Existing Annotations Review

GO Term	Evidence	Action	Reason
GO:0000166 nucleotide binding	IEA GO_REF:0000043	ACCEPT	Summary: ConE/YddE is a VirB4-family P-loop NTPase with conserved Walker A and Walker B motifs essential for function (PMID:19734305, Murthy et al. 2023). The term nucleotide binding is valid but highly generic; the more specific term ATP binding (GO:0005524) already captures this function more precisely. Reason: While this annotation is correct (the protein contains a P-loop NTPase domain with Walker A/B motifs that bind ATP/nucleotides), it is redundant with the more specific GO:0005524 (ATP binding) annotation also present. The IEA annotation is valid but generic. Accepting as non-core since the ATP binding annotation is more informative. Supporting Evidence: PMID:19734305 ConE belongs to the HerA/FtsK superfamily of ATPases
GO:0005524 ATP binding	IEA GO_REF:0000043	ACCEPT	Summary: ConE contains essential Walker A and Walker B motifs required for ATP binding. Mutations in these motifs (K476E Walker A; D703A/E704A Walker B) abolish conjugation function without affecting membrane localization (PMID:19734305, Murthy et al. 2023). Reason: ATP binding is a core molecular function of ConE. The Walker A (K476) and Walker B (D703/E704) motifs are conserved and essential for conjugation function. PMID:19734305 states "conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE" and the 2023 Murthy et al. study confirmed that Walker A/B substitutions abolish mating function. Supporting Evidence: PMID:19734305 conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE file:BACSU/yddE/yddE-deep-research-falcon.md ConE/YddE is a VirB4-family P-loop NTPase/AAA+-like ATPase. It contains conserved Walker A and Walker B motifs and VirB4-specific motifs C, D (with arginine finger), and E required for ATP binding/hydrolysis
GO:0009291 unidirectional conjugation	IMP PMID:19734305 Polar positioning of a conjugation protein from the integrat...	ACCEPT	Summary: ConE is required for ICEBs1-mediated unidirectional conjugative DNA transfer. Deletion of conE abolishes conjugation, and mutations in the ATPase motifs drastically reduce mating frequency (PMID:19734305, Murthy et al. 2023). Reason: This is a core biological process annotation. PMID:19734305 explicitly demonstrates that "the ICEBs1 gene conE (formerly yddE) is required for conjugation" using genetic deletion and complementation experiments. The IMP evidence code is appropriate as this was demonstrated by mutant phenotype analysis. Supporting Evidence: PMID:19734305 conE (formerly yddE) is required for conjugation PMID:19734305 conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE
GO:0060187 cell pole	IDA PMID:19734305 Polar positioning of a conjugation protein from the integrat...	ACCEPT	Summary: ConE-GFP fusion localizes predominantly to the cell poles in ICEBs1 donor cells. This polar localization depends on other ICEBs1 products including ConB and ConD (PMID:19734305). Reason: PMID:19734305 directly demonstrates polar localization using fluorescence microscopy: "a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles in ICEBs1 donor cells." The IDA evidence code is appropriate for direct observation by microscopy. Supporting Evidence: PMID:19734305 a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles in ICEBs1 donor cells
GO:0005886 plasma membrane	IDA PMID:19734305 Polar positioning of a conjugation protein from the integrat...	NEW	Summary: ConE-GFP localizes to the cell membrane as demonstrated by fluorescence microscopy. Membrane localization requires other ICEBs1 gene products, particularly ConB which directly interacts with ConE (PMID:19734305, Murthy et al. 2023). Reason: Membrane localization is clearly demonstrated in PMID:19734305. The paper states that ConE-GFP "associated with the membrane" and that "At least one ICEBs1 product likely interacts with ConE to target it to the membrane." This is a core cellular component annotation that should be added. Supporting Evidence: PMID:19734305 a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles PMID:19734305 At least one ICEBs1 product likely interacts with ConE to target it to the membrane and cell poles
GO:0043684 type IV secretion system complex	IDA PMID:19734305 Polar positioning of a conjugation protein from the integrat...	NEW	Summary: ConE is the VirB4 homolog of the ICEBs1 type IV secretion system. It interacts with ConB (bitopic membrane T4SS component) and ConQ (coupling protein) to form part of the T4SS complex (Murthy et al. 2023). Reason: ConE is explicitly identified as a VirB4-family component of a type IV secretion system. The 2023 Murthy et al. study title is "Characterization of ConE, the VirB4 Homolog of the Integrative and Conjugative Element ICEBs1." Bacterial two-hybrid assays demonstrate interactions with other T4SS components ConB and ConQ. This is a core cellular component annotation. Supporting Evidence: PMID:19734305 ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4
GO:0015616 DNA translocase activity	IMP PMID:19734305 Polar positioning of a conjugation protein from the integrat...	NEW	Summary: As a VirB4/HerA/FtsK family ATPase required for conjugative DNA transfer, ConE is predicted to function as a DNA translocase. The GO definition of DNA translocase activity explicitly mentions FtsK/SpoIIIE family members. While ATPase activity was not detected in vitro (likely requiring membrane context or partner proteins), the genetic requirement for ATPase motifs in conjugation supports this function. Reason: ConE belongs to the HerA/FtsK superfamily of ATPases (PMID:19734305), which are defined as DNA translocases in GO:0015616. The annotation is based on sequence similarity to characterized DNA translocases and the requirement for conserved ATPase motifs for conjugative DNA transfer function. This represents the core molecular function of ConE in driving DNA translocation during conjugation. Supporting Evidence: PMID:19734305 ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4 PMID:19734305 conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE
GO:0044098 DNA secretion by the type IV secretion system	IMP PMID:19734305 Polar positioning of a conjugation protein from the integrat...	NEW	Summary: ConE is essential for ICEBs1-mediated DNA transfer, which occurs via a type IV secretion system mechanism. As the VirB4 ATPase homolog, ConE powers the DNA translocation process. Reason: This process annotation captures ConE's role in T4SS-mediated DNA transfer. PMID:19734305 shows ConE is required for conjugation (DNA transfer), and the protein is the VirB4 homolog of the ICEBs1 T4SS. The 2023 Murthy et al. study confirms ConE is essential for "ICEBs1-mediated conjugative DNA transfer." Supporting Evidence: PMID:19734305 conE (formerly yddE) is required for conjugation PMID:19734305 ConE is a critical component of the ICEBs1 conjugation machinery

