Escherichia coli K-12 substr. MG1655 Enzyme: O-antigen ligase

Gene: waaL Accession Numbers: EG11424 (EcoCyc), b3622, ECK3612

Synonyms: rfaL

Regulation Summary Diagram: ?

Regulation summary diagram for waaL

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]. 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].

WaaL is thought to be the O-antigen ligase in the lipopolysaccharide synthesis pathway. Unlike most LPS core biosynthesis genes, waaL has little sequence similarity to the counterpart gene in Salmonella enterica [Klena92a]. This diversity is thought to play a role in generating core specificity and species-specific attachment of O antigen [Klena92]. WaaL may function together with WaaU [Heinrichs98]. Both WaaU and WaaL are required for the complementation of a waaK mutation in S. typhimurium LT2, suggesting an interaction between the two proteins [Klena92]. Colanic acid, or M-antigen, which is traditionally thought of as simply an exopolysaccharide can be attached to the LPS core by the O-antigen ligase, WaaL, at the same position that O-antigen is generally attached producing MLPS [Meredith07]. MLPS is the name given to the novel LPS glycoform that occurs in a highly mucoid strain of E. coli K-12 [Meredith07].

WaaL is an inner membrane protein with 12 predicted membrane-spanning regions. Its C terminus is located in the cytoplasm [Daley05]. Mutational studies indicate that conserved amino acids in the two adjacent periplasmic loops could interact with Und-PP, which is the common component in all WaaL substrates [Perez08a].

Inactivation of waaL does not generally cause a detectable morphological phenotype; this is not surprising, because the K-12 strain lacks the O antigen [Roncero92]. However, inactivation of waaL does prevent the formation of MLPS when colanic acid synthesis is induced [Meredith07]. Also, waaL appears to be required for core completion [Klena92]. A waaL mutant prevents core completion by rfp of Shigella dysenteriae 1, suggesting its own role in core completion [Klena92].

Reviews: [Raetz02, Whitfield97, Heinrichs98a, Schnaitman93]

Gene Citations: [Sirisena94]

Locations: inner membrane

Map Position: [3,794,971 -> 3,796,230] (81.79 centisomes, 294°)
Length: 1260 bp / 419 aa

Molecular Weight of Polypeptide: 46.878 kD (from nucleotide sequence)

Unification Links: ASAP:ABE-0011849 , CGSC:28438 , DIP:DIP-10673N , EchoBASE:EB1394 , EcoGene:EG11424 , EcoliWiki:b3622 , Mint:MINT-1286025 , OU-Microarray:b3622 , PortEco:rfaL , PR:PRO_000023723 , Pride:P27243 , Protein Model Portal:P27243 , RefSeq:NP_418079 , RegulonDB:EG11424 , String:511145.b3622 , UniProt:P27243

Relationship Links: Pfam:IN-FAMILY:PF04932

In Paralogous Gene Group: 409 (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, Klena92]
GO:0009103 - lipopolysaccharide biosynthetic process Inferred by computational analysis [UniProtGOA11]
Molecular Function: GO:0008754 - O antigen ligase activity Inferred from experiment [Klena92]
GO:0016874 - ligase activity Inferred by computational analysis [UniProtGOA11]
Cellular Component: GO:0005886 - plasma membrane Inferred from experiment Inferred by computational analysis [UniProtGOA11a, UniProtGOA11, DiazMejia09, Daley05]
GO:0016020 - membrane Inferred by computational analysis [UniProtGOA11]
GO:0016021 - integral component of membrane Inferred by computational analysis [UniProtGOA11]

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

Essentiality data for waaL 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 07-Feb-2006 by Keseler I , SRI International
Last-Curated ? 19-Oct-2007 by Johnson A , JCVI

Enzymatic reaction of: O-antigen ligase

lipid A-core[periplasmic space] + colanic acid[periplasmic space] <=> MLPS[periplasmic space] + H2O[periplasmic space]

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, is in accordance with the direction of enzyme catalysis.

