PilB (PA4526) is the type IV pilus extension (assembly) ATPase of Pseudomonas aeruginosa, a 566-residue cytoplasmic member of the GspE / secretion NTPase superfamily with an N-terminal MshEN domain, a Walker A/B P-loop ATPase domain with Asp and His boxes, and a tetracysteine zinc-binding motif. Like its homologues it forms a hexameric ring that docks on the cytoplasmic face of the inner-membrane platform protein PilC and uses ATP hydrolysis to drive polymerization of PilA pilin subunits into the pilus fibre. It is recruited to both cell poles in a PilC-dependent manner, and its activity is stimulated by the c-di-GMP effector FimX and blocked by the phage D3112 protein Tip. Loss of PilB abolishes surface piliation and therefore twitching motility, pilus-specific phage infection and pilus-dependent biofilm formation, while pilin synthesis is unaffected.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | EXP PMID:15659660 Disparate subcellular localization patterns of Pseudomonas a... | ACCEPT | Summary: PilB is a soluble cytoplasmic ATPase concentrated at the cell poles; a YFP-PilB fusion became diffusely cytoplasmic in a pilC mutant. Reason: Direct fluorescence-microscopy evidence in P. aeruginosa that PilB is cytoplasmic (polar, PilC-dependent). This is where the ATPase acts, on the cytoplasmic face of the assembly machinery. Supporting Evidence: PMID:15659660 YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant, suggesting that PilC is required for polar localization of PilB |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping to cytoplasm, based on the curated experimental location in the entry. Reason: Same claim as the EXP row, and the UniProt subcellular location it maps from is itself experimentally supported (PubMed:15659660). Supporting Evidence: PMID:15659660 YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant |
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: PAINT IBA from PTN001250205 (PilB/GspE-related subfamily of PTHR30258), seeded by two other family members, not by P. aeruginosa PilB. Reason: PilB is not an integral membrane protein, but it acts docked on the cytoplasmic face of the inner-membrane assembly platform: its polar localization depends on the integral inner-membrane protein PilC, with which it interacts. is_active_in plasma membrane is a fair description of where the extension motor works for a peripheral component of the inner membrane machinery, and the node lies within the GspE/PilB clade whose members function at the inner membrane. It is kept as non-core because the cytoplasmic location and the ATPase/assembly role carry the core claim. Supporting Evidence: PMID:15659660 YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant, suggesting that PilC is required for polar localization of PilB file:PSEAE/pilB/pilB-uniprot.txt Interacts with PilC (PubMed:23413032). |
| GO:0009297 pilus assembly | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from IPR013374 (type IV pilus assembly ATPase PilB). Reason: Correct but general. PilB is specifically the type IV pilus extension ATPase, and the gene already carries the more specific child term GO:0043683 type IV pilus assembly, which this row should be refined to. Proposed replacements: type IV pilus assembly Supporting Evidence: PMID:1971619 A 4.0-kilobase-pair region of DNA adjacent to the pilin structural gene was found to be essential for formation of pili. file:PSEAE/pilB/pilB-uniprot.txt Acts as a molecular motor to provide the energy that is required for biogenesis of the pilus |
| GO:0016887 ATP hydrolysis activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA from PTN001250205, an IBD whose only seed is the PseudoCAP IDA of this protein (PMID:18174131). PilB appears in its own WITH/FROM, which is expected for a seed and not circular. Reason: The seed is sound: purified P. aeruginosa PilB hydrolyses ATP in vitro, and Walker A/B and Asp/His box residues needed for that activity are essential for function. ATP hydrolysis is conserved across the GspE / PilB extension ATPases in this subfamily (the catalytic motifs are invariant), so placing it at this node is reasonable even with a single seed. Supporting Evidence: PMID:18174131 Assay of purified 6xHis-tagged PilB, PilT and PilU from P. aeruginosa showed that all three proteins have ATPase activities in vitro. |
| GO:0016887 ATP hydrolysis activity | IDA PMID:18174131 Functional role of conserved residues in the characteristic ... | ACCEPT | Summary: PseudoCAP IDA from Chiang et al. 2008, who purified His-tagged PilB, PilT and PilU and measured ATPase activity in vitro. Reason: Direct biochemical assay of the purified protein. This is the core molecular function of the extension motor. The reference is correct for this gene (the paper assays PilB) and is the sole seed of the PTN001250205 ATP hydrolysis IBD. Supporting Evidence: PMID:18174131 Assay of purified 6xHis-tagged PilB, PilT and PilU from P. aeruginosa showed that all three proteins have ATPase activities in vitro. PMID:18174131 Mutation of conserved WA or WB residues in any of the three ATPases abrogated twitching motility |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping from IPR013374 (type IV pilus assembly ATPase PilB). Reason: Consistent with the experimental activity; the PilB family signature covers the catalytic Walker and Asp/His box motifs. Supporting Evidence: PMID:18174131 Assay of purified 6xHis-tagged PilB, PilT and PilU from P. aeruginosa showed that all three proteins have ATPase activities in vitro. |