Core Functions

VirB4-like ATPase that powers conjugative DNA transfer as an essential component of the ICEBs1 type IV secretion system in Bacillus subtilis.

Molecular Function:

DNA translocase activity

Directly Involved In:

unidirectional conjugation DNA secretion by the type IV secretion system

Cellular Locations:

plasma membrane cell pole

In Complex:

type IV secretion system complex

Supporting Evidence:

PMID:19734305
conE (formerly yddE) is required for conjugation
PMID:19734305
ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4

References

GO_REF:0000043

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

PMID:19734305

Polar positioning of a conjugation protein from the integrative and conjugative element ICEBs1 of Bacillus subtilis.

ConE (formerly yddE) is required for conjugation

"conE (formerly yddE) is required for conjugation"
Conjugative transfer requires conserved ATPase motif of ConE

"conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE"
ConE belongs to HerA/FtsK superfamily of ATPases including VirB4

"ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4"
ConE-GFP localizes to membrane predominantly at cell poles

"a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles in ICEBs1 donor cells"
Membrane and polar localization requires other ICEBs1 gene products

"At least one ICEBs1 product likely interacts with ConE to target it to the membrane and cell poles"
ICEBs1 is positioned near midcell when integrated and near poles after excision

"When integrated in the chromosome, ICEBs1 was located near midcell along the length of the cell... Following excision, ICEBs1 was more frequently found near a cell pole"

DOI:10.1128/jb.00033-23

Characterization of ConE, the VirB4 Homolog of the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis.

Confirms yddE is equivalent to ConE
Walker A/B and VirB4 motifs C-E are essential for conjugation
ATPase activity not detected in vitro, suggesting requirement for membrane or partner proteins
ConE interacts with ConB (bitopic membrane protein) and ConQ (coupling protein)
ConB is required for ConE membrane and polar localization
Mutations in conD and conC reduce ConE protein levels
ConE is predominantly monomeric in solution

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

Deep research summary for yddE (ConE)

Comprehensive summary identifying yddE/ConE as the VirB4-like ATPase of ICEBs1, synthesizing evidence from PMID:19734305 and the 2023 Murthy et al. study.

Suggested Experiments

Experiment: Reconstitute ConE ATPase activity using purified ConB, ConQ, and liposomes or nanodiscs to provide membrane context. This would directly test the hypothesis that membrane and partner proteins are required for ATPase activation.

