FliH is a cytosolic component of the flagellar type III secretion system (fT3SS) export apparatus. It forms a complex with the FliI ATPase (FliH2-FliI1 stoichiometry) and acts as a negative regulator, inhibiting FliI ATPase activity until productive engagement with the export gate (FlhA/FlhB/FliPQR). FliH helps coordinate ATP hydrolysis with PMF-driven translocation of flagellar substrates and is essential for efficient and ordered export of flagellar proteins. Based primarily on conserved mechanism studies in Salmonella, with B. subtilis fliH expected to function similarly due to conserved architecture.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005829 cytosol | IBA GO_REF:0000033 | MODIFY | Summary: FliH is a soluble cytoplasmic protein that forms part of the ATPase module of the flagellar type III secretion system. The IBA annotation for cytosol localization is phylogenetically inferred and consistent with the known biochemistry of FliH across bacteria. However, this term is overly broad since FliH specifically localizes to the cytoplasmic face of the basal body at the C-ring/MS-ring interface as part of the export apparatus. A more specific cellular component term exists: GO:0120102 (bacterial-type flagellum secretion apparatus), which explicitly names FliH as a component. Reason: While FliH is indeed cytosolic, the term GO:0005829 (cytosol) is too broad. FliH specifically localizes to the flagellar secretion apparatus at the cytoplasmic face of the basal body. GO:0120102 (bacterial-type flagellum secretion apparatus) is a more specific and accurate term that explicitly mentions FliH as a component in its definition. Proposed replacements: bacterial-type flagellum secretion apparatus Supporting Evidence: PMID:33572887 The T3SS is composed of an export gate (FliPQR/FlhA/FlhB) located in the flagellar basal body and an associated soluble ATPase complex in the cytoplasm (FliHIJ). PMID:33572887 Associated at the cytoplasmic face is the ATPase complex constituted of FliHIJ. |
| GO:0015031 protein transport | IEA GO_REF:0000043 | MODIFY | Summary: This IEA annotation is derived from the UniProt keyword mapping (KW-0653 Protein transport). While technically correct that FliH is involved in protein transport (specifically flagellar protein export), this term is overly general. FliH functions specifically in the flagellar type III secretion system to export flagellar proteins. A more specific term exists: GO:0030254 (protein secretion by the type III secretion system). Reason: GO:0015031 (protein transport) is too general. FliH specifically functions in flagellar type III secretion, not general protein transport. The term GO:0030254 (protein secretion by the type III secretion system) is more accurate and specific for FliH function. Proposed replacements: protein secretion by the type III secretion system Supporting Evidence: PMID:33572887 The bacterial flagellum and the related virulence-associated injectisome system of pathogenic bacteria utilize a type III secretion system (T3SS) to export substrate proteins across the inner membrane in a proton motive force-dependent manner. PMID:33572887 The soluble ATPase complex made of FliHIJ is part of the export apparatus and thought to facilitate docking and unfolding of substrates [17]. |
| GO:0044781 bacterial-type flagellum organization | IEA GO_REF:0000043 | ACCEPT | Summary: This IEA annotation is derived from UniProt keyword mapping (KW-1005 Flagellum biogenesis). The term GO:0044781 (bacterial-type flagellum organization) is a parent term that encompasses flagellar assembly. While not wrong, the term GO:0044780 (bacterial-type flagellum assembly) is more specific and already annotated for this gene. This annotation is acceptable but redundant given the more specific assembly term. Reason: This is a broader term that is consistent with FliH function in flagellar biogenesis. While GO:0044780 (assembly) is more specific and already present, the IEA mapping to the broader organization term is not incorrect and provides useful context from the keyword mapping pipeline. Acceptable as a secondary annotation. Supporting Evidence: file:BACSU/fliH/fliH-deep-research-falcon.md Target is Bacillus subtilis (strain 168) fliH, UniProt P23449, annotated as a probable flagellar assembly protein FliH of the FliH family with Flagellar_FliH/T3SS_HrpE and related domains. |
