PA5458 is a polytopic integral inner-membrane protein of the acyltransferase-3 (AT3, Pfam PF01757) family, with nine predicted transmembrane helices. It is encoded in the pa5455-pa5459 operon next to the common polysaccharide antigen (CPA) biosynthesis locus pa5447-pa5454, and the CPA locus is now taken to span all 13 genes. CPA is the D-rhamnan O polysaccharide attached to P. aeruginosa lipopolysaccharide. An in-frame pa5458 deletion still makes CPA, but the polymer runs at shifted, more diffuse positions on SDS-PAGE and binds the CPA-specific monoclonal antibody N1F10 more weakly. Overexpression lowers CPA amount and shortens its chains slightly. PA5458 is therefore thought to be the membrane-bound O-acyltransferase (probably an O-acetyltransferase) that decorates CPA. Acetylation of CPA by PA5458 has not been shown chemically or enzymatically. Related AT3 proteins O-acetylate other cell-surface glycans, such as peptidoglycan (Staphylococcus aureus OatA) and O antigen (Salmonella OafA).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0009103 lipopolysaccharide biosynthetic process | IBA GO_REF:0000033 | MODIFY | Summary: IBA from the PAINT IBD at PTN000546841, whose only seed is PA5458's own PseudoCAP IMP row (PMID:23341552). The self-seed is expected, not circular. For PA5458 itself the claim is true: the deletion changes the structure of CPA, an O polysaccharide attached to LPS. The node placement is wrong, however. The clade includes peptidoglycan O-acetyltransferases (S. aureus and L. monocytogenes OatA) and mycobacterial and streptomycete members, and 15 of its 31 leaves are in organisms that make no LPS. Reason: For this gene the claim holds but is less specific than the evidence: the affected polymer is the CPA O antigen, so GO:0009243 O antigen biosynthetic process (is_a GO:0009103), which the gene already carries, is the right term. The action matches the IMP row on the same term. The IBD at PTN000546841 is reported upstream as wrong for the clade (source defect below). Propagation Review Root cause: PROPAGATION BAD Failure modes: LINEAGE OR TAXON MISMATCH FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000546841 · PTN000546841 SUPPORTS SOURCE BUT NOT TARGET The node term is not valid clade-wide: it spans Gram-positive peptidoglycan O-acetyltransferases (OatA) and LPS-less actinobacteria. It happens to hold for PA5458, the seed. UniProtKB:Q9HTB2 · PA5458 (self; sole IBD seed, IMP PMID:23341552) SUPPORTS TRANSFER Expected self-seed reflecting the target's own mutant evidence. Proposed replacements: O antigen biosynthetic process Supporting Evidence: PMID:23341552 its function is clearly not required for assembly and processing of the polysaccharide, although the mutant produces CPA with a different structure, which is consistent with previous reports of the presence of acetylation substitutions in the sugars of CPA file:PSEAE/PA5458/PA5458-bioinformatics/RESULTS.md So 15 of the 31 leaves are in organisms that make no LPS. PMID:15661003 the muramic acid of the oatA mutant lacked this modification |
| GO:0009103 lipopolysaccharide biosynthetic process | IMP PMID:23341552 Five new genes are important for common polysaccharide antig... | MODIFY | Summary: PseudoCAP IMP, and the sole seed of two IBDs: GO:0009103 at PTN000546841 and, generalised, GO:0000271 at PTN000546715. The in-frame deletion still makes CPA but changes its structure (shifted, diffuse bands and weaker MAb N1F10 binding), and complementation reverses the shift. This fits PA5458 modifying the polymer itself. It is more than a requirement phenotype, and the paper supports involvement in LPS O-antigen biosynthesis for this gene. Reason: The seed is sound for PA5458 but under-specific. The affected polymer is the CPA O polysaccharide, so GO:0009243 O antigen biosynthetic process (is_a GO:0009103), which the gene already carries from the same paper, is the precise term. Refining the row does not change the problems with the IBDs. Those concern node placement: one Gram-negative O-antigen seed propagated to LPS-less Gram-positive bacteria (PTN000546841) and to nematode and oomycete proteins (PTN000546715). They are recorded as GO_CENTRAL defects. Proposed replacements: O antigen biosynthetic process Supporting Evidence: PMID:23341552 When the run time of SDS-PAGE was increased, it was clear that the bands produced by the mutant were shifted relative to the positions of bands produced by the wild type, and the bands also appeared to be more diffuse PMID:23341552 its function is clearly not required for assembly and processing of the polysaccharide, although the mutant produces CPA with a different structure, which is consistent with previous reports of the presence of acetylation substitutions in the sugars of CPA |
