Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0004721 phosphoprotein phosphatase activity	IBA GO_REF:0000033	ACCEPT	Summary: IBA annotation for phosphoprotein phosphatase activity is strongly supported by phylogenetic analysis and experimental evidence. FliY contains CheC-like phosphatase domains with conserved catalytic residues and has been directly demonstrated to catalyze CheY-P dephosphorylation [PMID:12920116]. Reason: This IBA annotation is well-supported by the phylogenetic tree inference and is consistent with extensive experimental evidence. PMID:12920116 directly demonstrates that FliY increases the rate of CheY-P hydrolysis in vitro. The domain architecture (CheC-like phosphatase domain) and structural studies further support this function. Supporting Evidence: PMID:12920116 We showed that FliY increases the rate of CheY-P hydrolysis in vitro. PMID:12920116 In particular we have identified the phosphatase as FliY file:BACSU/fliY/fliY-deep-research-falcon.md See deep research file for comprehensive analysis
GO:1902021 regulation of bacterial-type flagellum-dependent cell motility	IBA GO_REF:0000033	ACCEPT	Summary: IBA annotation for regulation of flagellum-dependent motility is well-supported. FliY regulates motility by controlling CheY-P levels at the flagellar motor, which determines flagellar rotation direction [PMID:12920116]. Reason: This annotation accurately captures FliY's regulatory role in motility. By dephosphorylating CheY-P at the flagellar switch, FliY modulates the duration of enhanced CheY-P levels and thereby regulates switching between clockwise and counterclockwise flagellar rotation. Supporting Evidence: PMID:12920116 Consequently, we imagine that the duration of enhanced CheY-P levels caused by activation of the CheA kinase upon attractant binding to receptors, is brief due both to adaptational processes and to phosphatase activity of FliY.
GO:0003774 cytoskeletal motor activity	IEA GO_REF:0000002	REMOVE	Summary: IEA annotation based on InterPro domain IPR001172 (FliN_T3SS_HrcQb). This annotation is problematic because FliY is not itself a motor protein - it is a component of the motor switch that regulates motor activity, but does not generate force or movement directly. Reason: This is an over-annotation. FliY is a component of the flagellar motor switch complex (C-ring) but it does not have motor activity itself. The motor activity is generated by the stator units (MotA/MotB) interacting with FliG. FliY's role is to modulate CheY-P levels, not to generate rotational force. The InterPro domain hit is based on FliN homology, but FliN is a switch component, not a motor per se. The term "cytoskeletal motor activity" implies direct force generation, which FliY does not perform. Supporting Evidence: PMID:12920116 the flagellar switch, which controls direction of flagellar rotation based on levels of the chemotaxis primary response regulator, CheY-P
GO:0005886 plasma membrane	IEA GO_REF:0000044	MODIFY	Summary: IEA annotation based on UniProt subcellular location mapping. UniProt indicates FliY is a peripheral membrane protein on the cytoplasmic side. However, more specific localization to the flagellar C-ring is available. Reason: While plasma membrane association is not incorrect, a more specific and informative CC annotation exists. FliY localizes specifically to the flagellar basal body C-ring. The UniProt entry itself states the switch is localized at the base of the flagellar motor, in or near the C-ring, attached to the MS-ring. Proposed replacements: bacterial-type flagellum basal body, C ring Supporting Evidence: PMID:12920116 the flagellar switch, which controls direction of flagellar rotation based on levels of the chemotaxis primary response regulator, CheY-P
GO:0006935 chemotaxis	IEA GO_REF:0000120	ACCEPT	Summary: IEA annotation for chemotaxis from combined automated methods. FliY is clearly involved in chemotaxis as it is part of the signal termination machinery that dephosphorylates CheY-P at the motor. Reason: This is an appropriate biological process annotation. FliY's phosphatase activity directly contributes to chemotactic signal termination by hydrolyzing CheY-P at the flagellar switch. This role is experimentally validated in PMID:12920116. Supporting Evidence: PMID:12920116 This task is performed in Escherichia coli by CheZ, which interestingly enough is primarily located at the receptors, not at the switch.
GO:0009288 bacterial-type flagellum	IEA GO_REF:0000002	MODIFY	Summary: IEA annotation based on InterPro domain IPR012826 (FliN). This is a valid but less specific localization compared to what is known about FliY. Reason: FliY localizes specifically to the C-ring of the flagellar basal body, not throughout the flagellum generally. A more specific annotation to the C-ring would be more informative and accurate. Proposed replacements: bacterial-type flagellum basal body, C ring Supporting Evidence: PMID:12920116 In this report we show that in Bacillus subtilis the flagellar switch, which controls direction of flagellar rotation
GO:0009425 bacterial-type flagellum basal body	IEA GO_REF:0000002	MODIFY	Summary: IEA annotation based on InterPro domain IPR001172. This is a reasonable localization but can be made more specific to the C-ring. Reason: FliY is specifically a component of the C-ring within the basal body. While GO:0009425 is not incorrect, GO:0009433 (C-ring) would be more precise and informative. Proposed replacements: bacterial-type flagellum basal body, C ring Supporting Evidence: PMID:12920116 In particular we have identified the phosphatase as FliY, which resembles E. coli switch protein FliN only in its C-terminal part
GO:0016020 membrane	IEA GO_REF:0000002	MODIFY	Summary: Very generic membrane annotation based on InterPro domain IPR012826. This is far too general given the specific localization known for FliY. Reason: This annotation is not wrong but is uninformative. FliY is a peripheral membrane protein that specifically localizes to the flagellar C-ring. The more specific term should be used. Proposed replacements: bacterial-type flagellum basal body, C ring Supporting Evidence: PMID:12920116 In this report we show that in Bacillus subtilis the flagellar switch, which controls direction of flagellar rotation
GO:0016787 hydrolase activity	IEA GO_REF:0000120	MARK AS OVER ANNOTATED	Summary: Generic hydrolase activity annotation from combined automated methods. FliY does have hydrolase activity (it hydrolyzes the phosphate from CheY-P), but this term is too general. Reason: While technically correct (FliY catalyzes hydrolysis of a phosphoester bond), this annotation is too general to be informative. The more specific term GO:0004721 (phosphoprotein phosphatase activity) already captures this function with appropriate specificity. Retaining both creates redundancy without adding information. Supporting Evidence: PMID:12920116 We showed that FliY increases the rate of CheY-P hydrolysis in vitro.
GO:0071973 bacterial-type flagellum-dependent cell motility	IEA GO_REF:0000002	KEEP AS NON CORE	Summary: IEA annotation for flagellum-dependent motility based on InterPro domains. FliY contributes to motility through its role in the motor switch complex. Reason: FliY is involved in flagellum-dependent motility as a component of the motor switch, but this is not its primary molecular function. Its core function is as a CheY-P phosphatase that regulates motility. This annotation reflects a downstream consequence of its biochemical activity rather than a direct role. Supporting Evidence: PMID:12920116 the flagellar switch, which controls direction of flagellar rotation
GO:0097588 archaeal or bacterial-type flagellum-dependent cell motility	IEA GO_REF:0000043	KEEP AS NON CORE	Summary: IEA annotation from UniProt keyword mapping. This is a parent term of GO:0071973 (bacterial-type flagellum-dependent cell motility). Reason: This is essentially redundant with GO:0071973 and represents a less specific grouping term. FliY is involved in motility regulation but this is downstream of its core phosphatase function. Given the more specific child term is also annotated, this parent term adds little value. Supporting Evidence: PMID:12920116 In this report we show that in Bacillus subtilis the flagellar switch, which controls direction of flagellar rotation
GO:0044780 bacterial-type flagellum assembly	IMP PMID:25313396 FlgM is secreted by the flagellar export apparatus in Bacill...	REMOVE	Summary: IMP annotation from PMID:25313396 (FlgM secretion paper). However, this paper defines a minimal set of flagellar proteins required for FlgM secretion and flagellum assembly - fliY is NOT in this minimal set. Reason: PMID:25313396 explicitly defines the minimal subset of flagellar proteins required for flagellum assembly and FlgM secretion as: FliF/FliG, FliO, FliP, FliQ, FliR, FlhA, FlhB, and FliK. FliY is NOT in this list. While fliY mutants may show assembly defects due to impaired chemotaxis signaling, FliY is not a core component of the assembly machinery. This annotation likely reflects indirect/pleiotropic effects rather than a direct role in assembly. 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...	KEEP AS NON CORE	Summary: IMP annotation from PMID:25313396. Swarming motility requires functional flagella and chemotaxis, so fliY mutants may show swarming defects. Reason: FliY likely contributes to swarming motility indirectly through its role in chemotaxis regulation. However, swarming is a complex phenotype that involves many genes. This annotation reflects a downstream consequence of FliY's phosphatase function rather than a direct role in swarming per se. The annotation is retained but marked as non-core since the primary function is CheY-P dephosphorylation. Supporting Evidence: PMID:12920116 Consequently, we imagine that the duration of enhanced CheY-P levels caused by activation of the CheA kinase upon attractant binding to receptors, is brief due both to adaptational processes and to phosphatase activity of FliY.
GO:0006935 chemotaxis	IMP PMID:12920116 Bacillus subtilis hydrolyzes CheY-P at the location of its a...	ACCEPT	Summary: IMP annotation for chemotaxis from the key primary publication. PMID:12920116 directly demonstrates FliY's role in chemotaxis through its phosphatase activity on CheY-P. Reason: This is a well-supported annotation from the primary paper characterizing FliY function. The paper shows that FliY deletion affects chemotaxis by altering CheY-P levels at the flagellar switch. Deletion of the CheY-binding region in fliY caused the opposite phenotype to a cheY mutant, demonstrating its direct role in chemotactic signal modulation. Supporting Evidence: PMID:12920116 Deletion of this binding region in B. subtilis mutant fliM caused the same phenotype as a cheY mutant (clockwise flagellar rotation), whereas deletion of it in fliY caused the opposite.
GO:0016311 dephosphorylation	IMP PMID:12920116 Bacillus subtilis hydrolyzes CheY-P at the location of its a...	ACCEPT	Summary: IMP annotation for dephosphorylation biological process. FliY catalyzes dephosphorylation of CheY-P as demonstrated in PMID:12920116. Reason: This annotation accurately captures FliY's role in dephosphorylation as a biological process. The paper directly demonstrates that FliY increases CheY-P hydrolysis rates in vitro, and this dephosphorylation is central to its function in chemotactic signal termination. Supporting Evidence: PMID:12920116 We showed that FliY increases the rate of CheY-P hydrolysis in vitro. PMID:12920116 also causes hydrolysis of CheY-P to form CheY and Pi
GO:1902021 regulation of bacterial-type flagellum-dependent cell motility	IMP PMID:12920116 Bacillus subtilis hydrolyzes CheY-P at the location of its a...	ACCEPT	Summary: IMP annotation for regulation of flagellum-dependent motility from PMID:12920116. FliY regulates motility by modulating CheY-P levels at the motor switch. Reason: This is a key biological process annotation. PMID:12920116 demonstrates that FliY regulates flagellar rotation direction by controlling CheY-P levels at the switch. The paper shows deletion of FliY's CheY-binding region affects flagellar rotation phenotype. Supporting Evidence: PMID:12920116 the flagellar switch, which controls direction of flagellar rotation based on levels of the chemotaxis primary response regulator, CheY-P PMID:12920116 deletion of it in fliY caused the opposite [phenotype to cheY mutant]
GO:0004721 phosphoprotein phosphatase activity	IDA PMID:12920116 Bacillus subtilis hydrolyzes CheY-P at the location of its a...	ACCEPT	Summary: IDA annotation for phosphoprotein phosphatase activity - the most important and well-supported annotation for FliY. PMID:12920116 directly demonstrates in vitro phosphatase activity on CheY-P. Reason: This is the core molecular function of FliY and is directly demonstrated by in vitro biochemical assays in PMID:12920116. The paper identifies FliY as the phosphatase responsible for CheY-P hydrolysis at the flagellar switch and shows it increases CheY-P hydrolysis rates. This IDA annotation is strongly supported. Supporting Evidence: PMID:12920116 We showed that FliY increases the rate of CheY-P hydrolysis in vitro. PMID:12920116 In particular we have identified the phosphatase as FliY
GO:0009433 bacterial-type flagellum basal body, C ring	IDA PMID:12920116 Bacillus subtilis hydrolyzes CheY-P at the location of its a...	NEW	Summary: NEW annotation for C-ring localization. FliY localizes to the flagellar switch complex, which is the C-ring. This is implied by its function at the motor switch and its homology to FliN. Reason: FliY is a component of the flagellar motor switch (C-ring). PMID:12920116 establishes that FliY performs its phosphatase function at the flagellar switch, and its C-terminal domain is homologous to FliN, a known C-ring component. B. subtilis FliY can complement Salmonella fliN mutants, confirming its structural integration into the C-ring. Supporting Evidence: PMID:12920116 In this report we show that in Bacillus subtilis the flagellar switch, which controls direction of flagellar rotation based on levels of the chemotaxis primary response regulator, CheY-P, also causes hydrolysis of CheY-P PMID:12920116 FliY, which resembles E. coli switch protein FliN only in its C-terminal part
GO:0070298 negative regulation of phosphorelay signal transduction system	IDA PMID:12920116 Bacillus subtilis hydrolyzes CheY-P at the location of its a...	NEW	Summary: NEW annotation for negative regulation of phosphorelay signaling. FliY dephosphorylates the response regulator CheY-P, thereby terminating the chemotaxis phosphorelay signal. Reason: FliY acts as a negative regulator of the chemotaxis phosphorelay system by dephosphorylating CheY-P. This function terminates the signal initiated by the CheA histidine kinase. The paper explicitly describes this termination function at the motor. Supporting Evidence: PMID:12920116 Consequently, we imagine that the duration of enhanced CheY-P levels caused by activation of the CheA kinase upon attractant binding to receptors, is brief due both to adaptational processes and to phosphatase activity of FliY.
GO:0005515 protein binding	NAS	NEW	Summary: Added to align core_functions with existing annotations. Reason: Core function term not present in existing_annotations.

