PA4379 (warA) encodes WarA, a cytoplasmic class I SAM-dependent methyltransferase (Methyltransf_25, PF13649) that binds the second messenger cyclic-di-GMP. WarA was found as a partner of the SadC diguanylate cyclase and forms a complex with WarB (a putative kinase encoded by a convergently transcribed gene). Together they resemble the bifunctional E. coli O-antigen chain regulator WbdD. Purified WarA binds c-di-GMP specifically in the low micromolar range and shows SAM-dependent methyltransferase activity with lipopolysaccharide (LPS) as substrate, and c-di-GMP stimulates that activity. WarA influences the modal distribution of the common polysaccharide antigen (CPA) O antigen, interacts with LPS biogenesis proteins, and contributes to evasion of host immune detection in a zebrafish infection model.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | EXP PMID:28263305 Cyclic-di-GMP regulates lipopolysaccharide modification and ... | REMOVE | Summary: Bare protein binding for the reported WarA interactions with the SadC cytoplasmic domain and with WarB (two-hybrid and far-western assays). Reason: Generic protein binding carries no functional information, and no informative MF term describes these partner interactions. Removing the row does not mean the interactions are false; WarA's function is captured by its methyltransferase and c-di-GMP binding rows. Supporting Evidence: PMID:28263305 WarA was shown to specifically interact with the cytoplasmic part of SadC and form a complex with WarB encoded by a convergently transcribed gene. |
| GO:0008168 methyltransferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from PTN008676703, whose seeds are E. coli RlmA, M. tuberculosis Rv2952, human TMT1A and WarA itself. Consistent with WarA's own IDA. Reason: The generic term suits a node whose seeds methylate rRNA, a lipid and small thiols. WarA's appearance among the sources reflects its own experimental grounding. The substrate and product are not chemically characterised, so the generic term is as precise as the evidence allows. Supporting Evidence: PMID:28263305 Using a fluorescent-based methyltransferase activity assay, LPS was shown to be a substrate for WarA and that increasing the concentration of LPS leads to consequential increase in enzyme activity |
| GO:0008168 methyltransferase activity | IDA PMID:28263305 Cyclic-di-GMP regulates lipopolysaccharide modification and ... | ACCEPT | Summary: PseudoCAP IDA and one of four seeds of the PTN008676703 IBD. Purified WarA was tested in a SAM-donor methyltransferase kit, with LPS as acceptor. Reason: Full text read. The assay uses purified His-tagged WarA, SAM as methyl donor and purified LPS as substrate, and activity rises with LPS concentration. The kit reports SAM turnover (coupled fluorescence), so the methylated position on LPS is not identified. Generic methyltransferase activity is therefore the right level. Supporting Evidence: PMID:28263305 Methyltransferase activity assays were performed using a methyltransferase activity kit (Enzo Life Science, Farmingdale, NY) as per manufacturer’s specifications using S-adenosyl-L-methionine as the methyl group donor and measuring activity in relative fluorescent units. PMID:28263305 At every WarA concentration tested, the addition of c-di-GMP led to an increase in the enzymatic activity of WarA (Fig. 4d). |
| GO:0035438 cyclic-di-GMP binding | IDA PMID:28263305 Cyclic-di-GMP regulates lipopolysaccharide modification and ... | ACCEPT | Summary: DRaCALA with purified WarA shows specific c-di-GMP binding, competed only by unlabelled c-di-GMP. Binding stimulates methyltransferase activity. Reason: Direct binding assay with a specificity control. Binding regulates the enzyme, so it is a functional ligand-binding activity, part of the core function. Supporting Evidence: PMID:28263305 WarA was capable of binding to c-di-GMP (Fig. 4b) in the low micromolar range, which is consistent with known biologically relevant c-di-GMP binding concentrations |
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Download this section (compressed HTML)Q: Which residue or sugar of LPS does WarA methylate, and is the activity specific to the CPA O antigen, as for the WbdD-type O-antigen chain terminators it resembles?
Experiment: Identify the methylated LPS species from wild-type and warA mutant cells by mass spectrometry or NMR, with and without c-di-GMP, to define the reaction and allow a specific MF term.
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