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Escherichia coli K-12 substr. MG1655 Enzyme: lipopolysaccharide glucosyltransferase I

Gene: waaG Accession Numbers: EG11339 (EcoCyc), b3631, ECK3621

Synonyms: rfaG

Regulation Summary Diagram: ?

Regulation summary diagram for waaG

The lipopolysaccharide of E. coli K-12 consists of two major components: the hydrophobic lipid A moiety inserted into the outer membrane and the phosphorylated core oligosaccharide [Raetz02]. UDP-glucose:(heptosyl)lipopolysaccharide α-1,3-glucosyltransferase (WaaG) adds the first glucose (GlcI) of the outer core of LPS from UDP-glucose to the HepII residue of the inner core [Creeger79, Parker92]. WaaG is a glycosyltransferase family 4 enzyme.

Early studies characterized the rfa locus and identified the waaG gene [Austin90, Schnaitman91, Parker92a, Roncero92].

waaG+ restores flagella and pili to a waaGPSBI deletion mutant [Parker92]. Mutation of waaG reduces phosphorylation of the inner core heptoses HepI and HepII [Yethon00]. waaG mutants perturbed outer membrane vesicle production [McBroom06].

The chromosomal waa region (formerly rfa) contains the major core-oligosaccharide assembly operons in E. coli [Raetz02, Raetz07]. The current gene nomenclature system was proposed originally in [Reeves96] and [Heinrichs98] and followed thereafter.

Studies of waaG mutants showed altered behavior in biofilms [Agladze05, Genevaux99] and demonstrated an effect of LPS core synthesis on fimbrial biogenesis [Pilipcinec94]. In waaC, waaE, waaF and waaG mutants biofilm formation was significantly increased relative to the parental strain [Nakao12]. Mutants in waaG from strain Nissle1917 showed impaired colonization of mouse gut and increased susceptibility to bile acids [Nagy05].

Crystal structures of WaaG have been solved, providing insight into binding of the nucleotide-sugar substrate at the active site. WaaG is a retaining glycosyltransferase, catalyzing a reaction in which the α-stereochemistry of the donor glucose residue is retained in the product [MartinezFleites06].

NMR spectroscopy and molecular dynamics simulations of substrate binding have been performed. These techniques were used to investigate the binding of natural substrates, enzymatic activity, and donor substrate selectivity. Molecular docking studies identified ligands that compete with UDP-α-D-glucose for binding and can serve as scaffolds for further inhibitor design [Landstrom12].

WaaG from E. coli K-12 has been cloned, overexpressed and purified. For the in vitro assay an inner core analog [32P]heptose2-1-dephospho Kdo2-lipid A was prepared and used as the acceptor substrate. Several nucleotide sugar donors were tested and the reaction was shown to be specific for UDP-glucose as sugar donor. Triton X-100 at concentrations of 0-0.2% increased the activity although higher concentrations were inhibitory. Enzymatic activity decreased in the presence of a number of divalent cations. Similar studies were performed for WaaB and WaaO from this organism [Qian14].

In the absence of the lipid acceptor, WaaG can slowly hydrolyze the UDP-glucose sugar donor. It was not able to hydrolyze UDP-galactose [Qian14].

The chromosomal waa region (formerly rfa) contains the major core-oligosaccharide assembly operons in E. coli [Raetz02][Raetz07]. The current nomenclature system was proposed originally in [Reeves96] and [Heinrichs98] and followed thereafter.