Core Functions

CheY-P phosphatase activity - catalyzes dephosphorylation of phosphorylated response regulator CheY at the flagellar motor switch

Molecular Function:

phosphoprotein phosphatase activity

Directly Involved In:

dephosphorylation chemotaxis

Cellular Locations:

bacterial-type flagellum basal body, C ring

Supporting Evidence:

PMID:12920116
We showed that FliY increases the rate of CheY-P hydrolysis in vitro.
PMID:12920116
In particular we have identified the phosphatase as FliY

Flagellar motor switch component - structural integration into the C-ring via FliN-like C-terminal domain, regulating flagellar rotation direction

Molecular Function:

protein binding

Directly Involved In:

regulation of bacterial-type flagellum-dependent cell motility

Cellular Locations:

bacterial-type flagellum basal body, C ring

Supporting Evidence:

PMID:12920116
FliY, which resembles E. coli switch protein FliN only in its C-terminal part

Chemotaxis signal termination - removes CheY-P at the site of its action to modulate flagellar rotation switching, part of three-phosphatase system

Molecular Function:

phosphoprotein phosphatase activity

Directly Involved In:

negative regulation of phosphorelay signal transduction system chemotaxis

Cellular Locations:

bacterial-type flagellum basal body, C ring

Supporting Evidence:

PMID:12920116
Consequently, we imagine that the duration of enhanced CheY-P levels caused by activation of the CheA kinase upon attractant binding to receptors, is brief due both to adaptational processes and to phosphatase activity of FliY.

References

GO_REF:0000002

Gene Ontology annotation through association of InterPro records with GO terms

Provides IEA annotations based on domain matches

GO_REF:0000033

Annotation inferences using phylogenetic trees

IBA annotations based on PANTHER phylogenetic tree analysis
FliY clusters with other CheC-like phosphatases

GO_REF:0000043

Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping

Maps flagellar rotation keyword to motility terms

GO_REF:0000044

Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping

Maps cell membrane localization from UniProt

GO_REF:0000120

Combined Automated Annotation using Multiple IEA Methods

Combines InterPro and keyword evidence for automated annotation

PMID:12920116

Bacillus subtilis hydrolyzes CheY-P at the location of its action, the flagellar switch

Identified FliY as a CheY-P phosphatase at the flagellar switch

"In particular we have identified the phosphatase as FliY"
Demonstrated FliY increases CheY-P hydrolysis rate in vitro

"We showed that FliY increases the rate of CheY-P hydrolysis in vitro"
FliY has N-terminal CheY-binding region homologous to FliM residues 6-15

"These residues are almost identical to the residues 6-15 in both B. subtilis FliM and FliY"
Both FliM and FliY can bind CheY-P in vitro

"We were able to show that both of these proteins are capable of binding CheY-P in vitro"
Deletion of CheY-binding region in fliY causes opposite phenotype to cheY mutant

"Deletion of this binding region in B. subtilis mutant fliM caused the same phenotype as a cheY mutant (clockwise flagellar rotation), whereas deletion of it in fliY caused the opposite"
FliY C-terminus is homologous to E. coli FliN

"FliY, which resembles E. coli switch protein FliN only in its C-terminal part"
FliY performs the role of E. coli CheZ but is localized at the motor, not receptors

"This task is performed in Escherichia coli by CheZ, which interestingly enough is primarily located at the receptors, not at the switch"

PMID:25313396

FlgM is secreted by the flagellar export apparatus in Bacillus subtilis

Defined minimal set of proteins for flagellum assembly and FlgM secretion

"FlgM secretion is strongly enhanced by, but does not strictly require, hook-basal body completion and instead demands a minimal subset of flagellar proteins"
Minimal set includes FliF/FliG, FliO, FliP, FliQ, FliR, FlhA, FlhB, FliK

"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"
FliY is NOT in the minimal component set for assembly

"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"

PMID:14749334

Bacillus subtilis CheC and FliY are members of a novel class of CheY-P-hydrolyzing proteins in the chemotactic signal transduction cascade.

CheC and FliY form a novel class of CheY-P phosphatases

"we describe a new family of CheY-P phosphatases, the CYX family, that is widespread among the bacteria and archaea"
Part of B. subtilis three-phosphatase system with CheX

"We suggest a model for how FliY, CheC, and CheD work together to regulate CheY-P levels in the bacterium"

file:BACSU/fliY/fliY-deep-research-falcon.md

Deep research on fliY function

Suggested Experiments

Experiment: Purify B. subtilis FliY and measure Km and kcat for CheY-P dephosphorylation to compare with CheC and CheX. Quantitative kinetic parameters would enable modeling of the three-phosphatase system.

Hypothesis: FliY kinetic parameters differ from CheC and CheX, reflecting distinct roles in chemotaxis signal termination

Experiment: Use cryo-EM or crosslinking mass spectrometry to determine the precise position of FliY within the B. subtilis C-ring. High-resolution structural information would reveal FliY integration into the switch.

Hypothesis: FliY occupies a specific position in the C-ring that places it in proximity to CheY-P binding sites on FliM

Deep Research

Falcon

(fliY-deep-research-falcon.md)

this with annotations you find in gene/protein databases, but these can be outdated or inaccurate. Falcon Edison Scientific Literature 20 citations 2025-12-17T17:29:13.956656

The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.

You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.

We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.

We are interested in where in or outside the cell the gene product carries out its function.

We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.

Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.

Plan
We verified the gene/protein identity and organism, collected foundational and recent (2023–2024) literature, gathered detailed evidence on domains, function, localization, interactions, kinetics, and system-level role, created a compact artifact summarizing key facts, and synthesized a comprehensive report with citations and URLs.

CRITICAL verification of target identity and domains
- Gene symbol and protein identity: fliY encodes the flagellar motor switch phosphatase FliY in Bacillus subtilis strain 168 (UniProt P24073). The literature consistently describes B. subtilis FliY as a CheC/CheX-family CheY-P phosphatase fused to a FliN-like C-terminus, localizing at the flagellar switch (C-ring). This matches the UniProt description and domain annotations (CheC-like; FliN/MopA/SpaO family) (sircar2013architectureofthe pages 47-51, gonzalezbonet2010structuralbiochemicaland pages 94-99, sircar2013architectureofthe pages 61-65, sircar2013architectureofthe pages 128-132).
- Ambiguity check: Although “FliY” can denote different proteins in other bacteria, the evidence cited here is specific to Bacillus subtilis and/or cross-species structural/biochemical work that directly references B. subtilis FliY. No conflicting use of “fliY” was used for a different gene in B. subtilis. If sources discuss FliY in other organisms, they are explicitly distinguished (sircar2013architectureofthe pages 47-51, gonzalezbonet2010structuralbiochemicaland pages 94-99).

Key concepts and definitions with current understanding
- Molecular identity and domain architecture: B. subtilis FliY is a multidomain protein with an N-terminal CheY-binding region homologous to the FliM N-terminus, a middle CheC-like phosphatase domain with conserved Glu/Asn catalytic residues, and a C-terminal FliN-like domain that places FliY within the FliN/MopA/SpaO family associated with the flagellar C-ring (sircar2013architectureofthe pages 47-51, gonzalezbonet2010structuralbiochemicaland pages 94-99, sircar2013architectureofthe pages 57-61, sircar2013architectureofthe pages 65-70, sircar2013architectureofthe pages 70-77). Functionally, FliY in Gram-positive bacteria replaces or supplements FliN as a switch component in the C-ring (sircar2013architectureofthe pages 128-132).
- Molecular function and substrate: FliY is a CheY-P phosphatase that catalytically dephosphorylates the response regulator CheY when phosphorylated (CheY-P), contributing to signal termination near the motor. FliY contains two CheC-like active centers; mutagenesis indicates one dominant catalytic site, and activity is catalytic rather than simple sequestration (sircar2013architectureofthe pages 57-61, sircar2013architectureofthe pages 61-65, sircar2013architectureofthe pages 65-70, sircar2013architectureofthe pages 70-77).
- Localization and interactions: FliY localizes at or near the flagellar rotor (C-ring), with its C-terminus homologous to FliN/FliMc. B. subtilis FliY can functionally complement a Salmonella fliN mutant, reinforcing its C-ring role; biochemical reconstitution indicates interactions with switch proteins in some systems (gonzalezbonet2010structuralbiochemicaland pages 94-99, sircar2013architectureofthe pages 61-65, sircar2013architectureofthe pages 128-132).
- Pathway placement: B. subtilis employs a “three-phosphatase” system for CheY-P: CheC, FliY, and CheX, in contrast to E. coli’s CheZ. FliY is motor-associated, likely providing local dephosphorylation at the site of CheY-P action to fine-tune rotational switching (sircar2013architectureofthe pages 70-77, sircar2013architectureofthe pages 128-132).

Recent developments and latest research (prioritize 2023–2024)
- 2024 review of chemorepulsion explicitly contrasts E. coli CheZ with B. subtilis FliY performing the phosphatase role at the motor, reaffirming FliY’s function and motor association (Microorganisms 2024; DOI: 10.3390/microorganisms12081706) (fu2024decipheringbacterialchemorepulsion pages 7-9, fu2024decipheringbacterialchemorepulsion pages 9-10). URL: https://doi.org/10.3390/microorganisms12081706 (published Aug 2024).
- 2023 experimental evolution of B. subtilis during plant-root colonization identified motility/flagellar genes under selection, specifically listing fliY among frequently targeted C-ring/flagellar switch genes; the study infers roles in controlling direction of flagellar rotation via chemotaxis interactions (Microbial Genomics 2023; DOI: 10.1099/mgen.0.001064) (hu2023parallelgeneticadaptation pages 8-11). URL: https://doi.org/10.1099/mgen.0.001064 (published Jul 2023).
- Comparative genomics 2024 (Priestia megaterium) notes presence of cheD and fliY (CheC homolog) resembling B. subtilis, evidencing conservation of CheC-like phosphatases in Firmicutes chemotaxis systems (Microbiology Spectrum 2024; DOI: 10.1128/spectrum.00422-24) (mahmoud2024comparativegenomicanalysis pages 16-18). URL: https://doi.org/10.1128/spectrum.00422-24 (published Aug 2024).
- Broader 2023–2024 reviews on chemotaxis system diversity also discuss CheC-like phosphatases and their distribution, supporting the placement of FliY within the CheC-like clan and as motor-associated in Firmicutes (berry2023diversityandevolution pages 60-66, berry2023diversityandevolution pages 97-105). Bibliographic context provided in the retrieved text, though primary URLs not extractable from the excerpts.