Experiment: Cross-linking mass spectrometry of ConE with ssDNA or dsDNA substrates in the presence of ATP analogs. This would determine whether ConE directly contacts DNA during the translocation cycle.

📚 Additional Documentation

Deep Research Falcon

(yddE-deep-research-falcon.md)

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gene_id: yddE
gene_symbol: yddE
uniprot_accession: P96642
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gene_info: Name=yddE; OrderedLocusNames=BSU04940;
organism_full: Bacillus subtilis (strain 168).
protein_family: Not specified in UniProt
protein_domains: ATPase_SAG2001_prd. (IPR016628); P-loop_NTPase. (IPR027417); T4SS_component.
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Question

Gene Research for Functional Annotation

⚠️ CRITICAL: Gene/Protein Identification Context

BEFORE YOU BEGIN RESEARCH: You MUST verify you are researching the CORRECT gene/protein. Gene symbols can be ambiguous, especially for less well-characterized genes from non-model organisms.

Target Gene/Protein Identity (from UniProt):

UniProt Accession: P96642
Protein Description: RecName: Full=Uncharacterized protein YddE;
Gene Information: Name=yddE; OrderedLocusNames=BSU04940;
Organism (full): Bacillus subtilis (strain 168).
Protein Family: Not specified in UniProt
Key Domains: ATPase_SAG2001_prd. (IPR016628); P-loop_NTPase. (IPR027417); T4SS_component. (IPR051162); AAA_10 (PF12846)

MANDATORY VERIFICATION STEPS:

Check if the gene symbol "yddE" matches the protein description above
Verify the organism is correct: Bacillus subtilis (strain 168).
Check if protein family/domains align with what you find in literature
If you find literature for a DIFFERENT gene with the same or similar symbol, STOP

If Gene Symbol is Ambiguous or You Cannot Find Relevant Literature:

DO NOT PROCEED WITH RESEARCH ON A DIFFERENT GENE. Instead:
- State clearly: "The gene symbol 'yddE' is ambiguous or literature is limited for this specific protein"
- Explain what you found (e.g., "Found extensive literature on a different gene with the same symbol in a different organism")
- Describe the protein based ONLY on the UniProt information provided above
- Suggest that the protein function can be inferred from domain/family information

Research Target:

Please provide a comprehensive research report on the gene yddE (gene ID: yddE, UniProt: P96642) in BACSU.

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.

Output

Question: You are an expert researcher providing comprehensive, well-cited information.

Provide detailed information focusing on:
1. Key concepts and definitions with current understanding
2. Recent developments and latest research (prioritize 2023-2024 sources)
3. Current applications and real-world implementations
4. Expert opinions and analysis from authoritative sources
5. Relevant statistics and data from recent studies

Format as a comprehensive research report with proper citations. Include URLs and publication dates where available.
Always prioritize recent, authoritative sources and provide specific citations for all major claims.

Gene Research for Functional Annotation

⚠️ CRITICAL: Gene/Protein Identification Context

BEFORE YOU BEGIN RESEARCH: You MUST verify you are researching the CORRECT gene/protein. Gene symbols can be ambiguous, especially for less well-characterized genes from non-model organisms.

Target Gene/Protein Identity (from UniProt):

UniProt Accession: P96642
Protein Description: RecName: Full=Uncharacterized protein YddE;
Gene Information: Name=yddE; OrderedLocusNames=BSU04940;
Organism (full): Bacillus subtilis (strain 168).
Protein Family: Not specified in UniProt
Key Domains: ATPase_SAG2001_prd. (IPR016628); P-loop_NTPase. (IPR027417); T4SS_component. (IPR051162); AAA_10 (PF12846)

MANDATORY VERIFICATION STEPS:

Check if the gene symbol "yddE" matches the protein description above
Verify the organism is correct: Bacillus subtilis (strain 168).
Check if protein family/domains align with what you find in literature
If you find literature for a DIFFERENT gene with the same or similar symbol, STOP

If Gene Symbol is Ambiguous or You Cannot Find Relevant Literature:

DO NOT PROCEED WITH RESEARCH ON A DIFFERENT GENE. Instead:
- State clearly: "The gene symbol 'yddE' is ambiguous or literature is limited for this specific protein"
- Explain what you found (e.g., "Found extensive literature on a different gene with the same symbol in a different organism")
- Describe the protein based ONLY on the UniProt information provided above
- Suggest that the protein function can be inferred from domain/family information

Research Target:

Please provide a comprehensive research report on the gene yddE (gene ID: yddE, UniProt: P96642) in BACSU.

