WaaL of <i>Pseudomonas aeruginosa</i> utilizes ATP in <i>in vitro</i> ligation of O antigen onto lipid A‐core
Bibliographic record
Abstract
waaL has been implicated as the gene that encodes the O-antigen ligase. To date, in vitro biochemical evidence to prove that WaaL possesses ligase activity has been lacking due to the difficulty of purifying WaaL and unavailability of substrates. Here we describe the purification of WaaL, a membrane protein with 11 potential transmembrane segments from Pseudomonas aeruginosa, and the development of an in vitro O-antigen ligase assay. WaaL was expressed in a P. aeruginosa wbpL knockout strain, which is defective in its initial glycosyltransferase for O-antigen biosynthesis. This approach allowed the purification of WaaL without contaminating O-antigen-undecaprenol-phosphate (Und-P) molecules. Purified WaaL resolved to a monomer (35 kDa) and a dimer (70 kDa) band in SDS-PAGE. The substrates for the O-antigen ligase assay, O-antigen-Und-P and lipid A-core were prepared from a waaL mutant. ATP at 2-4 mM is optimum for the O-ligase activity, and ATP hydrolysis by WaaL follows Michaelis-Menten kinetics. Site-directed mutagenesis analysis indicated that the periplasmic loop region of WaaL is important for ligase activity. A waaL mutant of P. aeruginosa could not be cross-complemented by waaL of Escherichia coli, which suggested that each of these proteins has specificity for its cognate core oligosaccharide.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.001 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".