PilM (PA5044) is the cytoplasmic, actin-like component of the type IVa pilus alignment subcomplex of Pseudomonas aeruginosa, encoded first in the pilMNOPQ operon. The 354-residue protein has an FtsA/MreB-type actin fold that binds ATP and Mg2+ but has no detectable ATPase activity in orthologues. PilM associates with the inner membrane by binding the conserved cytoplasmic N-terminal tail of the bitopic membrane protein PilN; PilN binding monomerises PilM and closes its domains around the ATP site. Together with PilN, PilO and the lipoprotein PilP it forms the PilMNOP inner-membrane subcomplex, which, through PilP and the secretin PilQ, spans the cell envelope. PilM also binds the motor ATPases PilB and PilT and the platform protein PilC, connecting the alignment subcomplex to the motor. Loss of PilM destabilises the other subcomplex proteins and the PilQ secretin, abolishes surface pili, and therefore abolishes twitching motility and pilus-specific phage sensitivity.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | EXP PMID:19857645 PilM/N/O/P proteins form an inner membrane complex that affe... | ACCEPT | Summary: UniProt experimental location from Ayers et al. 2009. PilM has no transmembrane segment and is cytoplasmic, but co-fractionates with the inner membrane through PilN. Reason: PilM is a soluble protein acting on the cytoplasmic side of the inner membrane. The cytoplasm is where it resides; its membrane association is peripheral, via the PilN N-terminal tail, and is recorded separately as a NEW cytoplasmic side of plasma membrane row. Supporting Evidence: PMID:19857645 all four proteins, including the nominally cytoplasmic PilM, colocalized to the inner membrane file:PSEAE/pilM/pilM-uniprot.txt Cytoplasm {ECO:0000269|PubMed:19857645}. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping to cytoplasm. Reason: Same claim as the EXP row; the UniProt location it maps from is experimentally supported (PubMed:19857645). Supporting Evidence: file:PSEAE/pilM/pilM-uniprot.txt Note=Localizes to the inner membrane through interaction with PilN. |
| GO:0043107 type IV pilus-dependent motility | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA from PTN001286216, an IBD whose sole seed is the PseudoCAP IMP of this protein. PilM appearing in its own WITH/FROM is expected for a seed, not circular. Reason: Correct for PilM, which is a core component of the type IVa pilus machine and whose loss abolishes twitching (PMID:7565110). The node, however, is broader than the PilM clade: only 15 of its 23 leaves carry the PilM signatures that this gene carries (InterPro IPR005883, Pfam PF11104, NCBIfam TIGR01175), while four (E. coli P77277, Salmonella P0A206, Fusobacterium Q8RI50, Listeria Q8Y7V8) carry the InterPro IPR013366 EutJ signature instead, and four match neither. The row is accepted for this gene; the node placement is reported to GO Central. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: UniProtKB:G3XD28 · pilM PA5044 (self; sole IBD seed, IMP PMID:7565110 and PMID:19857645) SUPPORTS SOURCE BUT NOT TARGET The seed is sound for PilM, but does not support transfer to the EutJ-signature members of the node. PANTHER:PTN001286216 · PTN001286216 SUPPORTS SOURCE BUT NOT TARGET 23 leaves in 20 taxa within PTHR32432:SF3. Fifteen carry the PilM domain signature (IPR005883/PF11104), four carry the EutJ signature (IPR013366/PF14450) and lack all PilM signatures, and four carry neither. No leaf but the seed has an experimental annotation. Supporting Evidence: PMID:7565110 PilM-P were found to be required for fimbrial biogenesis by complementation studies using twitching motility and sensitivity to fimbrial-specific phage as indicators of the presence of functional fimbriae. |
| GO:0043107 type IV pilus-dependent motility | IMP PMID:19857645 PilM/N/O/P proteins form an inner membrane complex that affe... | ACCEPT | Summary: PseudoCAP IMP from Ayers et al. 2009, which characterised pilM/N/O/P mutants and the PilMNOP inner-membrane complex. Reason: PilM is a structural component of the pilus machine; deletion mutants lack surface pili and therefore twitching. As a part of the machine (binding PilN, PilB, PilT and PilC), PilM does some of the work of the process rather than merely being required for it. The abstract frames the cluster as required for twitching; full text not available here, deferred to the curator. Supporting Evidence: PMID:19857645 The highly conserved pilM/N/O/P/Q gene cluster is among the core set of genes required for cell surface expression of type IV pili and associated twitching motility. file:PSEAE/pilM/pilM-uniprot.txt Deletion mutants lack surface pili. |