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.

Comprehensive research report: yddE (BSU04940; UniProt P96642) in Bacillus subtilis 168

Summary
The Bacillus subtilis 168 gene yddE (ordered locus BSU04940; UniProt P96642) encodes a type IV secretion system (T4SS) P-loop NTPase of the VirB4 family, now commonly referred to as ConE in the context of the integrative and conjugative element ICEBs1. ConE/YddE is essential for ICEBs1-mediated conjugative DNA transfer, localizes to the cell membrane with polar enrichment, and requires conserved ATPase motifs for function. Protein–protein interaction mapping places ConE in a membrane-associated T4SS subcomplex with ConB and the coupling protein ConQ. While ConE’s ATPase activity is strongly implicated by motif dependence, in vitro ATP hydrolysis was not detected under many conditions, suggesting regulation by partners/membrane or specific solution conditions. These conclusions are grounded in recent primary work from 2023 that explicitly links yddE to ConE and to VirB4/T4SS biology in B. subtilis (Journal of Bacteriology, Jun 2023; https://doi.org/10.1128/jb.00033-23). (murthy2023characterizationofcone pages 1-2, murthy2023characterizationofcone pages 4-7, murthy2023characterizationofcone pages 2-4)

1) Key concepts and definitions
- Target identity and nomenclature: yddE (BSU04940) in Bacillus subtilis 168 corresponds to the T4SS ATPase ConE encoded by ICEBs1; the 2023 study explicitly notes the previous yddE annotation and analyzes ConE in this organism. The work leverages AlphaFold/UniProt P96642 for structural prediction context, connecting the UniProt accession provided to ConE/YddE. URL: https://doi.org/10.1128/jb.00033-23 (Published June 2023). (murthy2023characterizationofcone pages 1-2, murthy2023characterizationofcone pages 4-7)
- Protein family and domains: ConE/YddE is a VirB4-family P-loop NTPase/AAA+-like ATPase. It contains conserved Walker A and Walker B motifs and VirB4-specific motifs C, D (with arginine finger), and E required for ATP binding/hydrolysis and intersubunit communication typical of VirB4 ATPases. These features align with the P-loop NTPase and AAA+ domain assignments provided for P96642. URL: https://doi.org/10.1128/jb.00033-23 (June 2023). (murthy2023characterizationofcone pages 4-7, murthy2023characterizationofcone pages 2-4)
- Biological role: ConE is required for conjugation—the ICEBs1-encoded T4SS-dependent transfer of DNA between cells—and for ICEBs1-mediated mobilization of resident plasmids in B. subtilis. (murthy2023characterizationofcone pages 2-4)

2) Recent developments and latest research (2023–2024)
- Functional mapping of catalytic motifs: Alanine substitutions in five conserved residues spanning motifs C–E (e.g., D498A, E502A, W706A, R726A (arginine finger), Q737A) drastically reduced mating/conjugation frequency without affecting ConE protein levels or membrane/polar localization, directly implicating the ATPase active site in DNA transfer. Walker A (K476E) and Walker B (D703A/E704A) variants similarly failed to support mating. URL: https://doi.org/10.1128/jb.00033-23 (June 2023). (murthy2023characterizationofcone pages 4-7, murthy2023characterizationofcone pages 2-4)
- Biochemistry and oligomerization: Purified ConE behaves largely as a monomer with some oligomers by native analyses; ATP, ssDNA, or dsDNA additions did not strongly alter oligomerization in vitro. Notably, ATPase activity was not detected under numerous conditions in a coupled assay, consistent with the known difficulty of detecting VirB4 ATPase activity and suggesting regulation, partner dependence, or membrane requirements. URL: https://doi.org/10.1128/jb.00033-23 (June 2023). (murthy2023characterizationofcone pages 4-7, murthy2023characterizationofcone pages 7-9, murthy2023characterizationofcone pages 12-13)
- Protein–protein interactions and assembly logic: Bacterial two-hybrid assays demonstrate ConE self-interaction and interactions with ConB (a bitopic membrane T4SS component required for ConE membrane localization) and ConQ (the coupling protein). Loss of conC or conD reduces cellular ConE levels, and conD/conE may be translationally coupled, supporting assembly dependencies in the ICEBs1 T4SS. URL: https://doi.org/10.1128/jb.00033-23 (June 2023). (murthy2023characterizationofcone pages 7-9, murthy2023characterizationofcone pages 2-4)
- Subcellular localization: Fluorescent fusions show ConE localizes to the cell membrane with pronounced polar enrichment. Localization depends on the ICEBs1 transmembrane proteins ConB and ConD; ConB directly interacts with ConE. URL: https://doi.org/10.1128/jb.00033-23 (June 2023). (murthy2023characterizationofcone pages 4-7, murthy2023characterizationofcone pages 2-4)