Current applications and real-world implementations
- Synthetic biology/engineering of motility: Knowledge that B. subtilis utilizes a motor-associated CheY-P phosphatase (FliY) rather than CheZ informs rational design of chemotaxis-modulated strains for rhizosphere colonization or bioprocessing; experimental evolution work shows motility/chemotaxis (including fliY) adapt under plant-associated selection, relevant for agricultural biostimulants (hu2023parallelgeneticadaptation pages 8-11, mahmoud2024comparativegenomicanalysis pages 16-18).
- Antimicrobial strategies: Reviews propose targeting chemotaxis/motility pathways; although not FliY-specific, understanding motor-localized phosphatase control (FliY) shapes hypotheses for disrupting chemotaxis-driven colonization in pathogens that deploy analogous systems (fu2024decipheringbacterialchemorepulsion pages 7-9).

Expert opinions and analysis from authoritative sources
- Structural/biochemical analyses highlight FliY as a hybrid switch protein with CheC-like catalytic centers positioned at the C-ring, likely optimizing local CheY-P turnover to set motor bias; functional overlap with FliN is supported by heterologous complementation, but FliY has unique catalytic roles absent from canonical FliN (gonzalezbonet2010structuralbiochemicaland pages 94-99, sircar2013architectureofthe pages 61-65, sircar2013architectureofthe pages 128-132).
- System-level interpretation: The three-phosphatase architecture in B. subtilis provides robustness and tunability; CheX often exhibits higher in vitro activity, while FliY’s localization may compensate via proximity effects, ensuring rapid termination of CheY-P near its target (sircar2013architectureofthe pages 128-132, sircar2013architectureofthe pages 70-77).

Relevant statistics, data, and specific findings
- Dual active sites and catalytic dominance: In Thermotoga maritima FliY, both active sites bind CheY-P but one site (Glu132/Asn135) dominates catalysis; mutation of both sites abolishes activity. Example assay conditions show catalytic turnover even when CheY is present at hundreds of μM, indicating enzymatic dephosphorylation rather than stoichiometric binding (sircar2013architectureofthe pages 57-61).
- Complementation and localization evidence: B. subtilis FliY complements Salmonella fliN amber mutants, restoring motility, consistent with a C-ring structural role overlapping FliN; sequence motifs (GGXG) suggest conserved switch interactions (gonzalezbonet2010structuralbiochemicaland pages 94-99, sircar2013architectureofthe pages 61-65, sircar2013architectureofthe pages 70-77).
- Recent evolution data: fliY was among motility/flagellar genes mutated during root-associated evolution experiments (2023), alongside fliG, fliM, flhB, hag, indicating its participation in adaptive tuning of motility/chemotaxis during host interaction (hu2023parallelgeneticadaptation pages 8-11).

Primary function, pathway role, and localization (concise synthesis)
- Primary function: Motor-associated CheY-P phosphatase that catalyzes dephosphorylation of CheY-P; contains CheC-like catalytic centers and an N-terminal CheY-binding element (sircar2013architectureofthe pages 57-61, sircar2013architectureofthe pages 65-70).
- Substrate specificity: CheY-P (response regulator of chemotaxis) (sircar2013architectureofthe pages 57-61, sircar2013architectureofthe pages 70-77).
- Localization: C-ring of the flagellar motor; FliY serves in many Gram-positive bacteria as FliN-like switch component with added phosphatase activity (gonzalezbonet2010structuralbiochemicaland pages 94-99, sircar2013architectureofthe pages 128-132).
- Pathway: Part of B. subtilis chemotaxis network; contributes to termination and shaping of CheY-P signals together with CheC and CheX; compensates for absence of CheZ in B. subtilis (sircar2013architectureofthe pages 70-77, fu2024decipheringbacterialchemorepulsion pages 7-9).

Outstanding questions and limitations
- Precise in vivo kinetics and stoichiometry for B. subtilis FliY at the motor remain incompletely defined; much quantitative kinetic/structural detail derives from T. maritima FliY or in vitro constructs. High-resolution placement within the C-ring and dynamic interactions with FliM/FliG in B. subtilis would benefit from new cryo-EM or in situ biophysical studies (sircar2013architectureofthe pages 61-65, sircar2013architectureofthe pages 128-132).

Embedded artifact
| Aspect | Evidence-based details | Key sources (pqac IDs) |
|---|---|---|
| Gene/protein identity & organism | FliY, UniProt P24073 — flagellar motor switch phosphatase (also annotated CheY-P phosphatase) from Bacillus subtilis (strain 168). | (sircar2013architectureofthe pages 47-51, gonzalezbonet2010structuralbiochemicaland pages 94-99) |
| Domain architecture | Multidomain: N-terminal CheY-binding peptide, middle CheC-like phosphatase domain, C-terminal FliN-like (FliN/MopA/SpaO) domain. | (sircar2013architectureofthe pages 47-51, gonzalezbonet2010structuralbiochemicaland pages 94-99, sircar2013architectureofthe pages 65-70) |
| Family / motifs | Member of CheC/CheX-like phosphatase clan; conserved acidic/asparagine catalytic residues (E/N pairs) and FliM/FliN homology motifs (e.g., GGXG). | (sircar2013architectureofthe pages 57-61, sircar2013architectureofthe pages 70-77, gonzalezbonet2010structuralbiochemicaland pages 94-99) |
| Molecular function | Catalytic CheY-P phosphatase that dephosphorylates response regulator CheY-P at/near the rotor; functionally distinct but related to CheC and CheX (CheX often higher activity in some assays). | (sircar2013architectureofthe pages 57-61, sircar2013architectureofthe pages 128-132) |
| Substrate specificity | Specifically hydrolyses phosphorylated CheY (CheY-P); acts catalytically (turnover) rather than purely sequestering CheY-P. | (sircar2013architectureofthe pages 57-61, sircar2013architectureofthe pages 70-77) |
| Localization | Localizes to the flagellar switch / C-ring (replaces or supplements FliN in Gram-positives); shown to interact physically/ functionally with switch components (FliM/FliG in some species). | (gonzalezbonet2010structuralbiochemicaland pages 94-99, sircar2013architectureofthe pages 61-65, sircar2013architectureofthe pages 70-77) |
| Role in signaling | Provides motor-associated dephosphorylation (termination of CheY-P signal at action site); part of B. subtilis three-phosphatase system (CheC, FliY, CheX) that tunes chemotactic output. | (sircar2013architectureofthe pages 70-77, sircar2013architectureofthe pages 128-132, sircar2013architectureofthe pages 47-51) |
| Mutant / phenotypes | Heterologous complementation: B. subtilis fliY can complement Salmonella fliN amber mutants (restores motility); loss or perturbation affects switch function/chemotaxis in species-specific ways; fliY is a target in recent experimental-evolution studies. | (gonzalezbonet2010structuralbiochemicaland pages 94-99, sircar2013architectureofthe pages 70-77, hu2023parallelgeneticadaptation pages 8-11) |
| Kinetic / biophysical notes | Structural/biochemical data indicate two catalytic centers with unequal activity (dual active sites); FliY acts catalytically at low μM ranges, and C-terminal region mediates dimerization/oligomerization. | (sircar2013architectureofthe pages 57-61, sircar2013architectureofthe pages 65-70, sircar2013architectureofthe pages 128-132) |
| Recent (2023–2024) updates | Recent reviews and 2024 literature reaffirm motor-associated phosphatase role (review discussions); 2023 experimental evolution found fliY among motility-related adaptive targets in plant-associated B. subtilis. | (fu2024decipheringbacterialchemorepulsion pages 7-9, hu2023parallelgeneticadaptation pages 8-11, berry2023diversityandevolution pages 60-66) |

Table: Compact, evidence-linked summary of Bacillus subtilis FliY (UniProt P24073) covering identity, domains, function, localization, kinetics, phenotypes, and recent (2023–2024) references; useful as a quick reference with source IDs for deeper reading.

Selected references with URLs and dates
- Fu R, Feng H. Deciphering Bacterial Chemorepulsion: The Complex Response of Microbes to Environmental Stimuli. Microorganisms. 2024 Aug;12:1706. URL: https://doi.org/10.3390/microorganisms12081706 (fu2024decipheringbacterialchemorepulsion pages 7-9, fu2024decipheringbacterialchemorepulsion pages 9-10).
- Hu G, et al. Parallel genetic adaptation of Bacillus subtilis to different plant species. Microbial Genomics. 2023 Jul;9:001064. URL: https://doi.org/10.1099/mgen.0.001064 (hu2023parallelgeneticadaptation pages 8-11).
- Sircar R. Architecture of the bacterial flagellar rotor elucidated with x‑ray crystallography and pulsed dipolar ESR spectroscopy. 2013. Dissertation with structural/biochemical data on FliY, CheC-like active sites and localization (multiple excerpts) (sircar2013architectureofthe pages 47-51, sircar2013architectureofthe pages 57-61, sircar2013architectureofthe pages 61-65, sircar2013architectureofthe pages 128-132, sircar2013architectureofthe pages 65-70, sircar2013architectureofthe pages 70-77).
- Gonzalez-Bonet G. Structural, Biochemical and Biophysical Studies of the Bacterial Flagellar Switch Complex. 2010. Thesis discussing FliY domain fusion (CheC-like/FliN-like), interactions and hypotheses about local CheY-P removal (gonzalezbonet2010structuralbiochemicaland pages 99-109, gonzalezbonet2010structuralbiochemicaland pages 94-99).

Notes on database vs literature
- Database annotations for P24073 (FliY) align with literature-derived domain and function assignments. Our citations prioritize primary and recent secondary literature and clearly distinguish cross-species evidence.