| GO:0043107 type IV pilus-dependent motility | IMP PMID:7565110 Characterization of a five-gene cluster required for the bio... | ACCEPT | Summary: PseudoCAP IMP from Martin et al. 1995: transposon and deletion mutants in pilM-P lack the twitching colony morphology and are complemented. Reason: Direct mutant/complementation data for pilM in a twitching motility assay; PilM is a component of the pilus machine. Seed of the PTN001286216 motility IBD. Supporting Evidence: PMID:7565110 PilM-P were found to be required for fimbrial biogenesis by complementation studies using twitching motility and sensitivity to fimbrial-specific phage as indicators of the presence of functional fimbriae. |
| GO:0043683 type IV pilus assembly | IMP PMID:19857645 PilM/N/O/P proteins form an inner membrane complex that affe... | ACCEPT | Summary: PseudoCAP IMP: loss of PilM abolishes surface pili and destabilises the PilMNOP subcomplex and PilQ secretin. Reason: Core process. PilM is the cytoplasmic part of the alignment subcomplex of the assembly machine, binding PilN and the motor proteins PilB, PilT and PilC. Supporting Evidence: PMID:19857645 We provide evidence that an intact inner membrane complex is required for optimal formation of the outer membrane complex of the type IVa pilus system in P. aeruginosa PMID:27022027 Pull-down experiments demonstrated direct interactions of PilM with PilB and PilT. |
| GO:0043683 type IV pilus assembly | IMP PMID:7565110 Characterization of a five-gene cluster required for the bio... | ACCEPT | Summary: PseudoCAP IMP from Martin et al. 1995; pilM-P mutants lack fimbriae by phage sensitivity, twitching and electron microscopy. Reason: Mutant evidence for fimbrial biogenesis, confirmed by EM. Correct core process for this gene. Supporting Evidence: PMID:7565110 This was confirmed by electron microscopy. PMID:7565110 Deletion mutants of pilM and pilQ displayed a dominant negative phenotype when transformed into wild-type cells, suggesting that these genes encode proteins involved in multimeric structures. |
| GO:0044096 type IV pilus | IBA GO_REF:0000033 | REMOVE | Summary: PAINT IBA from PTN001286216, whose only seed is the PseudoCAP IMP located_in type IV pilus row of this protein. Reason: GO:0044096 is the surface filament ("A short filamentous structure on the surface of a bacterial cell"), built of pilins. PilM is a cytoplasmic protein docked on the inner membrane through PilN and is not part of the fibre. The only seed is a mutant phenotype (no fimbriae) annotated as a location, which cannot show where PilM is. The IBD is therefore unsupported, and it also propagates to EutJ proteins that have nothing to do with pili. This is an over-propagated phylogenetic inference that is contradicted on biological grounds. Propagation Review Root cause: SOURCE BAD Failure modes: COMPARTMENT OR COMPLEX MISMATCH ROLE CONFLATION WRONG ORTHOLOG OR PARALOG Sources checked: UniProtKB:G3XD28 · pilM PA5044 (self; sole IBD seed, PseudoCAP IMP PMID:7565110) SOURCE BAD The seed annotates a fimbria-less mutant phenotype as a location in the pilus fibre; PilM is cytoplasmic/inner-membrane-associated. PANTHER:PTN001286216 · PTN001286216 SOURCE BAD Node also covers four leaves with the EutJ domain signature and no PilM signature (E. coli P77277, Salmonella P0A206, Fusobacterium Q8RI50, Listeria Q8Y7V8), and four that match neither family. Supporting Evidence: PMID:19857645 all four proteins, including the nominally cytoplasmic PilM, colocalized to the inner membrane PMID:23457250 This complex has components in the cytoplasm (PilM), inner membrane (PilN, PilO, and PilP), and outer membrane (PilQ). |