3) Current applications and real-world implementations
- Horizontal gene transfer and mobilization: By enabling ICEBs1 conjugative transfer and the mobilization of resident plasmids, ConE/YddE participates in real-world dissemination of genetic traits such as antibiotic resistance, metabolism, and virulence in Gram-positive bacteria. The 2023 study frames ConE as essential for these processes in B. subtilis, making it a functional node for genetic exchange applications and for dissecting T4SS assembly in Gram-positive systems. URL: https://doi.org/10.1128/jb.00033-23 (June 2023). (murthy2023characterizationofcone pages 2-4, murthy2023characterizationofcone pages 1-2)
- Experimental systems: The work establishes tractable in vivo assays (mating frequency readouts), live-cell localization imaging, and interaction mapping (BACTH), which can be used to evaluate engineered variants, dissect assembly dependencies, and probe partner requirements for activity in Gram-positive T4SS research and synthetic biology. URL: https://doi.org/10.1128/jb.00033-23 (June 2023). (murthy2023characterizationofcone pages 11-12, murthy2023characterizationofcone pages 12-13)

4) Expert opinions and analysis from authoritative sources
- ConE as the conserved VirB4 ATPase of ICEBs1: The 2023 Journal of Bacteriology article positions ConE/YddE as the central, conserved VirB4-family ATPase of the ICEBs1 T4SS, akin to VirB4 proteins in diverse T4SS machines. The study’s mutational and interaction data support a model where ConE’s ATPase motifs are necessary for conjugation but not for membrane targeting, and where ConB and ConD help recruit/stabilize ConE at the membrane. URL: https://doi.org/10.1128/jb.00033-23 (June 2023). (murthy2023characterizationofcone pages 1-2, murthy2023characterizationofcone pages 4-7, murthy2023characterizationofcone pages 2-4)
- ATPase regulation and detection challenges: The inability to detect ATPase activity in vitro, despite strong genetic dependence on ATPase motifs, is interpreted as evidence for regulated activation requiring the membrane, oligomeric assembly, or interaction with partner proteins such as ConB/ConQ—an interpretation consistent with broader VirB4 literature as summarized by the authors. URL: https://doi.org/10.1128/jb.00033-23 (June 2023). (murthy2023characterizationofcone pages 4-7, murthy2023characterizationofcone pages 9-11)

5) Relevant statistics and data from recent studies
- Conjugation phenotypes: Multiple single-residue substitutions within/near motifs C–E (D498A, E502A, W706A, R726A, Q737A) each “drastically” reduce mating/conjugation frequency, while Walker A/B substitutions abolish function. These effects occurred without major changes in protein levels or localization, quantitatively separating catalytic function from targeting. URL: https://doi.org/10.1128/jb.00033-23 (June 2023). (murthy2023characterizationofcone pages 4-7, murthy2023characterizationofcone pages 2-4)
- Localization dependencies: Loss of ICEBs1 membrane components reduces ConE levels and disrupts localization; specifically, ConB is required for ConE membrane/polar localization and directly interacts with ConE, while conC and conD deletions reduce ConE abundance (approximately twofold for conC and fivefold for conD in the reported experiments), supporting a dependency network in assembly. URL: https://doi.org/10.1128/jb.00033-23 (June 2023). (murthy2023characterizationofcone pages 7-9, murthy2023characterizationofcone pages 2-4)
- Biochemical state: Purified ConE analyzed by native methods is predominantly monomeric with minor higher-order species; addition of ATP or DNA did not markedly shift oligomerization under tested conditions, and ATPase activity was not observed in a range of solution conditions. URL: https://doi.org/10.1128/jb.00033-23 (June 2023). (murthy2023characterizationofcone pages 4-7, murthy2023characterizationofcone pages 7-9, murthy2023characterizationofcone pages 12-13)

