis -phosphorylated lipid IV A precursor. The Kdo transferase WaaA (also known as KdtA) begins core OS synthesis via the transfer of Kdo residues using CMP-Kdo as the donor to form Kdo 2 -lipid IV A . Late acylation steps complete Kdo 2 -lipid A assembly using acyl-ACPs as donor substrates. The addition of the acyloxyacyl-linked fatty acid at the 2-position, however, is partial ( dashed bond) resulting in both hexa- and hepta-acylated Kdo 2 -lipid A species in A. baumannii . Core OS is extended via transfer of CMP-Kdo and UDP-GlcNAcA by two proteins, KdoT and GnaT, characterized here . The KdoIII residue added by KdoT diverges from inner core assembly in other bacteria, as WaaA is normally responsible for the transfer of all Kdo sugars. The core OS is then extended by a series of enzymes not shown in the schematic, completing LOS assembly. B , diagram of the complete LOS structure of A. baumannii ATCC 17978. Dashed lines indicate proteins predicted to transfer their corresponding residues based on knockout mutation analysis and prior literature ( , ). Note that deletion of either kdoT or gnaT results in an identical Kdo 2 –lipid A chemotype and expression of both proteins is required for KdoIII and GlcNAcA addition ( red box ) in whole cells. For most strains characterized to date, the inner core structure of A. baumannii LOS is conserved. Sugar residues are depicted using the official Symbol Nomenclature for Glycans (SNFG) .

Article Snippet: The A. baumannii ATCC 17978 KdoT protein sequence was queried against the NCBI nonredundant database using BLASTp and all available and complete homologs returned by the search were collected.

Techniques: Residue, Bacteria, Knock-Out, Mutagenesis, Expressing