The reaction is physiologically favored in the direction shown.

Sequence Features

Protein sequence of O-antigen ligase with features indicated

Feature Class Location Citations Comment
Transmembrane-Region 18 -> 38
UniProt: Helical;; Non-Experimental Qualifier: potential;
Transmembrane-Region 40 -> 60
UniProt: Helical;; Non-Experimental Qualifier: potential;
Transmembrane-Region 72 -> 92
UniProt: Helical;; Non-Experimental Qualifier: potential;
Transmembrane-Region 106 -> 126
UniProt: Helical;; Non-Experimental Qualifier: potential;
Transmembrane-Region 136 -> 156
UniProt: Helical;; Non-Experimental Qualifier: potential;
Transmembrane-Region 169 -> 189
UniProt: Helical;; Non-Experimental Qualifier: potential;
Transmembrane-Region 194 -> 214
UniProt: Helical;; Non-Experimental Qualifier: potential;
Sequence-Conflict 205 -> 224
[Klena92a, UniProt10a]
UniProt: (in Ref. 1; AAA24524);
Transmembrane-Region 216 -> 236
UniProt: Helical;; Non-Experimental Qualifier: potential;
Transmembrane-Region 242 -> 262
UniProt: Helical;; Non-Experimental Qualifier: potential;
Sequence-Conflict 307
[Klena92a, UniProt10a]
UniProt: (in Ref. 1; AAA24524);
Sequence-Conflict 344
[Klena92a, UniProt10a]
UniProt: (in Ref. 1; AAA24524);
Transmembrane-Region 347 -> 367
UniProt: Helical;; Non-Experimental Qualifier: potential;
Transmembrane-Region 370 -> 390
UniProt: Helical;; Non-Experimental Qualifier: potential;
Transmembrane-Region 391 -> 411
UniProt: Helical;; Non-Experimental Qualifier: potential;

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 b3622 from Blattner lab Genbank (v. M52) entry merged into EcoCyc gene EG11424; 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

Daley05: Daley DO, Rapp M, Granseth E, Melen K, Drew D, von Heijne G (2005). "Global topology analysis of the Escherichia coli inner membrane proteome." Science 308(5726);1321-3. PMID: 15919996

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

Heinrichs98: Heinrichs DE, Monteiro MA, Perry MB, Whitfield C (1998). "The assembly system for the lipopolysaccharide R2 core-type of Escherichia coli is a hybrid of those found in Escherichia coli K-12 and Salmonella enterica. Structure and function of the R2 WaaK and WaaL homologs." J Biol Chem 273(15);8849-59. PMID: 9535865

Heinrichs98a: 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, 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

Meredith07: Meredith TC, Mamat U, Kaczynski Z, Lindner B, Holst O, Woodard RW (2007). "Modification of lipopolysaccharide with colanic acid (M-antigen) repeats in Escherichia coli." J Biol Chem 282(11);7790-8. PMID: 17227761

Perez08a: Perez JM, McGarry MA, Marolda CL, Valvano MA (2008). "Functional analysis of the large periplasmic loop of the Escherichia coli K-12 WaaL O-antigen ligase." Mol Microbiol 70(6);1424-40. PMID: 19019161

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

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

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 2010-07 released on 2010-06-15 00:00:00." Database.

UniProt10a: UniProt Consortium (2010). "UniProt version 2010-11 released on 2010-11-02 00:00:00." Database.

UniProtGOA11: UniProt-GOA (2011). "Gene Ontology annotation based on manual assignment of UniProtKB keywords in UniProtKB/Swiss-Prot entries."

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

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

Whitfield97: Whitfield C, Amor PA, Koplin R (1997). "Modulation of the surface architecture of gram-negative bacteria by the action of surface polymer:lipid A-core ligase and by determinants of polymer chain length." Mol Microbiol 23(4);629-38. PMID: 9157235

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

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

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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