Escherichia coli K-12 substr. MG1655 Enzyme: ADP-heptose:LPS heptosyltransferase II

Gene: waaF Accession Numbers: EG12210 (EcoCyc), b3620, ECK3610

Synonyms: rfaF

Regulation Summary Diagram: ?

Regulation summary diagram for waaF

The lipopolysaccharide (LPS) 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]. E. coli K-12 does not produce O antigen to attach to the LPS core due to a defect in the rfb gene cluster which can be complemented with genes from a second, independent rfb mutant to produce an O16 type O antigen [Stevenson94]. E. coli K-12 may have two major pathways for LPS biosynthesis. One generates LPS cores suitable for O antigen attachment, and a second generates lipooligosaccharides (LOS) with modifications to the core structure which prevent O antigen attachment [Klena92].

RfaF is the enzyme responsible for transfer of the second heptose sugar onto the heptosyl-KDO2 moiety of the lipopolysaccharide inner core [Gronow00].

Gene waaF (rfaF) from E. coli K-12 W3110 has been expressed in the Gram-positive host Corynebacterium glutamicum strain R163 using a shuttle vector [Gronow00].

Truncation of the LPS inner core by defined mutations in hldD (rfaD), hldE (rfaE) and waaF resulted in high-level gab operon expression and a mucoid colony phenotype resulting from a colanic acid capsule [Joloba04].

In waaC, waaE, waaF and waaG mutants biofilm formation was significantly increased relative to the parental strain [Nakao12].

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: [Klena92a, Sirisena94]

Locations: cytosol

Map Position: [3,792,952 -> 3,793,998] (81.75 centisomes, 294°)
Length: 1047 bp / 348 aa

Molecular Weight of Polypeptide: 39.042 kD (from nucleotide sequence)

Unification Links: ASAP:ABE-0011845 , CGSC:28434 , DIP:DIP-10667N , EchoBASE:EB2124 , EcoGene:EG12210 , EcoliWiki:b3620 , Entrez-gene:948135 , ModBase:P37692 , OU-Microarray:b3620 , PortEco:rfaF , PR:PRO_000023718 , Pride:P37692 , Protein Model Portal:P37692 , RefSeq:NP_418077 , RegulonDB:EG12210 , SMR:P37692 , String:511145.b3620 , UniProt:P37692

Relationship Links: CAZy:IN-FAMILY:GT9 , InterPro:IN-FAMILY:IPR002201 , InterPro:IN-FAMILY:IPR011910 , PDB:Structure:1PSW , Pfam:IN-FAMILY:PF01075

In Paralogous Gene Group: 543 (2 members)

Gene-Reaction Schematic: ?

Gene-Reaction Schematic

GO Terms:

Biological Process: GO:0009244 - lipopolysaccharide core region biosynthetic process Inferred from experiment Inferred by computational analysis [UniProtGOA12, Joloba04, Gronow00]
GO:0008152 - metabolic process Inferred by computational analysis [GOA01a]
GO:0009103 - lipopolysaccharide biosynthetic process Inferred by computational analysis [UniProtGOA11a, GOA01a]
Molecular Function: GO:0008713 - ADP-heptose-lipopolysaccharide heptosyltransferase activity Inferred from experiment [Gronow00]
GO:0016757 - transferase activity, transferring glycosyl groups Inferred from experiment Inferred by computational analysis [UniProtGOA11a, GOA01a, Gronow00]
GO:0016740 - transferase activity Inferred by computational analysis [UniProtGOA11a]
Cellular Component: GO:0005829 - cytosol Inferred from experiment Inferred by computational analysis [DiazMejia09, Ishihama08, Zhang07]

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

Essentiality data for waaF 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 06-Feb-2006 by Keseler I , SRI International
Last-Curated ? 16-Jan-2015 by Fulcher C , SRI International

Enzymatic reaction of: ADP-heptose:LPS heptosyltransferase

EC Number: 2.-.-.-

heptosyl-Kdo2-lipid A + ADP-L-glycero-β-D-manno-heptose <=> (heptosyl)2-Kdo2-lipid A + ADP + 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.

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

WaaF prefers ADP-L,D-heptose as a substrate over ADP-D,D-heptose which is used at low efficiency. ADP-D-mannose can serve as an alternative substrate [Kadrmas96, Kadrmas98, Gronow00, Zamyatina00].

pH(opt): 5.5-10.5 [Gronow00]

Sequence Features

Protein sequence of ADP-heptose:LPS heptosyltransferase II with features indicated

Feature Class Location Citations Comment
Sequence-Conflict 16
[Pegues90, UniProt10]
UniProt: (in Ref. 4; M33577);
Sequence-Conflict 19 -> 20
[Pegues90, UniProt10]
UniProt: (in Ref. 4; M33577);
Sequence-Conflict 105
[Chen93, UniProt10]
UniProt: (in Ref. 5);
Sequence-Conflict 151
[Chen93, UniProt10]
UniProt: (in Ref. 5);
Sequence-Conflict 159
[Chen93, UniProt10]
UniProt: (in Ref. 5);
Sequence-Conflict 204 -> 205
[Chen93, UniProt10]
UniProt: (in Ref. 5);

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 b3620 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG12210; confirmed by SwissProt match.