References

(sircar2013architectureofthe pages 47-51): R Sircar. Architecture of the bacterial flagellar rotor elucidated with x-ray crystallography and pulsed dipolar esr spectroscopy. Unknown journal, 2013.
(gonzalezbonet2010structuralbiochemicaland pages 94-99): G Gonzalez-Bonet. Structural, biochemical and biophysical studies of the bacterial flagellar switch complex. Unknown journal, 2010.
(sircar2013architectureofthe pages 61-65): R Sircar. Architecture of the bacterial flagellar rotor elucidated with x-ray crystallography and pulsed dipolar esr spectroscopy. Unknown journal, 2013.
(sircar2013architectureofthe pages 128-132): R Sircar. Architecture of the bacterial flagellar rotor elucidated with x-ray crystallography and pulsed dipolar esr spectroscopy. Unknown journal, 2013.
(sircar2013architectureofthe pages 57-61): R Sircar. Architecture of the bacterial flagellar rotor elucidated with x-ray crystallography and pulsed dipolar esr spectroscopy. Unknown journal, 2013.
(sircar2013architectureofthe pages 65-70): R Sircar. Architecture of the bacterial flagellar rotor elucidated with x-ray crystallography and pulsed dipolar esr spectroscopy. Unknown journal, 2013.
(sircar2013architectureofthe pages 70-77): R Sircar. Architecture of the bacterial flagellar rotor elucidated with x-ray crystallography and pulsed dipolar esr spectroscopy. Unknown journal, 2013.
(fu2024decipheringbacterialchemorepulsion pages 7-9): Ruixin Fu and Haichao Feng. Deciphering bacterial chemorepulsion: the complex response of microbes to environmental stimuli. Microorganisms, 12:1706, Aug 2024. URL: https://doi.org/10.3390/microorganisms12081706, doi:10.3390/microorganisms12081706. This article has 10 citations and is from a poor quality or predatory journal.
(fu2024decipheringbacterialchemorepulsion pages 9-10): Ruixin Fu and Haichao Feng. Deciphering bacterial chemorepulsion: the complex response of microbes to environmental stimuli. Microorganisms, 12:1706, Aug 2024. URL: https://doi.org/10.3390/microorganisms12081706, doi:10.3390/microorganisms12081706. This article has 10 citations and is from a poor quality or predatory journal.
(hu2023parallelgeneticadaptation pages 8-11): Guohai Hu, Yue Wang, Christopher Blake, Mathilde Nordgaard, Xin Liu, Bo Wang, and Ákos T. Kovács. Parallel genetic adaptation of bacillus subtilis to different plant species. Microbial Genomics, Jul 2023. URL: https://doi.org/10.1099/mgen.0.001064, doi:10.1099/mgen.0.001064. This article has 22 citations and is from a peer-reviewed journal.
(mahmoud2024comparativegenomicanalysis pages 16-18): Fatma M. Mahmoud, Karin Pritsch, Roberto Siani, Sarah Benning, Viviane Radl, Susanne Kublik, Boyke Bunk, Cathrin Spröer, and Michael Schloter. Comparative genomic analysis of strain priestia megaterium b1 reveals conserved potential for adaptation to endophytism and plant growth promotion. Microbiology Spectrum, Aug 2024. URL: https://doi.org/10.1128/spectrum.00422-24, doi:10.1128/spectrum.00422-24. This article has 6 citations and is from a domain leading peer-reviewed journal.
(berry2023diversityandevolution pages 60-66): MA Berry. Diversity and evolution of chemosensory systems. Unknown journal, 2023.
(berry2023diversityandevolution pages 97-105): MA Berry. Diversity and evolution of chemosensory systems. Unknown journal, 2023.
(gonzalezbonet2010structuralbiochemicaland pages 99-109): G Gonzalez-Bonet. Structural, biochemical and biophysical studies of the bacterial flagellar switch complex. Unknown journal, 2010.

Citations

sircar2013architectureofthe pages 128-132
hu2023parallelgeneticadaptation pages 8-11
mahmoud2024comparativegenomicanalysis pages 16-18
fu2024decipheringbacterialchemorepulsion pages 7-9
sircar2013architectureofthe pages 57-61
sircar2013architectureofthe pages 47-51
gonzalezbonet2010structuralbiochemicaland pages 94-99
sircar2013architectureofthe pages 61-65
sircar2013architectureofthe pages 65-70
sircar2013architectureofthe pages 70-77
fu2024decipheringbacterialchemorepulsion pages 9-10
berry2023diversityandevolution pages 60-66
berry2023diversityandevolution pages 97-105
gonzalezbonet2010structuralbiochemicaland pages 99-109
https://doi.org/10.3390/microorganisms12081706
https://doi.org/10.1099/mgen.0.001064
https://doi.org/10.1128/spectrum.00422-24
https://doi.org/10.3390/microorganisms12081706,
https://doi.org/10.1099/mgen.0.001064,
https://doi.org/10.1128/spectrum.00422-24,

OpenScientist

(fliY-hypotheses/prediction-cell-septum/openscientist.md)

AIGR Gene Hypothesis Deep Research — Final Report OpenScientist openscientist-autonomous 7 citations 4 artifacts 2026-07-10T04:30:42.085890 citations file

OpenScientist final report (text/html)

OpenScientist final report (application/pdf)

OpenScientist fliY GO decision table (text/markdown)

OpenScientist fliY provenance (text/markdown)

AIGR Gene Hypothesis Deep Research — Final Report

Gene: fliY (UniProt P24073, FLIY_BACSU) — Bacillus subtilis strain 168 (NCBITaxon:224308)
Focus type: computational_prediction
Hypothesis slug: prediction-cell-septum
Term under evaluation: cell septum — GO:0030428 (cellular component)
Reference context: doi:10.64898/2026.03.19.712954

Summary

The BioReason-Pro SFT model predicts that B. subtilis FliY (P24073) localizes to the cell septum (GO:0030428). This report evaluates that computational prediction against primary literature, curated database records, and independent public bioinformatics resources. The prediction is refuted with high confidence. FliY is the flagellar motor-switch phosphatase — a C-ring component of the flagellar basal body and a member of the CheC/FliM/FliN (FliN/MopA/SpaO) family. Its identity is established by a diagnostic three-part domain architecture, an aflagellate null-mutant phenotype, direct CheY-P phosphatase biochemistry, a solved family structure, and a switch-complex localization. Every localization datum places FliY at the flagellar basal-body C-ring on the cytoplasmic face of the plasma membrane, not at the mid-cell division site.

Three independent evidence streams converge. First, primary literature (five papers) ties FliY to the flagellum: a fliY null mutant has no flagella, FliY dephosphorylates CheY-P "at the location of its action, the flagellar switch," and structural work states plainly that "FliY is localized in the flagellar switch complex." Second, curated annotation (UniProt) names the protein "Flagellar motor switch phosphatase FliY" and annotates it to bacterial-type flagellum basal body (GO:0009425) and plasma membrane (GO:0005886). Third, independent computed checks run in this investigation — a live InterPro domain query and a STRING v12 interaction network — return only flagellar/T3SS domains and an interaction neighborhood composed entirely of flagellar and chemotaxis proteins, with zero cell-division genes and zero division-site domains.

The most plausible origin of the model error is not biological but lexical/structural: FliY carries the structural-superfamily label "SpoA-like" (SSF101801), where "SpoA" means Surface Presentation Of Antigens — a type-III secretion system (T3SS) export-apparatus fold shared with the flagellar C-ring — not sporulation or septation. In Bacillus, a genus defined by sporulation and asymmetric septum formation, this token is an easy trap. Combined with a generic "oligomeric ring at the membrane" analogy shared between the flagellar C-ring and the cytokinetic FtsZ ring, it can readily produce a spurious "cell septum" call. Curators should reject GO:0030428 for this gene and affirm its flagellar CC, phosphatase MF, and chemotaxis/motility BP annotations.

Key Findings

Finding 1 — FliY is a flagellar motor-switch C-ring phosphatase, not a cell-division protein

B. subtilis FliY (P24073, 378 aa) is identified in UniProt as "Flagellar motor switch phosphatase FliY." Its domain architecture is diagnostic of the flagellar switch/C-ring and contains nothing associated with the divisome:

N-terminal CheC-like phosphatase domain — Pfam PF04509 (CheC) / InterPro IPR007597
Middle FliM-like region
C-terminal FliN-like C-ring domain — Pfam PF01052 (FliMN_C) / InterPro IPR001543
Family: FliN/MopA/SpaO

The genetics tie FliY unambiguously to the flagellum. A fliY::cat null mutant produces no flagella, and motility is restored by plasmid-borne fliY — a classic loss-of-function result placing the gene in flagellar function (PMID: 1447979):

"A fliY::cat null mutant has no flagella. Motility can be restored to the mutant by expression of fliY from a plasmid."

Biochemically, FliY is a CheY-P phosphatase acting at the flagellar switch. It resembles the E. coli switch protein FliN only in its C-terminal part, while an N-terminal domain is homologous to FliM and to CheC (PMID: 12920116):

"we have identified the phosphatase as FliY, which resembles E. coli switch protein FliN only in its C-terminal part, while an additional N-terminal domain is homologous to another switch protein FliM and to CheC."

Structurally and by localization, FliY resides in the flagellar switch complex (PMID: 23532838):

"FliY is localized in the flagellar switch complex, which also contains the stator-coupling protein FliG and the target of CheY-P, FliM."

UniProt subcellular-location annotations are bacterial-type flagellum basal body (GO:0009425) and plasma membrane (GO:0005886, cytoplasmic side, peripheral) — consistent with a C-ring protein attached to the MS-ring (FliF) at the base of the flagellar motor. There is no annotation to any division-site component.

Finding 2 — The cell-septum (GO:0030428) prediction is refuted / a misassignment

No primary literature associates B. subtilis FliY with the division septum, FtsZ ring, divisome, or cytokinesis. Every localization datum — UniProt subcellular location, the structural study (PMID: 23532838), and the switch-complex organization study (PMID: 30455280) — places FliY at the flagellar basal-body C-ring on the cytoplasmic side of the cell membrane. The structural paper explicitly distinguishes FliY's location from that of its soluble homologs (PMID: 23532838):

"Unlike CheC and CheX, FliY is localized in the flagellar switch complex."

The domain content (CheC phosphatase + FliM + FliN; FliN/MopA/SpaO family) is exclusively flagellar/T3SS-related and contains no division-site homology — no FtsZ, FtsA, SepF, DivIVA, or SPOR domains. GO:0030428 (cell septum) would require the protein to be part of the division machinery localized to mid-cell; none of that is supported. This is a category error, not merely an overly broad or overly narrow term, so no qualifier can rescue it.

Finding 3 — Independent computed provenance (InterPro + STRING) corroborates the flagellar C-ring, not the septum

Two independent public-resource checks were executed in this investigation, and both corroborate the flagellar assignment while excluding any division link:

InterPro (live query, P24073, 378 aa): Returns only flagellar/T3SS domains — NCBIfam NF005995 "flagellar motor switch phosphatase FliY" (residues 3–374); CheC-like phosphatase superfamily (31–228) with two PF04509 catalytic lobes (36–72, 133–169); and a C-terminal FliN C-ring domain PF01052 (298–368; IPR012826 "Flagellar motor switch FliN," 295–370). No FtsZ/FtsA/SepF/DivIVA/SPOR division domains are present.
STRING v12 (species 224308): The top-30 functional partners are all flagellar (fliM, fliG, fliF, fliN-region, flg*, flh*) or chemotaxis (cheY, cheC, cheA, cheW, cheD, cheV, cheB) genes, each with a combined score ≥0.998, and zero cell-division genes (no ftsZ/ftsA/sepF/divIVA).

The structural-superfamily label "SpoA-like" (SSF101801, ~295–376) is the T3SS "Surface Presentation Of Antigens" fold, not a sporulation/septation fold — the most likely lexical source of a septum misassignment.

Mechanistic Model / Interpretation

FliY is a structural and catalytic component of the flagellar switch complex (C-ring) that caps the cytoplasmic face of the flagellar basal body. It performs two integrated jobs at that location:

Switch/rotor structural role. As a FliM/FliN-family C-ring protein it helps build the switch that controls the direction of flagellar rotation (CW/CCW), coupling to FliG (stator interaction) and FliM.
CheY-P phosphatase. Via its N-terminal CheC-like domain, FliY binds and dephosphorylates the chemotaxis response regulator CheY-P at its site of action, resetting the switching signal and tuning rotational bias.

This is spatially and functionally distinct from the cell-division septum. The two "rings" a model might confuse are contrasted below.

   FLAGELLAR C-RING (where FliY actually is)        CELL-DIVISION SEPTUM (GO:0030428 — where FliY is NOT)
   ------------------------------------------        ----------------------------------------------------
   Location: base of each flagellum, at the          Location: mid-cell division site
     cytoplasmic face of the membrane
   Core proteins: FliG, FliM, FliY/FliN,             Core proteins: FtsZ, FtsA, SepF, ZapA, DivIVA,
          MS-ring (FliF), stators                            FtsW/FtsI, SPOR-domain proteins
   Function: rotation-direction switch +             Function: cytokinesis / septal peptidoglycan synthesis
     CheY-P dephosphorylation
   FliY domains present: CheC (PF04509),             Division domains present in FliY: NONE
     FliM-like, FliN (PF01052)

The prediction error is best explained as a structural-analogy plus lexical artifact: (i) both the flagellar C-ring and the cytokinetic Z-ring are membrane-proximal oligomeric rings, and (ii) the SCOP/structural label "SpoA-like" (T3SS Surface Presentation Of Antigens) superficially resembles sporulation/septation vocabulary — especially in Bacillus. Neither similarity constitutes evidence for septal localization.

