WaaL (PA4999) is the O-antigen ligase of Pseudomonas aeruginosa PAO1, a 401-residue polytopic inner-membrane protein of the Wzy_C/WaaL family (CAZy GT121) with its catalytic periplasmic loops facing the periplasm. It catalyses the final step of lipopolysaccharide assembly, transferring a completed O-antigen polysaccharide from its undecaprenyl-diphosphate carrier onto a terminal sugar of the lipid A-core oligosaccharide (EC 2.4.99.26), and is required to attach both the O-specific antigen (B-band) and the common polysaccharide antigen (A-band). Despite its name, WaaL needs neither ATP nor divalent metal ions and behaves as a metal-independent inverting glycosyltransferase; the conserved periplasmic His303 is essential. waaL mutants make only rough, O-antigen-free LPS and show secondary envelope and motility defects.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0016757 glycosyltransferase activity | IBA GO_REF:0000033 | MODIFY | Summary: IBA glycosyltransferase activity from the WaaL-like node PTN008076295 (3 seeds, including WaaL itself and enterobacterial WaaL proteins). Reason: The IBD is sound; WaaL's own appearance in WITH/FROM reflects its experimental annotation. Glycosyltransferase activity is an appropriate node-level term, but for WaaL the specific activity is GO:0008754 O-antigen ligase activity (is_a glycosyltransferase activity), which the gene carries by EXP, IMP and IEA. Refined for this gene; no change requested at the node. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q9HUG6 SUPPORTS TRANSFER In vitro O-antigen ligation by purified WaaL (PMID:17697256, PMID:30047569). Proposed replacements: O-antigen ligase activity Supporting Evidence: PMID:21983211 our biochemical and in silico data argue that WaaL proteins use a common reaction mechanism and share features of metal ion-independent inverting glycosyltransferases |
| GO:0000271 polysaccharide biosynthetic process | IBA GO_REF:0000033 | MODIFY | Summary: IBA polysaccharide biosynthetic process; the node's sole seed is the PseudoCAP IMP O antigen biosynthetic process row on WaaL. Reason: The node term is true for WaaL and suitably broad for a WaaL/TuaE-like clade, so the IBD stands. For WaaL itself the specific process is lipopolysaccharide biosynthesis (the ligation joins O antigen to lipid A-core), GO:0009103, an is_a descendant of polysaccharide biosynthetic process already carried by this gene (IEA, UniPathway UPA00030). Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q9HUG6 SUPPORTS TRANSFER Sole seed is the PseudoCAP IMP O antigen biosynthetic process row (PMID:15838026); knockout LPS lacks O antigen and is complemented. The seed term is arguably lateral (LPS assembly), but polysaccharide biosynthesis holds either way. Proposed replacements: lipopolysaccharide biosynthetic process Supporting Evidence: PMID:15838026 waaL is required for the attachment of O-antigen repeat units to the core |
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: IBA plasma membrane from the WaaL-like node. Reason: WaaL is a polytopic inner-membrane protein whose topology has been mapped experimentally; ligation occurs at the periplasmic face of the inner membrane. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q9HUG6 SUPPORTS TRANSFER Topology mapped in PAO1 (PMID:20824106, PMID:30047569). Supporting Evidence: PMID:30047569 WaaL is an inner membrane glycosyltransferase that catalyzes the transfer of O-antigen polysaccharide from its lipid-linked intermediate to a terminal sugar of the lipid A-core oligosaccharide |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular location mapping (cell inner membrane). Reason: Consistent with experimental topology. Supporting Evidence: file:PSEAE/waaL/waaL-uniprot.txt SUBCELLULAR LOCATION: Cell inner membrane {ECO:0000269|PubMed:20824106, |