| GO:0009243 O antigen biosynthetic process | IDA PMID:23341552 Five new genes are important for common polysaccharide antig... | ACCEPT | Summary: PseudoCAP row from the same paper. The term is the right one: the deletion alters the structure of CPA, a D-rhamnan O antigen, probably by removing an acyl (acetyl) substitution. It is a co-seed of both IBDs. The evidence code is wrong, though. No direct assay of PA5458 was done; the evidence is deletion, complementation and overexpression with LPS read out by SDS-PAGE and Western blot, which is IMP. Reason: Most specific supported process for this gene and its core process. The acceptance is of the claim, with the evidence-code error reported upstream. Supporting Evidence: PMID:23341552 its function is clearly not required for assembly and processing of the polysaccharide, although the mutant produces CPA with a different structure, which is consistent with previous reports of the presence of acetylation substitutions in the sugars of CPA PMID:23341552 Overexpression of the putative acetyltransferase resulted in a modest reduction in the amount of CPA produced and a subtle shift in the chain length distribution |
| GO:0016020 membrane | IBA GO_REF:0000033 | MODIFY | Summary: IBA from the membrane IBD at PTN000546715, seeded by a C. elegans AT3 protein. This IBD is independent of the PseudoCAP seed. Membrane location is sound for the whole AT3 family (polytopic integral membrane proteins), and PA5458 has nine predicted TM helices. The S. aureus homologue OatA is described as an integral membrane protein. Reason: Correct but general. In a Gram-negative bacterium a polytopic alpha-helical protein with no signal for the outer membrane sits in the cytoplasmic (inner) membrane. There the AT3 O-acyltransferases modify glycans on their way to the periplasm. So GO:0005886 plasma membrane is the supported specific term. Propagation Review Root cause: NO FAILURE NON CORE Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000546715 · PTN000546715 SUPPORTS TRANSFER Membrane location is a family-wide property of polytopic AT3 acyltransferases; appropriate at this deep node. WB:WBGene00044617 · C. elegans AT3 seed SUPPORTS TRANSFER Independent of the PseudoCAP seed; generic membrane term only. Proposed replacements: plasma membrane Supporting Evidence: file:PSEAE/PA5458/PA5458-uniprot.txt FT TRANSMEM 291..312 FT /note="Helical" FT /evidence="ECO:0000256|SAM:Phobius" PMID:15661003 the muramic acid of the oatA mutant lacked this modification |
| GO:0016747 acyltransferase activity, transferring groups other than amino-acyl groups | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO from the acyltransferase-3 domain (IPR002656, Pfam PF01757, residues 7-341). AT3 proteins O-acylate glycans, and the CPA structural change in the pa5458 mutant fits loss of an acyl (likely acetyl) substitution. Activity has not been assayed and the acyl donor and acceptor are unknown. Reason: As specific as the evidence allows. O-acetyltransferase activity is likely but not shown, so the domain-level acyltransferase term is kept as the core molecular function. Supporting Evidence: file:PSEAE/PA5458/PA5458-uniprot.txt FT DOMAIN 7..341 FT /note="Acyltransferase 3" FT /evidence="ECO:0000259|Pfam:PF01757" PMID:23341552 The pa5458 gene encodes a protein with a conserved acetyltransferase domain. |
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Download this section (compressed HTML)Q: Is the PTN000546841 node (which mixes Gram-negative O-antigen acyltransferases such as PA5458 and Salmonella OafA with Gram-positive peptidoglycan O-acetyltransferases such as OatA) better annotated with a cell-surface glycan modification term, with GO:0009103 restricted to a Gram-negative subclade?
Experiment: Compare CPA purified from wild type and the pa5458 deletion by NMR and mass spectrometry to identify the missing acyl substituent and its sugar position. Then test acyl-CoA dependent transfer by the purified or membrane-reconstituted protein to establish the activity directly.
Type: structural and biochemical analysis
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