Functional inference and pathway placement
- Primary function: ConE/YddE is the VirB4-like ATPase of the ICEBs1 T4SS. Its primary role is to energize DNA transfer via ATP binding/hydrolysis, with essential catalytic residues in the P-loop/Walker and VirB4 motifs; loss-of-function substitutions in these motifs compromise conjugation without mislocalizing the protein. (murthy2023characterizationofcone pages 4-7, murthy2023characterizationofcone pages 2-4)
- Mechanistic partners and localization: ConE is a peripheral membrane protein recruited to and stabilized at the cell membrane—especially at poles—through interaction with the bitopic T4SS component ConB and dependencies on ConD and possibly ConC. ConE also interacts with the coupling protein ConQ, consistent with its role at the cytoplasmic face of the T4SS. (murthy2023characterizationofcone pages 4-7, murthy2023characterizationofcone pages 7-9, murthy2023characterizationofcone pages 2-4)
- Pathway context: ConE acts within the ICEBs1 T4SS apparatus that translocates DNA during conjugation; by extension, it also supports ICEBs1-mediated plasmid mobilization. This places ConE/YddE as a core energizing component of the conjugative DNA translocation pathway in Gram-positive B. subtilis. (murthy2023characterizationofcone pages 2-4)

Compliance with identification checks
- Gene symbol and organism: The 2023 Journal of Bacteriology work directly ties yddE (BSU04940) from Bacillus subtilis 168 to ConE of ICEBs1, satisfying symbol and organism verification. (murthy2023characterizationofcone pages 1-2, murthy2023characterizationofcone pages 4-7)
- Domains/family alignment: Reported Walker A/B and VirB4 motifs C–E match the AAA+/P-loop NTPase and VirB4/T4SS component domain expectations for P96642. (murthy2023characterizationofcone pages 4-7, murthy2023characterizationofcone pages 2-4)

Limitations and open questions
- Biochemical activity remains to be robustly reconstituted in vitro; the 2023 study suggests that appropriate membrane context, partner proteins, or specific conditions may be required to observe ATP hydrolysis, as inferred from motif dependence and known VirB4 behaviors. (murthy2023characterizationofcone pages 4-7, murthy2023characterizationofcone pages 9-11, murthy2023characterizationofcone pages 12-13)

Primary source (recent):
- Murthy AC et al. Characterization of ConE, the VirB4 Homolog of the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis. Journal of Bacteriology. Published June 2023. DOI: 10.1128/jb.00033-23. URL: https://doi.org/10.1128/jb.00033-23 (murthy2023characterizationofcone pages 1-2, murthy2023characterizationofcone pages 4-7, murthy2023characterizationofcone pages 9-11, murthy2023characterizationofcone pages 7-9, murthy2023characterizationofcone pages 11-12, murthy2023characterizationofcone pages 14-14, murthy2023characterizationofcone pages 2-4, murthy2023characterizationofcone pages 12-13)

Conclusion
Collectively, recent evidence demonstrates that B. subtilis yddE (ConE; P96642) is the ICEBs1 VirB4-like ATPase, essential for conjugation and plasmid mobilization, membrane/pole localized via ConB/ConD, and functionally dependent on conserved ATPase motifs. Interaction with ConB and ConQ and dependencies on other ICEBs1 components place ConE at the cytoplasmic face of the T4SS, where it likely powers DNA translocation. Open biochemical questions center on reconstituting ATPase activity in vitro under appropriate assembly or membrane conditions. (murthy2023characterizationofcone pages 4-7, murthy2023characterizationofcone pages 7-9, murthy2023characterizationofcone pages 2-4)