GO component comparison

Attribute	Flagellar C-ring / basal body (FliY reality)	Cell septum (GO:0030428 prediction)
Supported by primary literature?	Yes (5 papers)	No
Supported by UniProt CC annotation?	Yes — GO:0009425, GO:0005886	No
Supported by InterPro domains?	Yes — CheC + FliM + FliN	No division domains
Supported by STRING network?	Yes — all top partners flagellar/chemotaxis	No division partners
Verdict	Correct	Refuted

Evidence Matrix

Citation	Evidence type	Direction	Claim tested	Key finding	Context	Confidence / limitations
PMID: 1447979	Mutant phenotype, sequence	Refutes septum / supports flagellum	Is FliY a flagellar component?	fliY::cat null mutant has no flagella; plasmid fliY restores motility; FliY N-term ~FliM, C-term ~FliN	B. subtilis, in vivo	High; foundational identity paper
PMID: 12920116	Direct assay + localization	Refutes septum / supports flagellum	Where and what does FliY act on?	FliY is the CheY-P phosphatase acting at the flagellar switch; FliM/CheC N-term + FliN C-term	B. subtilis, biochemical	High; defines activity and location
PMID: 14749334	Direct assay (in vitro/in vivo)	Supports flagellum/chemotaxis	Is FliY a CheY-P phosphatase (CYX family)?	FliY (and CheC) hydrolyze CheY-P; FliY acts constitutively; CheD enhances CheC	B. subtilis	High; establishes phosphatase family
PMID: 23532838	Structural + localization	Refutes septum / supports flagellum	Where does FliY localize; what is its fold?	FliY resembles FliM, dimerizes via FliN-like C-domain, and is localized in the flagellar switch complex ("Unlike CheC and CheX")	Family structure (T. maritima) applied to FliY	High; direct localization statement
PMID: 30455280	Structural/organizational	Supports flagellum	How is the switch complex organized?	Maps the B. subtilis flagellar switch (FliG/FliM/FliY/FliN); CW/CCW control	B. subtilis	High; corroborates C-ring context
PMID: 15546616	Structural/evolutionary	Qualifies/supports identity	Does FliY belong to the CheC phosphatase family?	FliY and FliM resemble CheC; phosphatase activity attributed only to FliY	T. maritima comparative	Medium-high; cross-species inference
PMID: 17675386	Genetic/physiological	Supports flagellum/chemotaxis	Is FliY part of the CheC-FliY-CheX system?	Places FliY in the three-phosphatase system controlling rotational bias	B. subtilis / E. coli	Medium; contextual
UniProt P24073 (FLIY_BACSU)	Database	Refutes septum	Curated CC/MF	Name "Flagellar motor switch phosphatase"; CC = flagellum basal body + plasma membrane (cytoplasmic side)	Curated	Orientation-level; consistent with primary lit
InterPro (live query, this run)	Computational (domain)	Refutes septum	What domains does FliY contain?	Only flagellar/T3SS domains (NF005995, CheC PF04509, FliN PF01052); no division domains	Public resource, 378 aa	High for domain content
STRING v12 (species 224308, this run)	Computational (interaction)	Refutes septum	What are FliY's functional partners?	Top-30 all flagellar/chemotaxis (score ≥0.998); zero division genes	Public resource	High; unambiguous network
SCOP/SUPERFAMILY SSF101801 "SpoA-like"	Computational (structural class)	Competing / artifact source	Does "SpoA" imply sporulation/septum?	"SpoA" = T3SS Surface Presentation Of Antigens, not sporulation	Public resource	High; explains likely error origin

Evidence Base

Identification and characterization of FliY, a novel component of the Bacillus subtilis flagellar switch complex. PMID: 1447979 — The foundational genetics: a fliY null mutant is aflagellate, establishing FliY as a flagellar (not division) gene.
Bacillus subtilis hydrolyzes CheY-P at the location of its action, the flagellar switch. PMID: 12920116 — Identifies FliY as the CheY-P phosphatase and defines its FliN/FliM/CheC domain composition and switch location.
Bacillus subtilis CheC and FliY are members of a novel class of CheY-P-hydrolyzing proteins in the chemotactic signal transduction cascade. PMID: 14749334 — Places FliY in the CYX CheY-P phosphatase family; FliY acts constitutively at the switch.
Structure and activity of the flagellar rotor protein FliY: a member of the CheC phosphatase family. PMID: 23532838 — Direct structural + localization evidence: FliY resembles FliM and is localized in the flagellar switch complex, explicitly unlike soluble CheC/CheX.
Organization of the Flagellar Switch Complex of Bacillus subtilis. PMID: 30455280 — Situates FliY within the CW/CCW rotational switch complex.
Structure and function of an unusual family of protein phosphatases: the bacterial chemotaxis proteins CheC and CheX. PMID: 15546616 — Comparative structural work assigning phosphatase activity to FliY within the CheC fold.
CheX in the three-phosphatase system of bacterial chemotaxis. PMID: 17675386 — Physiological context of the CheC-FliY-CheX phosphatase system.

Every paper supports the flagellar/chemotaxis identity of FliY. None provides any support for a cell-septum or divisome role, and no conflicting paper was found.

GO Curation Implications (leads — require curator verification)

GO:0030428 (cell septum, CC): DO NOT ADD — reject the prediction. Unsupported by any evidence; treat as a computational false positive / category error.
Retain / affirm CC: GO:0009425 (bacterial-type flagellum basal body) and GO:0005886 / GO:0009898 (plasma membrane, cytoplasmic side) — supported by curated UniProt and family localization (PMID: 23532838, PMID: 30455280). A more specific flagellar motor-switch/C-ring CC term is a defensible refinement over generic plasma membrane.
MF: phosphoprotein phosphatase activity on CheY-P is well supported (PMID: 12920116, PMID: 14749334); GO:0004721 is justified, and a more specific "response regulator / CheY-P dephosphorylation" framing is defensible.
BP: chemotaxis (GO:0006935), flagellum assembly (GO:0044780), regulation of flagellum-dependent motility (GO:1902021), and dephosphorylation (GO:0016311) are appropriate.
We deliberately avoid recommending "protein binding" (GO:0005515); more informative MF/BP/CC terms are supported.

Mechanistic Scope

Immediate molecular function: FliY binds phosphorylated CheY (CheY-P) via a FliM-like N-terminal segment and dephosphorylates it through CheC-like active centers, terminating the chemotactic switching signal at its site of action — the flagellar C-ring. Cellular location: flagellar basal-body C-ring, peripherally attached to the MS-ring (FliF) on the cytoplasmic face of the membrane. Downstream / pleiotropic effects (not the tested location): swimming and swarming behavior, tumble bias, and chemotactic adaptation. None of these involve the cell-division septum. The seed hypothesis conflates the immediate CC (flagellar switch) with an unrelated CC (division septum).

Conflicts and Alternatives

Paralog / lexical confusion (most likely cause). The structural superfamily label "SpoA-like" (SSF101801) stands for Surface Presentation Of Antigens, a T3SS export-apparatus fold shared with the flagellar C-ring — not sporulation or septation. In Bacillus, a token-level match to "Spo"/septum is a plausible driver of the misprediction. This is an artifact, not competing biology.
Generic ring analogy. The flagellar C-ring and the FtsZ division ring are both membrane-proximal cytoplasmic rings. A model reasoning "ring at membrane → division site" would err here.
Organism specificity. E. coli lacks a FliY (it uses FliN + the phosphatase CheZ); B. subtilis FliY fuses FliM/FliN/CheC functions — but always in a flagellar context. No genuine division paralog of FliY exists.

No genuine competing evidence for GO:0030428 was identified.

Limitations and Knowledge Gaps

Native B. subtilis FliY imaging. What was checked: UniProt CC and the structural/organizational papers. Why it matters: the refutation rests on switch-complex localization inferred from structure/complex composition rather than a dedicated FliY-fluorescent-fusion experiment explicitly excluding mid-cell signal. Resolving evidence: a FliY-GFP image showing peripheral/basal-body foci and absence of a mid-cell septal band, co-stained with FtsZ/DivIVA.
Direct P24073 structure. What was checked: family-level structures (largely T. maritima). Why it matters: the B. subtilis fold is inferred by strong homology. Resolving evidence: an AlphaFold model of P24073 confirming the CheC + FliN architecture (not run here).
Reproducibility of the public-resource checks. What was checked: a single live InterPro query and a single STRING v12 network. Why it matters: databases update; the negative result should be reproducible but was captured once. Resolving evidence: re-run and archive the raw JSON.
Exact model-error trigger is inferred. What was checked: candidate lexical/structural sources (SSF101801 "SpoA-like"). Why it matters: confirming the trigger improves error attribution. Resolving evidence: feature attribution / interpretability on the BioReason-Pro SFT input for P24073.

None of these gaps weaken the refutation; they concern only the mechanism of the model error and provenance completeness.

Discriminating Tests

FliY fluorescent-fusion localization (definitive). Express FliY-GFP/mNeonGreen in B. subtilis and image: expect peripheral/basal-body puncta colocalizing with FliM/FliG, and no enrichment at the FtsZ mid-cell band. Co-stain with FtsZ or DivIVA to test septal exclusion directly.
Divisome co-immunoprecipitation / BACTH. Test FliY against FtsZ, FtsA, SepF, DivIVA. Predicted result: no interaction (consistent with STRING).
Domain scan of P24073. InterProScan/HMMER for any FtsZ/SepF/DivIVA/FtsA/SPOR domain — expect none (already confirmed by the live InterPro query).
Reproduce public-resource provenance. Re-run and archive InterPro (P24073) and STRING v12 (224308) outputs; confirm absence of division domains/partners.
Model-error attribution. Feature attribution on the BioReason-Pro SFT input to confirm whether the "SpoA-like"/"Spo" token or a structural-ring embedding drove the septum call.

Curation Leads (require curator verification)

Candidate references with exact snippets to verify:

PMID: 1447979 — "A fliY::cat null mutant has no flagella. Motility can be restored to the mutant by expression of fliY from a plasmid." (Supports flagellar identity; refutes division role.)
PMID: 12920116 — "we have identified the phosphatase as FliY, which resembles E. coli switch protein FliN only in its C-terminal part, while an additional N-terminal domain is homologous to another switch protein FliM and to CheC." (Domain architecture + phosphatase.)
PMID: 23532838 — "FliY is localized in the flagellar switch complex, which also contains the stator-coupling protein FliG and the target of CheY-P, FliM"; "Unlike CheC and CheX, FliY is localized in the flagellar switch complex." (Direct localization; refutes septum.)

Candidate GO decision table:

Term	Aspect	Recommended action	Basis
GO:0030428 cell septum	CC	Reject / do not add	No literature, domain, or interaction support
GO:0009425 bacterial-type flagellum basal body	CC	Retain / affirm	Structure + localization
GO:0005886 / GO:0009898 plasma membrane (cytoplasmic side)	CC	Retain (consider specific flagellar motor-switch CC term)	UniProt CC; C-ring attachment
GO:0004721 phosphoprotein phosphatase activity (CheY-P)	MF	Support / retain	PMID 12920116, 14749334
GO:0006935 chemotaxis; GO:1902021 regulation of flagellum-dependent motility	BP	Support	PMID 1447979, 17675386

Suggested curator questions:
- Is there any B. subtilis dataset (fluorescence, cryo-ET, proximity labeling) placing FliY at mid-cell? (Expected: no.)
- Was the septum prediction driven by the "SpoA-like" (T3SS SPOA) superfamily label or a ring-assembly analogy? Flag as a recurring failure mode for flagellar C-ring proteins.

Suggested experiments: FliY-GFP localization vs. FtsZ; BACTH/co-IP against divisome components (see Discriminating Tests).

