waaL (PA4999, Q9HUG6) notes

No deep-research provider was available; literature research done manually (PubMed MCP + cached
publications).

Identity

UniProt Q9HUG6 (Swiss-Prot), 401 aa, P. aeruginosa PAO1, locus PA4999. O-antigen ligase,
EC 2.4.99.26; CAZy GT121; Pfam PF04932 (Wzy_C); PANTHER PTHR37422 (TEICHURONIC ACID BIOSYNTHESIS
PROTEIN TUAE), SF13. Polytopic inner-membrane protein, 12 TM helices in the PEG-maleimide
topology (UniProt FT, PMID:30047569); essential periplasmic His303 (HX10G motif).

Ligates completed undecaprenyl-PP-linked O antigen (both B-band OSA and A-band CPA) to lipid
A-core at the periplasmic face [PMID:15838026 "The completed chains are covalently attached to
lipid A core by the O-antigen ligase, WaaL"; "waaL is required for the attachment of O-antigen
repeat units to the core"].
In vitro ligation with purified WaaL PMID:17697256. That paper proposed ATP-dependence, later refuted: ligation needs no ATP or
Mg2+, and WaaL does not hydrolyse ATP; WaaL behaves like a metal-independent inverting
glycosyltransferase [PMID:21983211 "E. coli and Pseudomonas aeruginosa WaaL proteins cannot
catalyze ATP hydrolysis in vitro"; "share features of metal ion-independent inverting
glycosyltransferases"]. PMID:30047569 confirmed this for P. aeruginosa WaaL per UniProt.
GO:0008754 O-antigen ligase activity is defined by the transfer reaction and is_a
glycosyltransferase activity; GO:0016874 ligase activity (EC 6) requires energy from NTP
hydrolysis, which WaaL does not use, despite its name.
Topology: GFP and PhoA-LacZ fusions PMID:20824106; PEG-Mal labeling of intact
protein PMID:30047569.

PMID:15838026 reported the waaL mutation "drastically affected the swimming and twitching
motilities". This is a pleiotropic consequence of O-antigen-less (rough) LPS / cell-envelope
defects; WaaL acts in the periplasmic leaflet on LPS and has no known role in pilus or flagellum
assembly. In PA14 and PAK, waaL deletion did not abolish swimming PMID:29059582. Annotations to pilus assembly, flagellum assembly and the two motility processes are
indirect phenotypes.
Audit 2026-10-29: the full text of PMID:15838026 (PMC1082828, PMC HTML) was read. Its pilus,
flagellum and motility data are all mutant phenotypes (TEM counts of piliated/flagellated cells,
twitching and swimming assays, complementation), and the Discussion attributes them to outer
membrane disturbance and reduced surface wettability. The four PSEUDOCAP WRONG_TERM defects on
these rows are therefore raised to CONFIRMED. The ligase activity defect (PMID:17697256) is
lowered to PROBABLE: it rests on the later abstract (PMID:21983211) contradicting the 2007 ATP
hydrolysis claim, and neither full text was read.

O antigen biosynthetic process (GO:0009243) is the formation of the O side chain; the chain is
completed (Wzy polymerization) before WaaL acts, and WaaL forms the bond between O antigen and
core. The step belongs to lipopolysaccharide biosynthesis (GO:0009103), which UniProt/UniPathway
also use (UPA00030, not the O-antigen pathway UPA00281 used for e.g. WbpD). GO:0009103 is_a
polysaccharide biosynthetic process (GO:0000271).

PseudoCAP IBA-seed assessment

PTN node	node term	n_seeds	leaf IBAs	seed annotation (term, evidence, PMID)	verdict	rationale
PTN008076295	GO:0016757 glycosyltransferase activity	3 (WaaL, P26471, P27243)	64 (31 taxa)	GO:0016757 glycosyltransferase activity, IDA, PMID:17697256 (plus UniProt EXP O-antigen ligase rows)	SOUND	In vitro ligation assay with purified WaaL; O-antigen ligase activity is_a glycosyltransferase activity, and WaaL is an inverting GT (PMID:21983211). Broad GT term suits a Wzy_C/WaaL-like clade.
PTN008076295	GO:0005886 plasma membrane	2 (WaaL, P27243)	64 (31 taxa)	GO:0005886 plasma membrane, IDA, PMID:17697256; GO:0098567 periplasmic side of plasma membrane, IDA, PMID:20824106 (plus UniProt EXP rows)	SOUND	Polytopic inner-membrane protein; topology mapped by reporter fusions and PEG-Mal labeling.
PTN008076295	GO:0000271 polysaccharide biosynthetic process	1 (sole seed)	64 (31 taxa)	GO:0009243 O antigen biosynthetic process, IMP, PMID:15838026	SOUND	The knockout lacks O antigen on LPS and is complemented; WaaL catalyses the step that attaches O antigen to core. The seed term is arguably lateral (ligation is LPS assembly rather than O-chain formation; GO:0009103 would be better), but the node term polysaccharide biosynthetic process holds either way and suits a clade that includes TuaE-like teichuronic acid proteins. Re-seeding with an LPS biosynthetic process row or the EXP ligase evidence would be cleaner.