| GO:0008754 O-antigen ligase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC 2.4.99.26 mapping. Reason: Consistent with the experimental ligation assays. Supporting Evidence: file:PSEAE/waaL/waaL-uniprot.txt Catalyzes the transfer of a polymerized O-antigen molecule from its polyprenyl diphosphate membrane anchor to a terminal sugar of the lipid A-core oligosaccharide |
| GO:0016757 glycosyltransferase activity | IEA GO_REF:0000117 | MODIFY | Summary: ARBA glycosyltransferase activity. Reason: Correct but general; refined to GO:0008754 O-antigen ligase activity, the specific activity supported experimentally. Proposed replacements: O-antigen ligase activity Supporting Evidence: PMID:30047569 WaaL is an inner membrane glycosyltransferase that catalyzes the transfer of O-antigen polysaccharide from its lipid-linked intermediate to a terminal sugar of the lipid A-core oligosaccharide |
| GO:0009103 lipopolysaccharide biosynthetic process | IEA GO_REF:0000041 | ACCEPT | Summary: UniPathway mapping (lipopolysaccharide biosynthesis). Reason: WaaL catalyses the step that completes LPS by attaching O antigen to lipid A-core; this is the core process. Supporting Evidence: PMID:15838026 waaL is required for the attachment of O-antigen repeat units to the core PMID:21983211 WaaL is a membrane enzyme that catalyzes a key step in lipopolysaccharide (LPS) synthesis |
| GO:0005886 plasma membrane | EXP PMID:20824106 Membrane topology mapping of the O-antigen flippase (Wzx), p... | ACCEPT | Summary: Plasma membrane from reporter-fusion topology mapping of WaaL in PAO1. Reason: PhoA-LacZ truncation fusions and C-terminal GFP fusions place WaaL in the inner membrane with defined cytoplasmic and periplasmic loops. Supporting Evidence: PMID:20824106 Protein orientation in the inner membrane was confirmed via C-terminal fusion to green fluorescent protein. |
| GO:0005886 plasma membrane | EXP PMID:30047569 Escherichia coli and Pseudomonas aeruginosa lipopolysacchari... | ACCEPT | Summary: Plasma membrane from PEG-maleimide accessibility topology of intact WaaL. Reason: Direct topology analysis of full-length P. aeruginosa WaaL in the inner membrane. Supporting Evidence: PMID:30047569 The accessibility of PEG-Mal to targeted engineered cysteine residues in both E. coli and P. aeruginosa WaaL proteins demonstrates that both ligases share similar membrane topology. |
| GO:0008754 O-antigen ligase activity | EXP PMID:17697256 WaaL of Pseudomonas aeruginosa utilizes ATP in in vitro liga... | ACCEPT | Summary: O-antigen ligase activity from the first in vitro ligation assay with purified WaaL. Reason: Purified WaaL ligated O-antigen-Und-PP onto lipid A-core in vitro. The paper's claim of ATP dependence was later refuted, but the ligase (transfer) activity itself stands. Supporting Evidence: PMID:17697256 the development of an in vitro O-antigen ligase assay |
| GO:0008754 O-antigen ligase activity | EXP PMID:30047569 Escherichia coli and Pseudomonas aeruginosa lipopolysacchari... | ACCEPT | Summary: O-antigen ligase activity of P. aeruginosa WaaL confirmed in vitro without ATP. Reason: Core activity; P. aeruginosa WaaL shares the ATP- and metal-independent properties of E. coli WaaL. Supporting Evidence: PMID:30047569 we also demonstrate that P. aeruginosa WaaL shares similar functional properties with E. coli WaaL file:PSEAE/waaL/waaL-uniprot.txt Activity does not require ATP and magnesium ions. |
| GO:0005886 plasma membrane | IDA PMID:17697256 WaaL of Pseudomonas aeruginosa utilizes ATP in in vitro liga... | ACCEPT | Summary: PseudoCAP IDA plasma membrane from purification of membrane-embedded WaaL; seed of the plasma membrane IBD at PTN008076295. Reason: WaaL was purified as a multi-TM membrane protein; consistent with later topology studies. Seed is sound. Supporting Evidence: PMID:17697256 Here we describe the purification of WaaL, a membrane protein with 11 potential transmembrane segments from Pseudomonas aeruginosa |