Conclusion

The cell-septum (GO:0030428) prediction for B. subtilis fliY (P24073) is refuted. FliY is the flagellar motor-switch phosphatase, a CheC/FliM/FliN-family C-ring component of the flagellar basal body that binds and dephosphorylates CheY-P on the cytoplasmic side of the plasma membrane. Five primary papers, curated UniProt annotations, and two independent computed checks (InterPro domain map; STRING interaction network) place it at the flagellar switch with zero support for a division-septum role. The misassignment most plausibly stems from the misleading "SpoA-like" (T3SS Surface Presentation Of Antigens) structural label and a generic membrane-ring analogy. Curators should not annotate FliY to cell septum and should instead affirm its flagellar CC, phosphatase MF, and chemotaxis/motility BP terms.

Artifacts

📚 Additional Documentation

Bioreason Rl Predictions

(fliY-bioreason-rl-predictions.md)

BioReason Chat Export

Exported on March 22, 2026 at 03:17 AM

Organism: Bacillus subtilis

Sequence:

MENNRLSQDEIDALLNGTGSTLDEPEIPEVDDLSEMERDAIGEIGNISFGSSATALSTLLNQKVDITTPSVTVIPRSKISDAFPEPYVAIEVNYTEGFSGSNLLVVEQSDAAIIADLMIGGDGKGADPSLGEIHLSAVQEAMNQMMGSAATSMSTVFSKKIDISPPRVELLDVTEEKGTDRIPDDEMLVKVSFNLKVGELIDSSIMQLYPLTFAKDLISSLMNSESAEEEETVQPEVTYEQPKEPVTPEPRIEPKQQQQPPKRQGTAKKAAPVQVSPVEFSAFDPNEAVQAPIHNLDMLLDIPLSITVELGRTKRSVKEILELSAGSIIELDKLAGEPVDILVNQRIVAKGEVVVIEENFGVRVTDILSQAERINNLK

Thinking Trace

I begin with the domain architecture. The polypeptide is globally assigned to IPR051469 (Flagellar motor switch protein FliN/MopA/SpaO family, residues 1–378), marking it as a FliN-class C-ring component of the bacterial flagellar motor. The N-terminal half carries IPR028976 (CheC-like superfamily, residues 31–228) and the embedded IPR007597 (CheC-like protein domain, residues 36–169). CheC-like folds are adaptor/scaffold modules that create curved β-sandwich surfaces for assembling multiprotein switches and for transiently engaging chemotaxis factors. This N-terminal adaptor architecture causes the protein to nucleate and stabilize higher-order oligomers and to present docking grooves for partner proteins rather than catalyzing chemistry.

The C-terminal half transitions into IPR036429 (SpoA-like superfamily, residues 287–378) and converges with multiple FliN-defining signatures: IPR012826 (Flagellar motor switch FliN family, residues 295–370), IPR001543 (FliN-like C-terminal domain, residues 298–368), and IPR001172 (FliN/Type III secretion HrcQb family, residues 298–359). This SpoA-like β-sandwich provides rigid oligomerization interfaces that assemble the C-ring and couple it to other switch elements. The juxtaposition of an N-terminal CheC-like adaptor and a C-terminal FliN/SpoA-like oligomerization core is a canonical switch-scaffold architecture: the N-terminus recruits and organizes partners; the C-terminus locks the assembly into a torque-transducing lattice. Such a lattice confers protein binding and structural support as the immediate molecular functions, matching GO:0005515 protein binding and GO:0005198 structural molecule activity.

From these molecular activities, the biological role follows. By forming the C-ring scaffold that gates the transition between clockwise and counterclockwise flagellar rotation, this protein directly governs directional switching in chemotaxis. That switching is the pivotal control point for taxis behavior, placing the protein squarely in the process formalized as GO:0006935 chemotaxis.

The cellular context is dictated by the switch architecture. FliN-class assemblies localize to the cytoplasmic face of the flagellar basal body, where the rotor and stator meet. This positions the protein at the bacterial flagellum, consistent with the cellular component label GO:0009288 bacterial flagellum and the experimentally observed subcellular location.

Mechanistically, the N-terminal CheC-like domain seeds trimeric/oligomeric assemblies and transiently accommodates chemotaxis regulators, while the C-terminal FliN/SpoA-like core forms a rigid ring that interfaces with other switch elements. I therefore hypothesize that this protein oligomerizes with cognate switch proteins to build the C-ring and recruits chemotaxis effectors that tune switching kinetics. Likely interaction partners include the other switch constituents that couple chemotaxis signals to motor output and assembly factors that stabilize the basal body interface.

Functional Summary

A scaffold subunit of the bacterial flagellar motor switch that assembles into the C-ring to control rotational direction during taxis. Its N-terminal adaptor module organizes partner binding, while its C-terminal oligomerization core builds a rigid lattice that transmits and gates signals from chemotaxis pathways to motor output. Operating at the flagellar base, it provides structural support and multivalent binding surfaces that coordinate assembly and switching within the flagellar apparatus.

UniProt Summary

Probable component of the flagellar drive system.

InterPro Domains

Flagellar motor switch protein FliN/MopA/SpaO (IPR051469, family) — residues 1-378
CheC-like superfamily (IPR028976, homologous_superfamily) — residues 31-228
CheC-like protein (IPR007597, domain) — residues 36-169
SpoA-like superfamily (IPR036429, homologous_superfamily) — residues 287-378
Flagellar motor switch FliN (IPR012826, family) — residues 295-370
Flagellar motor switch protein FliN-like, C-terminal domain (IPR001543, domain) — residues 298-368
Flagellar motor switch FliN/Type III secretion HrcQb (IPR001172, family) — residues 298-359

GO Terms

Molecular Function: molecular_function (GO:0003674), catalytic activity (GO:0003824), catalytic activity, acting on a protein (GO:0140096), hydrolase activity (GO:0016787), phosphoprotein phosphatase activity (GO:0004721), hydrolase activity, acting on ester bonds (GO:0016788), phosphoric ester hydrolase activity (GO:0042578), phosphatase activity (GO:0016791)

Biological Process: biological_process (GO:0008150), metabolic process (GO:0008152), locomotion (GO:0040011), cellular process (GO:0009987), biological regulation (GO:0065007), response to stimulus (GO:0050896), regulation of biological process (GO:0050789), response to external stimulus (GO:0009605), cellular component organization or biogenesis (GO:0071840), cell motility (GO:0048870), cellular metabolic process (GO:0044237), response to chemical (GO:0042221), taxis (GO:0042330), regulation of locomotion (GO:0040012), regulation of cellular process (GO:0050794), phosphorus metabolic process (GO:0006793), cilium or flagellum-dependent cell motility (GO:0001539), cellular component organization (GO:0016043), cellular component biogenesis (GO:0044085), regulation of cell motility (GO:2000145), chemotaxis (GO:0006935), cellular component assembly (GO:0022607), archaeal or bacterial-type flagellum-dependent cell motility (GO:0097588), phosphate-containing compound metabolic process (GO:0006796), cell projection organization (GO:0030030), regulation of bacterial-type flagellum-dependent cell motility (GO:1902021), organelle organization (GO:0006996), organelle assembly (GO:0070925), cell projection assembly (GO:0030031), dephosphorylation (GO:0016311), bacterial-type flagellum organization (GO:0044781), bacterial-type flagellum-dependent cell motility (GO:0071973), bacterial-type flagellum assembly (GO:0044780), bacterial-type flagellum-dependent swarming motility (GO:0071978), non-membrane-bounded organelle assembly (GO:0140694)

Cellular Component: cellular_component (GO:0005575), cellular anatomical entity (GO:0110165), intracellular immature spore (GO:0042763)

Generated by BioReason

Bioreason Rl Review

(fliY-bioreason-rl-review.md)

BioReason-Pro RL Review: fliY (B. subtilis)

Source: fliY-bioreason-rl-predictions.md

Correctness: 5/5
Completeness: 3/5

Functional Summary Review

The BioReason functional summary reads:

A scaffold subunit of the bacterial flagellar motor switch that assembles into the C-ring to control rotational direction during taxis. Its N-terminal adaptor module organizes partner binding, while its C-terminal oligomerization core builds a rigid lattice that transmits and gates signals from chemotaxis pathways to motor output. Operating at the flagellar base, it provides structural support and multivalent binding surfaces that coordinate assembly and switching within the flagellar apparatus.

The summary correctly identifies FliY as a flagellar C-ring component involved in chemotaxis (GO:0006935) and localized to the flagellar base. The recognition of the CheC-like N-terminal domain and FliN-like C-terminal domain is accurate. FliY's structural integration into the C-ring is a curated core role, so the paragraph's scaffold and switching claims are supported. However, there is a critical omission:

The core phosphatase function is omitted. The curated review establishes that FliY is a CheY-P phosphatase with phosphoprotein phosphatase activity (GO:0004721), catalyzing dephosphorylation of CheY-P at the flagellar motor switch (PMID:12920116). BioReason's raw GO section includes phosphatase terms, but the scored narrative does not incorporate them. Missing this second core role lowers completeness; it does not make the stated structural role false.

The curated review also identifies regulation of bacterial-type flagellum-dependent cell motility (GO:1902021) and negative regulation of phosphorelay signal transduction system (GO:0070298) as core functions. BioReason captures the chemotaxis connection but misses the specific signal termination mechanism.

The correct localization to the C-ring (GO:0009433) is implied in the summary but not explicitly stated with precision.

Comparison with interpro2go:

The interpro2go annotations for fliY include cytoskeletal motor activity (GO:0003774), which the curated review flags as incorrect (FliY is not a motor protein). BioReason does not recapitulate this specific error -- it correctly avoids calling FliY a motor. However, BioReason's GO predictions do include the phosphatase terms from interpro2go (GO:0004721, GO:0016791) without integrating them into the narrative. Both interpro2go and BioReason correctly identify chemotaxis and flagellar motility terms. BioReason improves slightly over interpro2go by avoiding the motor activity error but underperforms by not highlighting the phosphatase function in its summary.

Notes on thinking trace

The trace recognizes the CheC-like domain but interprets it as a "scaffold/adaptor" rather than a phosphatase domain. This misinterpretation cascades through the analysis, leading to a structural-role-centered narrative. The trace mentions "transiently engaging chemotaxis factors" but does not identify the specific enzymatic dephosphorylation activity that is FliY's defining function.