Reviews: [Raetz02, Heinrichs98, Schnaitman93]

Gene Citations: [Klena92, Klena92a]

Map Position: [3,803,966 <- 3,805,090] (81.99 centisomes, 295°)
Length: 1125 bp / 374 aa

Molecular Weight of Polypeptide: 42.284 kD (from nucleotide sequence)

Unification Links: ASAP:ABE-0011868 , CGSC:15583 , EchoBASE:EB1315 , EcoGene:EG11339 , EcoliWiki:b3631 , Entrez-gene:948149 , ModBase:P25740 , OU-Microarray:b3631 , PortEco:rfaG , PR:PRO_000023719 , Pride:P25740 , Protein Model Portal:P25740 , RefSeq:NP_418088 , RegulonDB:EG11339 , SMR:P25740 , String:511145.b3631 , UniProt:P25740

Relationship Links: CAZy:IN-FAMILY:GT4 , InterPro:IN-FAMILY:IPR001296 , PDB:Structure:2IV7 , PDB:Structure:2IW1 , Pfam:IN-FAMILY:PF00534

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

Genetic Regulation Schematic: ?

Genetic regulation schematic for waaG

GO Terms:

Biological Process: GO:0009244 - lipopolysaccharide core region biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, Parker92]
GO:0009058 - biosynthetic process Inferred by computational analysis [GOA01a]
GO:0009103 - lipopolysaccharide biosynthetic process Inferred by computational analysis [UniProtGOA11a]
Molecular Function: GO:0008919 - lipopolysaccharide glucosyltransferase I activity Inferred from experiment [Qian14, Parker92, Creeger79]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a]
GO:0016757 - transferase activity, transferring glycosyl groups Inferred by computational analysis [UniProtGOA11a]

MultiFun Terms: cell structure surface antigens (ECA, O antigen of LPS)
metabolism biosynthesis of macromolecules (cellular constituents) lipopolysaccharide core region

Essentiality data for waaG knockouts: ?

Growth Medium Growth? T (°C) O2 pH Osm/L Growth Observations
LB enriched Yes 37 Aerobic 6.95   Yes [Gerdes03, Comment 1]
LB Lennox Yes 37 Aerobic 7   Yes [Baba06, Comment 2]
M9 medium with 1% glycerol Yes 37 Aerobic 7.2 0.35 Yes [Joyce06, Comment 3]
MOPS medium with 0.4% glucose Yes 37 Aerobic 7.2 0.22 Yes [Baba06, Comment 2]
Yes [Feist07, Comment 4]

Curated 05-Mar-2014 by Keseler I , SRI International
Last-Curated ? 19-Jan-2015 by Fulcher C , SRI International

Enzymatic reaction of: lipopolysaccharide glucosyltransferase

EC Number: 2.4.1.-

UDP-α-D-glucose + (heptosyl)2-Kdo2-lipid A <=> glucosyl-(heptosyl)2-Kdo2-lipid A + UDP + H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction in which it was curated.

The reaction is favored in the direction shown.

Alternative Substrates for (heptosyl)2-Kdo2-lipid A: H2O [Qian14 ]

In Pathways: superpathway of lipopolysaccharide biosynthesis , Lipid A-core biosynthesis

Inhibitors (Unknown Mechanism): Cu2+ [Qian14] , Ca2+ [Qian14] , Zn2+ [Qian14] , Co2+ [Qian14] , Ni2+ [Qian14] , Mg2+ [Qian14]

Kinetic Parameters:

Km (μM)
kcat (sec-1)
kcat/Km (sec-1 μM-1)
(heptosyl)2-Kdo2-lipid A

Gene Local Context (not to scale): ?

Gene local context diagram

Transcription Units:

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram

Transcription-unit diagram


10/20/97 Gene b3631 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11339; confirmed by SwissProt match.


Agladze05: Agladze K, Wang X, Romeo T (2005). "Spatial periodicity of Escherichia coli K-12 biofilm microstructure initiates during a reversible, polar attachment phase of development and requires the polysaccharide adhesin PGA." J Bacteriol 187(24);8237-46. PMID: 16321928

Austin90: Austin EA, Graves JF, Hite LA, Parker CT, Schnaitman CA (1990). "Genetic analysis of lipopolysaccharide core biosynthesis by Escherichia coli K-12: insertion mutagenesis of the rfa locus." J Bacteriol 172(9);5312-25. PMID: 2168379

Baba06: Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006). "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection." Mol Syst Biol 2;2006.0008. PMID: 16738554