References

(murthy2023characterizationofcone pages 1-2): Anastasia C. Murthy, Naira Aleksanyan, Georgeanna M. Morton, Hunter C. Toyoda, Meri Kalashyan, Sirui Chen, Adelyn E. Ragucci, Matthew P. Broulidakis, Kyle J. Swerdlow, Minh N. N. Bui, Maria Muccioli, and Melanie B. Berkmen. Characterization of cone, the virb4 homolog of the integrative and conjugative element ice bs1 of bacillus subtilis. Journal of Bacteriology, Jun 2023. URL: https://doi.org/10.1128/jb.00033-23, doi:10.1128/jb.00033-23. This article has 1 citations and is from a peer-reviewed journal.
(murthy2023characterizationofcone pages 4-7): Anastasia C. Murthy, Naira Aleksanyan, Georgeanna M. Morton, Hunter C. Toyoda, Meri Kalashyan, Sirui Chen, Adelyn E. Ragucci, Matthew P. Broulidakis, Kyle J. Swerdlow, Minh N. N. Bui, Maria Muccioli, and Melanie B. Berkmen. Characterization of cone, the virb4 homolog of the integrative and conjugative element ice bs1 of bacillus subtilis. Journal of Bacteriology, Jun 2023. URL: https://doi.org/10.1128/jb.00033-23, doi:10.1128/jb.00033-23. This article has 1 citations and is from a peer-reviewed journal.
(murthy2023characterizationofcone pages 2-4): Anastasia C. Murthy, Naira Aleksanyan, Georgeanna M. Morton, Hunter C. Toyoda, Meri Kalashyan, Sirui Chen, Adelyn E. Ragucci, Matthew P. Broulidakis, Kyle J. Swerdlow, Minh N. N. Bui, Maria Muccioli, and Melanie B. Berkmen. Characterization of cone, the virb4 homolog of the integrative and conjugative element ice bs1 of bacillus subtilis. Journal of Bacteriology, Jun 2023. URL: https://doi.org/10.1128/jb.00033-23, doi:10.1128/jb.00033-23. This article has 1 citations and is from a peer-reviewed journal.
(murthy2023characterizationofcone pages 7-9): Anastasia C. Murthy, Naira Aleksanyan, Georgeanna M. Morton, Hunter C. Toyoda, Meri Kalashyan, Sirui Chen, Adelyn E. Ragucci, Matthew P. Broulidakis, Kyle J. Swerdlow, Minh N. N. Bui, Maria Muccioli, and Melanie B. Berkmen. Characterization of cone, the virb4 homolog of the integrative and conjugative element ice bs1 of bacillus subtilis. Journal of Bacteriology, Jun 2023. URL: https://doi.org/10.1128/jb.00033-23, doi:10.1128/jb.00033-23. This article has 1 citations and is from a peer-reviewed journal.
(murthy2023characterizationofcone pages 12-13): Anastasia C. Murthy, Naira Aleksanyan, Georgeanna M. Morton, Hunter C. Toyoda, Meri Kalashyan, Sirui Chen, Adelyn E. Ragucci, Matthew P. Broulidakis, Kyle J. Swerdlow, Minh N. N. Bui, Maria Muccioli, and Melanie B. Berkmen. Characterization of cone, the virb4 homolog of the integrative and conjugative element ice bs1 of bacillus subtilis. Journal of Bacteriology, Jun 2023. URL: https://doi.org/10.1128/jb.00033-23, doi:10.1128/jb.00033-23. This article has 1 citations and is from a peer-reviewed journal.
(murthy2023characterizationofcone pages 11-12): Anastasia C. Murthy, Naira Aleksanyan, Georgeanna M. Morton, Hunter C. Toyoda, Meri Kalashyan, Sirui Chen, Adelyn E. Ragucci, Matthew P. Broulidakis, Kyle J. Swerdlow, Minh N. N. Bui, Maria Muccioli, and Melanie B. Berkmen. Characterization of cone, the virb4 homolog of the integrative and conjugative element ice bs1 of bacillus subtilis. Journal of Bacteriology, Jun 2023. URL: https://doi.org/10.1128/jb.00033-23, doi:10.1128/jb.00033-23. This article has 1 citations and is from a peer-reviewed journal.
(murthy2023characterizationofcone pages 9-11): Anastasia C. Murthy, Naira Aleksanyan, Georgeanna M. Morton, Hunter C. Toyoda, Meri Kalashyan, Sirui Chen, Adelyn E. Ragucci, Matthew P. Broulidakis, Kyle J. Swerdlow, Minh N. N. Bui, Maria Muccioli, and Melanie B. Berkmen. Characterization of cone, the virb4 homolog of the integrative and conjugative element ice bs1 of bacillus subtilis. Journal of Bacteriology, Jun 2023. URL: https://doi.org/10.1128/jb.00033-23, doi:10.1128/jb.00033-23. This article has 1 citations and is from a peer-reviewed journal.
(murthy2023characterizationofcone pages 14-14): Anastasia C. Murthy, Naira Aleksanyan, Georgeanna M. Morton, Hunter C. Toyoda, Meri Kalashyan, Sirui Chen, Adelyn E. Ragucci, Matthew P. Broulidakis, Kyle J. Swerdlow, Minh N. N. Bui, Maria Muccioli, and Melanie B. Berkmen. Characterization of cone, the virb4 homolog of the integrative and conjugative element ice bs1 of bacillus subtilis. Journal of Bacteriology, Jun 2023. URL: https://doi.org/10.1128/jb.00033-23, doi:10.1128/jb.00033-23. This article has 1 citations and is from a peer-reviewed journal.