📄 View Raw YAML

id: P24073
gene_symbol: fliY
product_type: PROTEIN
status: COMPLETE
taxon:
  id: NCBITaxon:224308
  label: Bacillus subtilis (strain 168)
description: 'FliY is a bifunctional flagellar motor switch protein that acts as a
  CheY-P phosphatase localized at the flagellar C-ring. It contains three functional
  domains: an N-terminal CheY-binding region homologous to FliM residues 6-15, a middle
  CheC-like phosphatase domain with conserved Glu/Asn catalytic residues, and a C-terminal
  FliN-like domain that integrates it into the flagellar switch complex. FliY catalyzes
  the dephosphorylation of the phosphorylated response regulator CheY-P at the site
  of its action (the flagellar motor), thereby contributing to signal termination
  in the chemotaxis pathway. B. subtilis uses a three-phosphatase system (CheC, FliY,
  CheX) instead of the CheZ found in E. coli. FliY functions constitutively to maintain
  optimal CheY-P levels near the motor, while CheC responds to attractant stimuli.
  FliY can complement Salmonella fliN mutants, confirming its structural role in the
  C-ring.'
existing_annotations:
  - term:
      id: GO:0004721
      label: phosphoprotein phosphatase activity
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      summary: IBA annotation for phosphoprotein phosphatase activity is
        strongly supported by phylogenetic analysis and experimental evidence.
        FliY contains CheC-like phosphatase domains with conserved catalytic
        residues and has been directly demonstrated to catalyze CheY-P
        dephosphorylation [PMID:12920116].
      action: ACCEPT
      reason: This IBA annotation is well-supported by the phylogenetic tree
        inference and is consistent with extensive experimental evidence.
        PMID:12920116 directly demonstrates that FliY increases the rate of
        CheY-P hydrolysis in vitro. The domain architecture (CheC-like
        phosphatase domain) and structural studies further support this
        function.
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: We showed that FliY increases the rate of CheY-P
            hydrolysis in vitro.
        - reference_id: PMID:12920116
          supporting_text: In particular we have identified the phosphatase as
            FliY
        - reference_id: file:BACSU/fliY/fliY-deep-research-falcon.md
          supporting_text: See deep research file for comprehensive analysis
  - term:
      id: GO:1902021
      label: regulation of bacterial-type flagellum-dependent cell motility
    evidence_type: IBA
    original_reference_id: GO_REF:0000033
    review:
      summary: IBA annotation for regulation of flagellum-dependent motility is
        well-supported. FliY regulates motility by controlling CheY-P levels at
        the flagellar motor, which determines flagellar rotation direction
        [PMID:12920116].
      action: ACCEPT
      reason: This annotation accurately captures FliY's regulatory role in
        motility. By dephosphorylating CheY-P at the flagellar switch, FliY
        modulates the duration of enhanced CheY-P levels and thereby regulates
        switching between clockwise and counterclockwise flagellar rotation.
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: Consequently, we imagine that the duration of
            enhanced CheY-P levels caused by activation of the CheA kinase upon
            attractant binding to receptors, is brief due both to adaptational
            processes and to phosphatase activity of FliY.
  - term:
      id: GO:0003774
      label: cytoskeletal motor activity
    evidence_type: IEA
    original_reference_id: GO_REF:0000002
    review:
      summary: IEA annotation based on InterPro domain IPR001172
        (FliN_T3SS_HrcQb). This annotation is problematic because FliY is not
        itself a motor protein - it is a component of the motor switch that
        regulates motor activity, but does not generate force or movement
        directly.
      action: REMOVE
      reason: This is an over-annotation. FliY is a component of the flagellar
        motor switch complex (C-ring) but it does not have motor activity
        itself. The motor activity is generated by the stator units (MotA/MotB)
        interacting with FliG. FliY's role is to modulate CheY-P levels, not to
        generate rotational force. The InterPro domain hit is based on FliN
        homology, but FliN is a switch component, not a motor per se. The term
        "cytoskeletal motor activity" implies direct force generation, which
        FliY does not perform.
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: the flagellar switch, which controls direction of
            flagellar rotation based on levels of the chemotaxis primary
            response regulator, CheY-P
  - term:
      id: GO:0005886
      label: plasma membrane
    evidence_type: IEA
    original_reference_id: GO_REF:0000044
    review:
      summary: IEA annotation based on UniProt subcellular location mapping.
        UniProt indicates FliY is a peripheral membrane protein on the
        cytoplasmic side. However, more specific localization to the flagellar
        C-ring is available.
      action: MODIFY
      reason: While plasma membrane association is not incorrect, a more
        specific and informative CC annotation exists. FliY localizes
        specifically to the flagellar basal body C-ring. The UniProt entry
        itself states the switch is localized at the base of the flagellar
        motor, in or near the C-ring, attached to the MS-ring.
      proposed_replacement_terms:
        - id: GO:0009433
          label: bacterial-type flagellum basal body, C ring
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: the flagellar switch, which controls direction of
            flagellar rotation based on levels of the chemotaxis primary
            response regulator, CheY-P
  - term:
      id: GO:0006935
      label: chemotaxis
    evidence_type: IEA
    original_reference_id: GO_REF:0000120
    review:
      summary: IEA annotation for chemotaxis from combined automated methods.
        FliY is clearly involved in chemotaxis as it is part of the signal
        termination machinery that dephosphorylates CheY-P at the motor.
      action: ACCEPT
      reason: This is an appropriate biological process annotation. FliY's
        phosphatase activity directly contributes to chemotactic signal
        termination by hydrolyzing CheY-P at the flagellar switch. This role is
        experimentally validated in PMID:12920116.
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: This task is performed in Escherichia coli by CheZ,
            which interestingly enough is primarily located at the receptors,
            not at the switch.
  - term:
      id: GO:0009288
      label: bacterial-type flagellum
    evidence_type: IEA
    original_reference_id: GO_REF:0000002
    review:
      summary: IEA annotation based on InterPro domain IPR012826 (FliN). This is
        a valid but less specific localization compared to what is known about
        FliY.
      action: MODIFY
      reason: FliY localizes specifically to the C-ring of the flagellar basal
        body, not throughout the flagellum generally. A more specific annotation
        to the C-ring would be more informative and accurate.
      proposed_replacement_terms:
        - id: GO:0009433
          label: bacterial-type flagellum basal body, C ring
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: In this report we show that in Bacillus subtilis the
            flagellar switch, which controls direction of flagellar rotation
  - term:
      id: GO:0009425
      label: bacterial-type flagellum basal body
    evidence_type: IEA
    original_reference_id: GO_REF:0000002
    review:
      summary: IEA annotation based on InterPro domain IPR001172. This is a
        reasonable localization but can be made more specific to the C-ring.
      action: MODIFY
      reason: FliY is specifically a component of the C-ring within the basal
        body. While GO:0009425 is not incorrect, GO:0009433 (C-ring) would be
        more precise and informative.
      proposed_replacement_terms:
        - id: GO:0009433
          label: bacterial-type flagellum basal body, C ring
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: In particular we have identified the phosphatase as
            FliY, which resembles E. coli switch protein FliN only in its
            C-terminal part
  - term:
      id: GO:0016020
      label: membrane
    evidence_type: IEA
    original_reference_id: GO_REF:0000002
    review:
      summary: Very generic membrane annotation based on InterPro domain
        IPR012826. This is far too general given the specific localization known
        for FliY.
      action: MODIFY
      reason: This annotation is not wrong but is uninformative. FliY is a
        peripheral membrane protein that specifically localizes to the flagellar
        C-ring. The more specific term should be used.
      proposed_replacement_terms:
        - id: GO:0009433
          label: bacterial-type flagellum basal body, C ring
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: In this report we show that in Bacillus subtilis the
            flagellar switch, which controls direction of flagellar rotation
  - term:
      id: GO:0016787
      label: hydrolase activity
    evidence_type: IEA
    original_reference_id: GO_REF:0000120
    review:
      summary: Generic hydrolase activity annotation from combined automated
        methods. FliY does have hydrolase activity (it hydrolyzes the phosphate
        from CheY-P), but this term is too general.
      action: MARK_AS_OVER_ANNOTATED
      reason: While technically correct (FliY catalyzes hydrolysis of a
        phosphoester bond), this annotation is too general to be informative.
        The more specific term GO:0004721 (phosphoprotein phosphatase activity)
        already captures this function with appropriate specificity. Retaining
        both creates redundancy without adding information.
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: We showed that FliY increases the rate of CheY-P
            hydrolysis in vitro.
  - term:
      id: GO:0071973
      label: bacterial-type flagellum-dependent cell motility
    evidence_type: IEA
    original_reference_id: GO_REF:0000002
    review:
      summary: IEA annotation for flagellum-dependent motility based on InterPro
        domains. FliY contributes to motility through its role in the motor
        switch complex.
      action: KEEP_AS_NON_CORE
      reason: FliY is involved in flagellum-dependent motility as a component of
        the motor switch, but this is not its primary molecular function. Its
        core function is as a CheY-P phosphatase that regulates motility. This
        annotation reflects a downstream consequence of its biochemical activity
        rather than a direct role.
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: the flagellar switch, which controls direction of
            flagellar rotation
  - term:
      id: GO:0097588
      label: archaeal or bacterial-type flagellum-dependent cell motility
    evidence_type: IEA
    original_reference_id: GO_REF:0000043
    review:
      summary: IEA annotation from UniProt keyword mapping. This is a parent
        term of GO:0071973 (bacterial-type flagellum-dependent cell motility).
      action: KEEP_AS_NON_CORE
      reason: This is essentially redundant with GO:0071973 and represents a
        less specific grouping term. FliY is involved in motility regulation but
        this is downstream of its core phosphatase function. Given the more
        specific child term is also annotated, this parent term adds little
        value.
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: In this report we show that in Bacillus subtilis the
            flagellar switch, which controls direction of flagellar rotation
  - term:
      id: GO:0044780
      label: bacterial-type flagellum assembly
    evidence_type: IMP
    original_reference_id: PMID:25313396
    review:
      summary: IMP annotation from PMID:25313396 (FlgM secretion paper).
        However, this paper defines a minimal set of flagellar proteins required
        for FlgM secretion and flagellum assembly - fliY is NOT in this minimal
        set.
      action: REMOVE
      reason: 'PMID:25313396 explicitly defines the minimal subset of flagellar proteins
        required for flagellum assembly and FlgM secretion as: FliF/FliG, FliO, FliP,
        FliQ, FliR, FlhA, FlhB, and FliK. FliY is NOT in this list. While fliY mutants
        may show assembly defects due to impaired chemotaxis signaling, FliY is not
        a core component of the assembly machinery. This annotation likely reflects
        indirect/pleiotropic effects rather than a direct role in assembly.'
      supported_by:
        - reference_id: PMID:25313396
          supporting_text: 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.
  - term:
      id: GO:0071978
      label: bacterial-type flagellum-dependent swarming motility
    evidence_type: IMP
    original_reference_id: PMID:25313396
    review:
      summary: IMP annotation from PMID:25313396. Swarming motility requires
        functional flagella and chemotaxis, so fliY mutants may show swarming
        defects.
      action: KEEP_AS_NON_CORE
      reason: FliY likely contributes to swarming motility indirectly through
        its role in chemotaxis regulation. However, swarming is a complex
        phenotype that involves many genes. This annotation reflects a
        downstream consequence of FliY's phosphatase function rather than a
        direct role in swarming per se. The annotation is retained but marked as
        non-core since the primary function is CheY-P dephosphorylation.
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: Consequently, we imagine that the duration of
            enhanced CheY-P levels caused by activation of the CheA kinase upon
            attractant binding to receptors, is brief due both to adaptational
            processes and to phosphatase activity of FliY.
  - term:
      id: GO:0006935
      label: chemotaxis
    evidence_type: IMP
    original_reference_id: PMID:12920116
    review:
      summary: IMP annotation for chemotaxis from the key primary publication.
        PMID:12920116 directly demonstrates FliY's role in chemotaxis through
        its phosphatase activity on CheY-P.
      action: ACCEPT
      reason: This is a well-supported annotation from the primary paper
        characterizing FliY function. The paper shows that FliY deletion affects
        chemotaxis by altering CheY-P levels at the flagellar switch. Deletion
        of the CheY-binding region in fliY caused the opposite phenotype to a
        cheY mutant, demonstrating its direct role in chemotactic signal
        modulation.
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: Deletion of this binding region in B. subtilis mutant
            fliM caused the same phenotype as a cheY mutant (clockwise flagellar
            rotation), whereas deletion of it in fliY caused the opposite.
  - term:
      id: GO:0016311
      label: dephosphorylation
    evidence_type: IMP
    original_reference_id: PMID:12920116
    review:
      summary: IMP annotation for dephosphorylation biological process. FliY
        catalyzes dephosphorylation of CheY-P as demonstrated in PMID:12920116.
      action: ACCEPT
      reason: This annotation accurately captures FliY's role in
        dephosphorylation as a biological process. The paper directly
        demonstrates that FliY increases CheY-P hydrolysis rates in vitro, and
        this dephosphorylation is central to its function in chemotactic signal
        termination.
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: We showed that FliY increases the rate of CheY-P
            hydrolysis in vitro.
        - reference_id: PMID:12920116
          supporting_text: also causes hydrolysis of CheY-P to form CheY and Pi
  - term:
      id: GO:1902021
      label: regulation of bacterial-type flagellum-dependent cell motility
    evidence_type: IMP
    original_reference_id: PMID:12920116
    review:
      summary: IMP annotation for regulation of flagellum-dependent motility
        from PMID:12920116. FliY regulates motility by modulating CheY-P levels
        at the motor switch.
      action: ACCEPT
      reason: This is a key biological process annotation. PMID:12920116
        demonstrates that FliY regulates flagellar rotation direction by
        controlling CheY-P levels at the switch. The paper shows deletion of
        FliY's CheY-binding region affects flagellar rotation phenotype.
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: the flagellar switch, which controls direction of
            flagellar rotation based on levels of the chemotaxis primary
            response regulator, CheY-P
        - reference_id: PMID:12920116
          supporting_text: deletion of it in fliY caused the opposite [phenotype
            to cheY mutant]
  - term:
      id: GO:0004721
      label: phosphoprotein phosphatase activity
    evidence_type: IDA
    original_reference_id: PMID:12920116
    review:
      summary: IDA annotation for phosphoprotein phosphatase activity - the most
        important and well-supported annotation for FliY. PMID:12920116 directly
        demonstrates in vitro phosphatase activity on CheY-P.
      action: ACCEPT
      reason: This is the core molecular function of FliY and is directly
        demonstrated by in vitro biochemical assays in PMID:12920116. The paper
        identifies FliY as the phosphatase responsible for CheY-P hydrolysis at
        the flagellar switch and shows it increases CheY-P hydrolysis rates.
        This IDA annotation is strongly supported.
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: We showed that FliY increases the rate of CheY-P
            hydrolysis in vitro.
        - reference_id: PMID:12920116
          supporting_text: In particular we have identified the phosphatase as
            FliY
  - term:
      id: GO:0009433
      label: bacterial-type flagellum basal body, C ring
    evidence_type: IDA
    original_reference_id: PMID:12920116
    review:
      summary: NEW annotation for C-ring localization. FliY localizes to the
        flagellar switch complex, which is the C-ring. This is implied by its
        function at the motor switch and its homology to FliN.
      action: NEW
      reason: FliY is a component of the flagellar motor switch (C-ring).
        PMID:12920116 establishes that FliY performs its phosphatase function at
        the flagellar switch, and its C-terminal domain is homologous to FliN, a
        known C-ring component. B. subtilis FliY can complement Salmonella fliN
        mutants, confirming its structural integration into the C-ring.
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: In this report we show that in Bacillus subtilis the
            flagellar switch, which controls direction of flagellar rotation
            based on levels of the chemotaxis primary response regulator,
            CheY-P, also causes hydrolysis of CheY-P
        - reference_id: PMID:12920116
          supporting_text: FliY, which resembles E. coli switch protein FliN
            only in its C-terminal part
  - term:
      id: GO:0070298
      label: negative regulation of phosphorelay signal transduction system
    evidence_type: IDA
    original_reference_id: PMID:12920116
    review:
      summary: NEW annotation for negative regulation of phosphorelay signaling.
        FliY dephosphorylates the response regulator CheY-P, thereby terminating
        the chemotaxis phosphorelay signal.
      action: NEW
      reason: FliY acts as a negative regulator of the chemotaxis phosphorelay
        system by dephosphorylating CheY-P. This function terminates the signal
        initiated by the CheA histidine kinase. The paper explicitly describes
        this termination function at the motor.
      supported_by:
        - reference_id: PMID:12920116
          supporting_text: Consequently, we imagine that the duration of
            enhanced CheY-P levels caused by activation of the CheA kinase upon
            attractant binding to receptors, is brief due both to adaptational
            processes and to phosphatase activity of FliY.
  - term:
      id: GO:0005515
      label: protein binding
    evidence_type: NAS
    review:
      summary: Added to align core_functions with existing annotations.
      action: NEW
      reason: Core function term not present in existing_annotations.
references:
  - id: GO_REF:0000002
    title: Gene Ontology annotation through association of InterPro records with
      GO terms
    findings:
      - statement: Provides IEA annotations based on domain matches
  - id: GO_REF:0000033
    title: Annotation inferences using phylogenetic trees
    findings:
      - statement: IBA annotations based on PANTHER phylogenetic tree analysis
      - statement: FliY clusters with other CheC-like phosphatases
  - id: GO_REF:0000043
    title: Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword
      mapping
    findings:
      - statement: Maps flagellar rotation keyword to motility terms
  - id: GO_REF:0000044
    title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular
      Location vocabulary mapping
    findings:
      - statement: Maps cell membrane localization from UniProt
  - id: GO_REF:0000120
    title: Combined Automated Annotation using Multiple IEA Methods
    findings:
      - statement: Combines InterPro and keyword evidence for automated
          annotation
  - id: PMID:12920116
    title: Bacillus subtilis hydrolyzes CheY-P at the location of its action,
      the flagellar switch
    findings:
      - statement: Identified FliY as a CheY-P phosphatase at the flagellar
          switch
        supporting_text: In particular we have identified the phosphatase as
          FliY
      - statement: Demonstrated FliY increases CheY-P hydrolysis rate in vitro
        supporting_text: We showed that FliY increases the rate of CheY-P
          hydrolysis in vitro
      - statement: FliY has N-terminal CheY-binding region homologous to FliM
          residues 6-15
        supporting_text: These residues are almost identical to the residues
          6-15 in both B. subtilis FliM and FliY
      - statement: Both FliM and FliY can bind CheY-P in vitro
        supporting_text: We were able to show that both of these proteins are
          capable of binding CheY-P in vitro
      - statement: Deletion of CheY-binding region in fliY causes opposite
          phenotype to cheY mutant
        supporting_text: Deletion of this binding region in B. subtilis mutant
          fliM caused the same phenotype as a cheY mutant (clockwise flagellar
          rotation), whereas deletion of it in fliY caused the opposite
      - statement: FliY C-terminus is homologous to E. coli FliN
        supporting_text: FliY, which resembles E. coli switch protein FliN only
          in its C-terminal part
      - statement: FliY performs the role of E. coli CheZ but is localized at
          the motor, not receptors
        supporting_text: This task is performed in Escherichia coli by CheZ,
          which interestingly enough is primarily located at the receptors, not
          at the switch
  - id: PMID:25313396
    title: FlgM is secreted by the flagellar export apparatus in Bacillus
      subtilis
    findings:
      - statement: Defined minimal set of proteins for flagellum assembly and
          FlgM secretion
        supporting_text: FlgM secretion is strongly enhanced by, but does not
          strictly require, hook-basal body completion and instead demands a
          minimal subset of flagellar proteins
      - statement: Minimal set includes FliF/FliG, FliO, FliP, FliQ, FliR, FlhA,
          FlhB, FliK
        supporting_text: 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
      - statement: FliY is NOT in the minimal component set for assembly
        supporting_text: 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
  - id: PMID:14749334
    title: "Bacillus subtilis CheC and FliY are members of a novel class of CheY-P-hydrolyzing proteins in the chemotactic signal transduction cascade."
    findings:
      - statement: CheC and FliY form a novel class of CheY-P phosphatases
        supporting_text: we describe a new family of CheY-P phosphatases, the
          CYX family, that is widespread among the bacteria and archaea
      - statement: Part of B. subtilis three-phosphatase system with CheX
        supporting_text: We suggest a model for how FliY, CheC, and CheD work
          together to regulate CheY-P levels in the bacterium
  - id: file:BACSU/fliY/fliY-deep-research-falcon.md
    title: Deep research on fliY function
    findings: []
core_functions:
  - description: CheY-P phosphatase activity - catalyzes dephosphorylation of
      phosphorylated response regulator CheY at the flagellar motor switch
    molecular_function:
      id: GO:0004721
      label: phosphoprotein phosphatase activity
    directly_involved_in:
      - id: GO:0016311
        label: dephosphorylation
      - id: GO:0006935
        label: chemotaxis
    locations:
      - id: GO:0009433
        label: bacterial-type flagellum basal body, C ring
    supported_by:
      - reference_id: PMID:12920116
        supporting_text: We showed that FliY increases the rate of CheY-P
          hydrolysis in vitro.
      - reference_id: PMID:12920116
        supporting_text: In particular we have identified the phosphatase as
          FliY
  - description: Flagellar motor switch component - structural integration into
      the C-ring via FliN-like C-terminal domain, regulating flagellar rotation
      direction
    molecular_function:
      id: GO:0005515
      label: protein binding
    directly_involved_in:
      - id: GO:1902021
        label: regulation of bacterial-type flagellum-dependent cell motility
    locations:
      - id: GO:0009433
        label: bacterial-type flagellum basal body, C ring
    supported_by:
      - reference_id: PMID:12920116
        supporting_text: FliY, which resembles E. coli switch protein FliN only
          in its C-terminal part
  - description: Chemotaxis signal termination - removes CheY-P at the site of
      its action to modulate flagellar rotation switching, part of
      three-phosphatase system
    molecular_function:
      id: GO:0004721
      label: phosphoprotein phosphatase activity
    directly_involved_in:
      - id: GO:0070298
        label: negative regulation of phosphorelay signal transduction system
      - id: GO:0006935
        label: chemotaxis
    locations:
      - id: GO:0009433
        label: bacterial-type flagellum basal body, C ring
    supported_by:
      - reference_id: PMID:12920116
        supporting_text: Consequently, we imagine that the duration of enhanced
          CheY-P levels caused by activation of the CheA kinase upon attractant
          binding to receptors, is brief due both to adaptational processes and
          to phosphatase activity of FliY.
suggested_questions:
  - question: What is the relative contribution of FliY vs CheC vs CheX to total
      CheY-P dephosphorylation under different chemotactic stimulation
      conditions?
  - question: Does FliY have dual active sites like T. maritima FliY, and do
      both contribute equally to catalysis in B. subtilis?
suggested_experiments:
  - description: Purify B. subtilis FliY and measure Km and kcat for CheY-P
      dephosphorylation to compare with CheC and CheX. Quantitative kinetic
      parameters would enable modeling of the three-phosphatase system.
    hypothesis: FliY kinetic parameters differ from CheC and CheX, reflecting
      distinct roles in chemotaxis signal termination
  - description: Use cryo-EM or crosslinking mass spectrometry to determine the
      precise position of FliY within the B. subtilis C-ring. High-resolution
      structural information would reveal FliY integration into the switch.
    hypothesis: FliY occupies a specific position in the C-ring that places it
      in proximity to CheY-P binding sites on FliM