Creeger79: Creeger ES, Rothfield LI (1979). "Cloning of genes for bacterial glycosyltransferases. I. Selection of hybrid plasmids carrying genes for two glucosyltransferases." J Biol Chem 254(3);804-10. PMID: 368061

Feist07: Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007). "A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information." Mol Syst Biol 3;121. PMID: 17593909

Genevaux99: Genevaux P, Bauda P, DuBow MS, Oudega B (1999). "Identification of Tn10 insertions in the rfaG, rfaP, and galU genes involved in lipopolysaccharide core biosynthesis that affect Escherichia coli adhesion." Arch Microbiol 172(1);1-8. PMID: 10398745

Gerdes03: Gerdes SY, Scholle MD, Campbell JW, Balazsi G, Ravasz E, Daugherty MD, Somera AL, Kyrpides NC, Anderson I, Gelfand MS, Bhattacharya A, Kapatral V, D'Souza M, Baev MV, Grechkin Y, Mseeh F, Fonstein MY, Overbeek R, Barabasi AL, Oltvai ZN, Osterman AL (2003). "Experimental determination and system level analysis of essential genes in Escherichia coli MG1655." J Bacteriol 185(19);5673-84. PMID: 13129938

GOA01a: GOA, DDB, FB, MGI, ZFIN (2001). "Gene Ontology annotation through association of InterPro records with GO terms."

Heinrichs98: Heinrichs DE, Yethon JA, Whitfield C (1998). "Molecular basis for structural diversity in the core regions of the lipopolysaccharides of Escherichia coli and Salmonella enterica." Mol Microbiol 30(2);221-32. PMID: 9791168

Joyce06: Joyce AR, Reed JL, White A, Edwards R, Osterman A, Baba T, Mori H, Lesely SA, Palsson BO, Agarwalla S (2006). "Experimental and computational assessment of conditionally essential genes in Escherichia coli." J Bacteriol 188(23);8259-71. PMID: 17012394

Klena92: Klena JD, Pradel E, Schnaitman CA (1992). "Comparison of lipopolysaccharide biosynthesis genes rfaK, rfaL, rfaY, and rfaZ of Escherichia coli K-12 and Salmonella typhimurium." J Bacteriol 174(14);4746-52. PMID: 1624462

Klena92a: Klena JD, Ashford RS, Schnaitman CA (1992). "Role of Escherichia coli K-12 rfa genes and the rfp gene of Shigella dysenteriae 1 in generation of lipopolysaccharide core heterogeneity and attachment of O antigen." J Bacteriol 174(22);7297-307. PMID: 1385388

Landstrom12: Landstrom J, Persson K, Rademacher C, Lundborg M, Wakarchuk W, Peters T, Widmalm G (2012). "Small molecules containing hetero-bicyclic ring systems compete with UDP-Glc for binding to WaaG glycosyltransferase." Glycoconj J 29(7);491-502. PMID: 22711644

MartinezFleites06: Martinez-Fleites C, Proctor M, Roberts S, Bolam DN, Gilbert HJ, Davies GJ (2006). "Insights into the synthesis of lipopolysaccharide and antibiotics through the structures of two retaining glycosyltransferases from family GT4." Chem Biol 13(11);1143-52. PMID: 17113996

McBroom06: McBroom AJ, Johnson AP, Vemulapalli S, Kuehn MJ (2006). "Outer membrane vesicle production by Escherichia coli is independent of membrane instability." J Bacteriol 188(15);5385-92. PMID: 16855227

Nagy05: Nagy G, Dobrindt U, Grozdanov L, Hacker J, Emody L (2005). "Transcriptional regulation through RfaH contributes to intestinal colonization by Escherichia coli." FEMS Microbiol Lett 244(1);173-80. PMID: 15727837

Nakao12: Nakao R, Ramstedt M, Wai SN, Uhlin BE (2012). "Enhanced biofilm formation by Escherichia coli LPS mutants defective in Hep biosynthesis." PLoS One 7(12);e51241. PMID: 23284671