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

Chen93: Chen L, Coleman WG (1993). "Cloning and characterization of the Escherichia coli K-12 rfa-2 (rfaC) gene, a gene required for lipopolysaccharide inner core synthesis." J Bacteriol 175(9);2534-40. PMID: 8478319

DiazMejia09: Diaz-Mejia JJ, Babu M, Emili A (2009). "Computational and experimental approaches to chart the Escherichia coli cell-envelope-associated proteome and interactome." FEMS Microbiol Rev 33(1);66-97. PMID: 19054114

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

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."

Gronow00: Gronow S, Brabetz W, Brade H (2000). "Comparative functional characterization in vitro of heptosyltransferase I (WaaC) and II (WaaF) from Escherichia coli." Eur J Biochem 267(22);6602-11. PMID: 11054112

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

Ishihama08: Ishihama Y, Schmidt T, Rappsilber J, Mann M, Hartl FU, Kerner MJ, Frishman D (2008). "Protein abundance profiling of the Escherichia coli cytosol." BMC Genomics 9;102. PMID: 18304323

Joloba04: Joloba ML, Clemmer KM, Sledjeski DD, Rather PN (2004). "Activation of the gab operon in an RpoS-dependent manner by mutations that truncate the inner core of lipopolysaccharide in Escherichia coli." J Bacteriol 186(24);8542-6. PMID: 15576807

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

Kadrmas96: Kadrmas JL, Brozek KA, Raetz CR (1996). "Lipopolysaccharide core glycosylation in Rhizobium leguminosarum. An unusual mannosyl transferase resembling the heptosyl transferase I of Escherichia coli." J Biol Chem 271(50);32119-25. PMID: 8943265

Kadrmas98: Kadrmas JL, Raetz CR (1998). "Enzymatic synthesis of lipopolysaccharide in Escherichia coli. Purification and properties of heptosyltransferase i." J Biol Chem 273(5);2799-807. PMID: 9446588

Klena92: 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

Klena92a: 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

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

Pegues90: Pegues JC, Chen LS, Gordon AW, Ding L, Coleman WG (1990). "Cloning, expression, and characterization of the Escherichia coli K-12 rfaD gene." J Bacteriol 172(8);4652-60. PMID: 2198271

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

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

Sirisena94: Sirisena DM, MacLachlan PR, Liu SL, Hessel A, Sanderson KE (1994). "Molecular analysis of the rfaD gene, for heptose synthesis, and the rfaF gene, for heptose transfer, in lipopolysaccharide synthesis in Salmonella typhimurium." J Bacteriol 1994;176(8);2379-85. PMID: 8157607

Stevenson94: Stevenson G, Neal B, Liu D, Hobbs M, Packer NH, Batley M, Redmond JW, Lindquist L, Reeves P (1994). "Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster." J Bacteriol 1994;176(13);4144-56. PMID: 7517391

UniProt10: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

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."

Zamyatina00: Zamyatina A, Gronow S, Oertelt C, Puchberger M, Brade H, Kosma P (2000). "Efficient Chemical Synthesis of the Two Anomers of ADP-L-glycero- and D-glycero-D-manno-Heptopyranose Allows the Determination of the Substrate Specificities of Bacterial Heptosyltransferases This work was supported by grants from FWF (grant nos. P11449-MOB and P13843-CHE)." Angew Chem Int Ed Engl 39(22);4150-4153. PMID: 11093236

Zhang07: Zhang N, Chen R, Young N, Wishart D, Winter P, Weiner JH, Li L (2007). "Comparison of SDS- and methanol-assisted protein solubilization and digestion methods for Escherichia coli membrane proteome analysis by 2-D LC-MS/MS." Proteomics 7(4);484-93. PMID: 17309111

Other References Related to Gene Regulation

Dartigalongue01: Dartigalongue C, Missiakas D, Raina S (2001). "Characterization of the Escherichia coli sigma E regulon." J Biol Chem 276(24);20866-75. PMID: 11274153

Raina91: Raina S, Georgopoulos C (1991). "The htrM gene, whose product is essential for Escherichia coli viability only at elevated temperatures, is identical to the rfaD gene." Nucleic Acids Res 19(14);3811-9. PMID: 1861974

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Please cite the following article in publications resulting from the use of EcoCyc: Nucleic Acids Research 41:D605-12 2013
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