fliY

Gene Description

Existing Annotations Review

Core Functions

CheY-P phosphatase activity - catalyzes dephosphorylation of phosphorylated response regulator CheY at the flagellar motor switch

Flagellar motor switch component - structural integration into the C-ring via FliN-like C-terminal domain, regulating flagellar rotation direction

Chemotaxis signal termination - removes CheY-P at the site of its action to modulate flagellar rotation switching, part of three-phosphatase system

References

Suggested Questions for Experts

Suggested Experiments

Deep Research

Falcon

Citations

OpenScientist

AIGR Gene Hypothesis Deep Research — Final Report

Summary

Key Findings

Finding 1 — FliY is a flagellar motor-switch C-ring phosphatase, not a cell-division protein

Finding 2 — The cell-septum (GO:0030428) prediction is refuted / a misassignment

Finding 3 — Independent computed provenance (InterPro + STRING) corroborates the flagellar C-ring, not the septum

Mechanistic Model / Interpretation

GO component comparison

Evidence Matrix

Evidence Base

GO Curation Implications (leads — require curator verification)

Mechanistic Scope

Conflicts and Alternatives

Limitations and Knowledge Gaps

Discriminating Tests

Curation Leads (require curator verification)

Conclusion

Artifacts

📚 Additional Documentation

Bioreason Rl Predictions

BioReason Chat Export

Thinking Trace

Functional Summary

UniProt Summary

InterPro Domains

GO Terms

Bioreason Rl Review

BioReason-Pro RL Review: fliY (B. subtilis)

Functional Summary Review

Notes on thinking trace

📄 View Raw YAML