| GO:0008754 O-antigen ligase activity | IMP PMID:15838026 Functional characterization of WaaL, a ligase associated wit... | ACCEPT | Summary: PseudoCAP IMP O-antigen ligase activity from the waaL knockout (LPS lacks O antigen, complemented in trans). Reason: The knockout and complementation show WaaL is required for attaching O antigen to core; on its own this is genetic, but the activity was later demonstrated in vitro (PMID:17697256, PMID:30047569), so the claim is correct. Supporting Evidence: PMID:15838026 These results demonstrate that waaL in P. aeruginosa encodes a functional O-antigen ligase |
| GO:0009243 O antigen biosynthetic process | IMP PMID:15838026 Functional characterization of WaaL, a ligase associated wit... | MODIFY | Summary: PseudoCAP IMP O antigen biosynthetic process; sole seed of the polysaccharide biosynthetic process IBD at PTN008076295. Reason: The knockout result is sound (O antigen is absent from LPS and restored by complementation), but the O side chain is assembled and polymerized before WaaL acts; WaaL attaches the finished chain to lipid A-core, which is a step of lipopolysaccharide biosynthesis (GO:0009103, of which O antigen biosynthetic process is a part). UniProt likewise places WaaL in LPS biosynthesis (UPA00030), not in the O-antigen pathway. The row is modified laterally to the parent process. The IBD built on it (polysaccharide biosynthetic process) is unaffected, because LPS biosynthetic process is_a polysaccharide biosynthetic process. Proposed replacements: lipopolysaccharide biosynthetic process Supporting Evidence: PMID:15838026 The completed chains are covalently attached to lipid A core by the O-antigen ligase, WaaL. file:PSEAE/waaL/waaL-uniprot.txt PATHWAY: Bacterial outer membrane biogenesis; lipopolysaccharide biosynthesis. |
| GO:0009297 pilus assembly | IMP PMID:15838026 Functional characterization of WaaL, a ligase associated wit... | MARK AS OVER ANNOTATED | Summary: PseudoCAP IMP pilus assembly from reduced surface pili/twitching of the waaL mutant. Reason: WaaL acts on LPS in the periplasmic leaflet of the inner membrane and does no part of pilus assembly. Reduced pili and twitching in the mutant are pleiotropic consequences of rough LPS and a compromised envelope; in the full text (PMC1082828) the authors attribute them to outer-membrane disturbance and reduced surface wettability. Indirect phenotype, not participation. Supporting Evidence: PMID:15838026 encodes a functional O-antigen ligase that is important for cell wall integrity and motility of the bacteria |
| GO:0016757 glycosyltransferase activity | IDA PMID:17697256 WaaL of Pseudomonas aeruginosa utilizes ATP in in vitro liga... | MODIFY | Summary: PseudoCAP IDA glycosyltransferase activity from the in vitro ligation assay; seed of the glycosyltransferase IBD at PTN008076295. Reason: Correct and directly supported (in vitro ligation is a glycosyl transfer, and WaaL is an inverting GT), so the seed is sound; the specific term GO:0008754 O-antigen ligase activity is a descendant already carried by this gene. Proposed replacements: O-antigen ligase activity Supporting Evidence: PMID:17697256 the development of an in vitro O-antigen ligase assay PMID:21983211 our biochemical and in silico data argue that WaaL proteins use a common reaction mechanism and share features of metal ion-independent inverting glycosyltransferases |
| GO:0016874 ligase activity | IDA PMID:17697256 WaaL of Pseudomonas aeruginosa utilizes ATP in in vitro liga... | REMOVE | Summary: PseudoCAP IDA ligase activity, based on the 2007 report that WaaL hydrolyses ATP during ligation. Reason: GO:0016874 ligase activity (EC 6) is bond formation driven by NTP hydrolysis. WaaL is an EC 2.4.99.26 glycosyltransferase whose name, not chemistry, says ligase. The ATP dependence reported in PMID:17697256 was contradicted by later work showing ligation without ATP or Mg2+ and no ATP hydrolysis by either E. coli or P. aeruginosa WaaL, and GO:0008754 O-antigen ligase activity is classified under glycosyltransferase activity, not ligase activity. The claim is genuinely contradicted, not merely unverified. Supporting Evidence: PMID:21983211 E. coli and Pseudomonas aeruginosa WaaL proteins cannot catalyze ATP hydrolysis in vitro file:PSEAE/waaL/waaL-uniprot.txt However, it was shown later that ligation occurs without ATP and magnesium ions, and that WaaL cannot catalyze ATP hydrolysis in vitro |