| GO:0043107 type IV pilus-dependent motility | IDA PMID:1671384 Multiple roles of the pilus biogenesis protein pilD: involve... | ACCEPT | Summary: PseudoCAP row citing Strom et al. 1991, a paper about PilD. Its abstract describes pilB only as required for pilus biogenesis (citing Nunn et al. 1990) and reports that pilB mutants have no protein export defect; it does not mention twitching motility. Reason: The claim is correct and central: PilB drives the pilus extension step of the extension/retraction cycle that is twitching motility, and loss or inhibition of PilB abolishes twitching (PMID:25049409, PMID:18174131). The full text of PMID:1671384 is not available here, so the reference and the IDA code cannot be checked against what the curator saw; a query to PseudoCAP is recorded rather than overruling the row. Supporting Evidence: PMID:18174131 PilB and PilT are bipolar proteins belonging to the secretion NTPase superfamily, and power pilus extension and retraction, respectively PMID:25049409 Tip interacts with and blocks the activity of bacterial-encoded PilB, the TFP assembly/extension ATPase |
| GO:0043107 type IV pilus-dependent motility | IMP PMID:25049409 A phage protein that inhibits the bacterial ATPase required ... | ACCEPT | Summary: CACAO IMP from Chung et al. 2014: the phage protein Tip binds PilB, blocks its activity and inhibits twitching motility. Reason: Inhibition of PilB abolishes twitching and surface piliation. Because PilB itself powers the pilus extension step, this is participation in the process, not mere necessity. Supporting Evidence: PMID:25049409 Tip interacts with and blocks the activity of bacterial-encoded PilB, the TFP assembly/extension ATPase, at an internal 40-aa region unique to PilB. Tip expression results in the loss of surface piliation. |
| GO:0043683 type IV pilus assembly | IDA PMID:1671384 Multiple roles of the pilus biogenesis protein pilD: involve... | ACCEPT | Summary: PseudoCAP row citing Strom et al. 1991 (pilD paper). The abstract states that pilB, pilC and pilD are needed for assembly of pilin into pili and that PilB function is restricted to pilus biogenesis. Reason: Core process. PilB is the extension ATPase that powers polymerization of pilin into the type IV pilus; pilB mutants make pilin but no pili. The cited abstract supports the claim, although the primary pilB mutant data are in Nunn et al. 1990 (PMID:1971619), and a mutant phenotype is IMP rather than IDA. Deferred to the curator, with a query recorded. Supporting Evidence: PMID:1671384 In Pseudomonas aeruginosa, the genes pilB, pilC, and pilD encode proteins necessary for posttranslational modification and assembly of pilin monomers into pilus organelles PMID:1971619 A 4.0-kilobase-pair region of DNA adjacent to the pilin structural gene was found to be essential for formation of pili. |
| GO:0048870 cell motility | IMP PMID:25049409 A phage protein that inhibits the bacterial ATPase required ... | MODIFY | Summary: CACAO IMP for the general term cell motility, from the same Tip/PilB inhibition experiment as the type IV pilus-dependent motility row. Reason: Correct but too general. The motility affected is twitching, i.e. type IV pilus-dependent motility (GO:0043107), which the gene already carries from the same paper. Proposed replacements: type IV pilus-dependent motility Supporting Evidence: PMID:25049409 whose expression is necessary and sufficient to mediate the inhibition of twitching motility |
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Download this section (compressed HTML)Q: GO has no term for the pilin-polymerizing mechanochemical activity of extension ATPases (or for the depolymerizing activity of retraction ATPases), so PilB and PilT both carry only ATP hydrolysis activity. Should GO add terms that distinguish type IV pilus extension from retraction (for example a "type IV pilus retraction" process), so that the motors are not annotated identically?
Q: Does P. aeruginosa PilB bind c-di-GMP directly through its MshEN domain, as shown for the Vibrio MshE N-terminal domain, in addition to being regulated by the c-di-GMP effector FimX?
Experiment: Measure ATP hydrolysis by purified P. aeruginosa PilB hexamers alone and with FimX, the PilC cytoplasmic domain and c-di-GMP, to define how each partner regulates the extension motor's activity.
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