| GO:0044780 bacterial-type flagellum assembly | IMP PMID:25313396 FlgM is secreted by the flagellar export apparatus in Bacill... | UNDECIDED | Summary: This annotation cites PMID:25313396, which is titled "FlgM is secreted by the flagellar export apparatus in Bacillus subtilis." Critical review of this paper reveals that it focuses on FlgM secretion and identifies the minimal components required for secretion as FliO, FliP, FliQ, FliR, FlhA, FlhB, FliF, FliG, and FliK. Importantly, FliH is NOT mentioned in the abstract or as a required component. The paper does not provide direct experimental evidence for FliH involvement. Reason: PMID:25313396 does NOT provide direct experimental evidence for FliH function. The paper is about FlgM secretion and explicitly lists the minimal required components, which do not include FliH. While FliH likely does function in flagellar assembly based on conserved mechanism studies in Salmonella, this specific reference does not support the annotation. The annotation should be re-evaluated with appropriate evidence. Supporting Evidence: PMID:25313396 FlgM secretion is strongly enhanced by, but does not strictly require, hook-basal body completion and instead demands a minimal subset of flagellar proteins that includes the FliF/FliG basal body proteins, the flagellar type III export apparatus components FliO, FliP, FliQ, FliR, FlhA, and FlhB, and the substrate specificity switch regulator FliK. |
| GO:0071978 bacterial-type flagellum-dependent swarming motility | IMP PMID:25313396 FlgM is secreted by the flagellar export apparatus in Bacill... | UNDECIDED | Summary: This annotation to swarming motility also cites PMID:25313396. As noted above, this paper focuses on FlgM secretion and does not provide direct experimental evidence for FliH. The paper does not specifically test fliH mutants for swarming motility phenotypes. While loss of FliH would likely affect motility indirectly through impaired flagellar assembly and export efficiency, this specific reference does not document FliH-dependent swarming defects. Reason: PMID:25313396 does not provide direct evidence for FliH involvement in swarming motility. The paper is focused on FlgM secretion mechanism, not motility phenotypes. The annotation requires better supporting evidence - either from this paper (if experiments on fliH were performed but not mentioned in abstract) or from alternative references that specifically tested fliH mutant motility phenotypes in B. subtilis. Supporting Evidence: PMID:25313396 FlgM is secreted by the flagellar export apparatus in Bacillus subtilis. |
| GO:0042030 ATPase inhibitor activity | ISS PMID:10998179 FliH, a soluble component of the type III flagellar export a... | NEW | Summary: FliH acts as a negative regulator of the FliI ATPase, inhibiting its ATPase activity until productive engagement with the export gate. This biochemical function has been demonstrated in Salmonella (PMID:10998179) and is expected to be conserved in B. subtilis based on sequence homology. GO:0042030 (ATPase inhibitor activity) accurately describes this core molecular function. Reason: This annotation is proposed based on conserved biochemistry from Salmonella FliH, which has been shown to inhibit FliI ATPase activity. The Minamino and Macnab 2000 paper demonstrates that FliH inhibits FliI ATPase activity approximately 10-fold. This is a core molecular function that should be annotated. Evidence code ISS (inferred from sequence similarity) is appropriate given the conservation of FliH function across bacteria. Supporting Evidence: PMID:10998179 The ATPase activity of the FliH-FliI complex was about 10-fold lower than that of FliI alone; the presence or absence of ATP did not affect the formation of the complex. PMID:10998179 We propose that FliH functions as a negative regulator to prevent FliI from hydrolysing ATP until the flagellar export apparatus is competent to link this hydrolysis to the translocation of export substrates across the plane of the cytoplasmic membrane into the lumen of the nascent flagellar structure. |
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Download this section (compressed HTML)Q: Does B. subtilis fliH show the same ATPase inhibitory activity toward FliI as demonstrated in Salmonella? Direct biochemical characterization in B. subtilis is lacking.
Suggested experts: Kearns DB
Q: What is the phenotype of a B. subtilis fliH deletion mutant with respect to flagellar assembly, motility, and flagellar protein export?
Suggested experts: Kearns DB, Calvo RA
Experiment: Express and purify B. subtilis FliH and FliI proteins. Measure FliI ATPase activity in the presence and absence of FliH to confirm inhibitory function.
Hypothesis: B. subtilis FliH inhibits FliI ATPase activity similar to Salmonella FliH.
Type: biochemical assay
Experiment: Construct clean fliH deletion in B. subtilis 168. Assess swarming motility on soft agar plates and flagellar assembly by electron microscopy or flagellin secretion assays.
Hypothesis: fliH deletion in B. subtilis results in reduced motility and flagellar assembly defects.
Type: genetic/phenotypic analysis
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