| GO:0043107 type IV pilus-dependent motility | IMP PMID:15838026 Functional characterization of WaaL, a ligase associated wit... | MARK AS OVER ANNOTATED | Summary: PseudoCAP IMP type IV pilus-dependent (twitching) motility from the waaL mutant. Reason: Indirect phenotype of O-antigen-less LPS, as for the pilus assembly row; WaaL performs no step of twitching motility. Supporting Evidence: PMID:15838026 The waaL mutation also drastically affected the swimming and twitching motilities of the bacteria. |
| GO:0044780 bacterial-type flagellum assembly | IMP PMID:15838026 Functional characterization of WaaL, a ligase associated wit... | MARK AS OVER ANNOTATED | Summary: PseudoCAP IMP flagellum assembly from fewer surface flagella on the waaL mutant. Reason: Indirect phenotype; WaaL has no role in flagellar export or assembly. In PA14 and PAK waaL deletion did not abolish swimming, consistent with a modest secondary effect. Supporting Evidence: PMID:29059582 ΞwaaL mutation in P. aeruginosa did not abrogate swimming motility nor ability of the bacteria to form biofilms |
| GO:0071977 bacterial-type flagellum-dependent swimming motility | IMP PMID:15838026 Functional characterization of WaaL, a ligase associated wit... | MARK AS OVER ANNOTATED | Summary: PseudoCAP IMP swimming motility from the waaL mutant. Reason: Indirect phenotype of rough LPS; WaaL performs no step of flagellum-dependent swimming, and waaL deletion did not abolish swimming in PA14 or PAK. Supporting Evidence: PMID:15838026 The waaL mutation also drastically affected the swimming and twitching motilities of the bacteria. PMID:29059582 ΞwaaL mutation in P. aeruginosa did not abrogate swimming motility nor ability of the bacteria to form biofilms |
| GO:0098567 periplasmic side of plasma membrane | IDA PMID:20824106 Membrane topology mapping of the O-antigen flippase (Wzx), p... | KEEP AS NON CORE | Summary: PseudoCAP IDA periplasmic side of plasma membrane from topology mapping; seed of the plasma membrane IBD. Reason: WaaL is an integral polytopic protein spanning both leaflets, so plasma membrane is the primary location; the large catalytic periplasmic loop (containing His303) and the ligation reaction are on the periplasmic face, which this term captures. Correct but secondary to the plasma membrane location. Supporting Evidence: PMID:30047569 Ligation occurs at the periplasmic side of the bacterial cell membrane, suggesting the catalytic region of WaaL faces the periplasm. PMID:20824106 In WaaL from P. aeruginosa PAO1 (WaaLPa), the periplasmic His residue of the HX10G motif (H303) was found to be critically important for function |
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Download this section (compressed HTML)Q: Should GO's PAINT IBD for polysaccharide biosynthetic process at PTN008076295 be re-seeded from an LPS biosynthetic process or O-antigen ligase EXP row rather than the PseudoCAP IMP O antigen biosynthetic process row?
Q: Is the motility defect reported for PAO1 waaL (but not seen in PA14/PAK) strain-specific, and does it reflect loss of O antigen per se or altered envelope stress signalling?
Experiment: Determine a structure of P. aeruginosa WaaL with lipid A-core and Und-PP-O antigen analogs to define the roles of His303 and the conserved periplasmic Arg in the inverting glycosyl transfer.
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