Parker92: Parker CT, Kloser AW, Schnaitman CA, Stein MA, Gottesman S, Gibson BW (1992). "Role of the rfaG and rfaP genes in determining the lipopolysaccharide core structure and cell surface properties of Escherichia coli K-12." J Bacteriol 174(8);2525-38. PMID: 1348243

Parker92a: Parker CT, Pradel E, Schnaitman CA (1992). "Identification and sequences of the lipopolysaccharide core biosynthetic genes rfaQ, rfaP, and rfaG of Escherichia coli K-12." J Bacteriol 174(3);930-4. PMID: 1732225

Pilipcinec94: Pilipcinec E, Huisman TT, Willemsen PT, Appelmelk BJ, de Graaf FK, Oudega B (1994). "Identification by Tn10 transposon mutagenesis of host factors involved in the biosynthesis of K99 fimbriae of Escherichia coli: effect of LPS core mutations." FEMS Microbiol Lett 123(1-2);201-6. PMID: 7988890

Qian14: Qian J, Garrett TA, Raetz CR (2014). "In vitro assembly of the outer core of the lipopolysaccharide from Escherichia coli K-12 and Salmonella typhimurium." Biochemistry 53(8);1250-62. PMID: 24479701

Raetz02: Raetz CR, Whitfield C (2002). "Lipopolysaccharide endotoxins." Annu Rev Biochem 71;635-700. PMID: 12045108

Raetz07: Raetz CR, Reynolds CM, Trent MS, Bishop RE (2007). "Lipid A modification systems in gram-negative bacteria." Annu Rev Biochem 76;295-329. PMID: 17362200

Reeves96: Reeves PR, Hobbs M, Valvano MA, Skurnik M, Whitfield C, Coplin D, Kido N, Klena J, Maskell D, Raetz CR, Rick PD (1996). "Bacterial polysaccharide synthesis and gene nomenclature." Trends Microbiol 4(12);495-503. PMID: 9004408

Roncero92: Roncero C, Casadaban MJ (1992). "Genetic analysis of the genes involved in synthesis of the lipopolysaccharide core in Escherichia coli K-12: three operons in the rfa locus." J Bacteriol 174(10);3250-60. PMID: 1577693

Schnaitman91: Schnaitman CA, Parker CT, Klena JD, Pradel EL, Pearson NB, Sanderson KE, MacClachlan PR (1991). "Physical maps of the rfa loci of Escherichia coli K-12 and Salmonella typhimurium." J Bacteriol 173(23);7410-1. PMID: 1938935

Schnaitman93: Schnaitman CA, Klena JD (1993). "Genetics of lipopolysaccharide biosynthesis in enteric bacteria." Microbiol Rev 57(3);655-82. PMID: 7504166

UniProtGOA11a: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

UniProtGOA12: UniProt-GOA (2012). "Gene Ontology annotation based on UniPathway vocabulary mapping."

Yethon00: Yethon JA, Vinogradov E, Perry MB, Whitfield C (2000). "Mutation of the lipopolysaccharide core glycosyltransferase encoded by waaG destabilizes the outer membrane of Escherichia coli by interfering with core phosphorylation." J Bacteriol 182(19);5620-3. PMID: 10986272

Other References Related to Gene Regulation

Clementz92: Clementz T (1992). "The gene coding for 3-deoxy-manno-octulosonic acid transferase and the rfaQ gene are transcribed from divergently arranged promoters in Escherichia coli." J Bacteriol 1992;174(23);7750-6. PMID: 1447141

Maciag11: Maciag A, Peano C, Pietrelli A, Egli T, De Bellis G, Landini P (2011). "In vitro transcription profiling of the {sigma}S subunit of bacterial RNA polymerase: re-definition of the {sigma}S regulon and identification of {sigma}S-specific promoter sequence elements." Nucleic Acids Res 39(13);5338-55. PMID: 21398637

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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
Page generated by SRI International Pathway Tools version 19.0 on Wed Sep 2, 2015, biocyc12.