| GO:0044096 type IV pilus | IMP PMID:7565110 Characterization of a five-gene cluster required for the bio... | REMOVE | Summary: PseudoCAP located_in type IV pilus, IMP, from the Martin et al. 1995 mutant screen. The same paper and code were used for pilN, pilO and pilP. Reason: The IMP code and the abstract both point to a mutant phenotype: pilM-P mutants lack functional fimbriae (twitching, phage, EM); the full text (not available here) was not read. A loss of pili does not locate the gene product in the pilus, and an IMP cannot support a located_in claim of this kind. PilM is cytoplasmic and inner-membrane associated (PMID:19857645), not part of the fibre that GO:0044096 defines. This row is the sole seed of the PTN001286216 type IV pilus IBD. Supporting Evidence: PMID:7565110 PilM-P were found to be required for fimbrial biogenesis by complementation studies using twitching motility and sensitivity to fimbrial-specific phage as indicators of the presence of functional fimbriae. PMID:19857645 all four proteins, including the nominally cytoplasmic PilM, colocalized to the inner membrane |
| GO:0051301 cell division | IEA GO_REF:0000002 | REMOVE | Summary: InterPro2GO mapping from IPR003494 (SHS2 domain of FtsA), which PilM shares with the cell division protein FtsA. Reason: The SHS2/actin-like fold is shared by FtsA, MreB, EutJ and PilM, but only FtsA functions in cell division. PilM is a type IV pilus machine protein; there is no evidence that it participates in cell division. The domain-to-process mapping is wrong for this family member. Supporting Evidence: PMID:7565110 PilM has shared sequence motifs with the cell division protein FtsA from Bacillus subtilis and Escherichia coli, as well as the rod-shape-determining protein MreB PMID:23457250 PilM is a cytoplasmic actin-like protein that has been shown in Thermus thermophilus to bind the N terminus of PilN |
| GO:0005524 ATP binding | IDA PMID:27022027 PilN Binding Modulates the Structure and Binding Partners of... | NEW | Summary: Proposed: PilM crystal structures with bound ATP/Mg2+, and PilN binding kinetics that depend on ATP. Reason: P. aeruginosa PilM was crystallised with ATP and Mg2+ (PDB 5EOU, 5EOY), and PilN association is faster with ATP than ADP. Orthologues bind but do not hydrolyse ATP (Thermus, Myxococcus), so ATP binding, not ATP hydrolysis, is the supported activity. Supporting Evidence: PMID:27022027 Using biolayer interferometry, we found that the association rate of PilN with PilM is higher in the presence of ATP compared with ADP. file:PSEAE/pilM/pilM-uniprot.txt /ligand="ATP" PMID:21596754 We find no evidence for the catalysis of ATP hydrolysis by PilM |
| GO:0009898 cytoplasmic side of plasma membrane | IC PMID:19857645 PilM/N/O/P proteins form an inner membrane complex that affe... | NEW | Summary: Proposed: PilM is peripherally associated with the cytoplasmic face of the inner membrane through the PilN N-terminal tail. Reason: Two independent lines agree: PilM co-fractionates with the inner membrane in P. aeruginosa (PMID:19857645), and it binds the cytoplasmic N-terminal tail of the bitopic inner-membrane protein PilN (PMID:23457250, PMID:27022027). PilM has no transmembrane segment, so the association is peripheral, on the cytoplasmic side. Added beside the cytoplasm rows rather than replacing them. Supporting Evidence: PMID:19857645 all four proteins, including the nominally cytoplasmic PilM, colocalized to the inner membrane PMID:23457250 the N-terminal cytoplasmic tail of PilN binds to the actin-like protein PilM |
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Download this section (compressed HTML)Q: GO has no cellular component for the type IV pilus assembly machine (the PilBTCMNOPQ envelope-spanning apparatus) distinct from GO:0044096, the surface fibre. Should a "type IV pilus machine" complex term be added, so that machine components such as PilM, PilN, PilO, PilP and PilQ are not annotated to the fibre?
Q: What is the role of ATP binding by PilM, given that it does not appear to hydrolyse ATP: does nucleotide state switch PilM between PilB- and PilT-bound conformations during extension and retraction?
Experiment: Test whether ATP-binding-site mutants of P. aeruginosa PilM (P-loop 18-23) still bind PilN, PilB and PilT and support twitching, to establish whether nucleotide binding is required for motor coupling.
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