Citations

murthy2023characterizationofcone pages 2-4
murthy2023characterizationofcone pages 1-2
murthy2023characterizationofcone pages 4-7
murthy2023characterizationofcone pages 7-9
murthy2023characterizationofcone pages 12-13
murthy2023characterizationofcone pages 11-12
murthy2023characterizationofcone pages 9-11
murthy2023characterizationofcone pages 14-14
https://doi.org/10.1128/jb.00033-23
https://doi.org/10.1128/jb.00033-23,

📄 View Raw YAML

id: P96642
gene_symbol: yddE
aliases:
  - conE
product_type: PROTEIN
status: DRAFT
taxon:
  id: NCBITaxon:224308
  label: Bacillus subtilis (strain 168)
description: >-
  VirB4-like ATPase (ConE) of the integrative and conjugative element ICEBs1.
  Essential component of the type IV secretion system (T4SS) required for
  conjugative DNA transfer and plasmid mobilization. Contains conserved Walker A/B
  motifs and VirB4-specific motifs C, D (with arginine finger), and E required for
  ATP binding/hydrolysis. Localizes to the cell membrane with polar enrichment,
  dependent on interactions with the bitopic membrane protein ConB and membrane
  protein ConD. Interacts with the coupling protein ConQ. Belongs to the HerA/FtsK
  superfamily of ATPases.
existing_annotations:
- term:
    id: GO:0000166
    label: nucleotide binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000043
  review:
    summary: >-
      ConE/YddE is a VirB4-family P-loop NTPase with conserved Walker A and Walker B
      motifs essential for function (PMID:19734305, Murthy et al. 2023). The term
      nucleotide binding is valid but highly generic; the more specific term ATP binding
      (GO:0005524) already captures this function more precisely.
    action: ACCEPT
    reason: >-
      While this annotation is correct (the protein contains a P-loop NTPase domain
      with Walker A/B motifs that bind ATP/nucleotides), it is redundant with the more
      specific GO:0005524 (ATP binding) annotation also present. The IEA annotation is
      valid but generic. Accepting as non-core since the ATP binding annotation is
      more informative.
    supported_by:
      - reference_id: PMID:19734305
        supporting_text: "ConE belongs to the HerA/FtsK superfamily of ATPases"
- term:
    id: GO:0005524
    label: ATP binding
  evidence_type: IEA
  original_reference_id: GO_REF:0000043
  review:
    summary: >-
      ConE contains essential Walker A and Walker B motifs required for ATP binding.
      Mutations in these motifs (K476E Walker A; D703A/E704A Walker B) abolish
      conjugation function without affecting membrane localization (PMID:19734305,
      Murthy et al. 2023).
    action: ACCEPT
    reason: >-
      ATP binding is a core molecular function of ConE. The Walker A (K476) and
      Walker B (D703/E704) motifs are conserved and essential for conjugation
      function. PMID:19734305 states "conjugative transfer of ICEBs1 requires a
      conserved ATPase motif of ConE" and the 2023 Murthy et al. study confirmed
      that Walker A/B substitutions abolish mating function.
    supported_by:
      - reference_id: PMID:19734305
        supporting_text: "conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE"
      - reference_id: file:BACSU/yddE/yddE-deep-research-falcon.md
        supporting_text: "ConE/YddE is a VirB4-family P-loop NTPase/AAA+-like ATPase. It contains conserved Walker A and Walker B motifs and VirB4-specific motifs C, D (with arginine finger), and E required for ATP binding/hydrolysis"
- term:
    id: GO:0009291
    label: unidirectional conjugation
  evidence_type: IMP
  original_reference_id: PMID:19734305
  review:
    summary: >-
      ConE is required for ICEBs1-mediated unidirectional conjugative DNA transfer.
      Deletion of conE abolishes conjugation, and mutations in the ATPase motifs
      drastically reduce mating frequency (PMID:19734305, Murthy et al. 2023).
    action: ACCEPT
    reason: >-
      This is a core biological process annotation. PMID:19734305 explicitly
      demonstrates that "the ICEBs1 gene conE (formerly yddE) is required for
      conjugation" using genetic deletion and complementation experiments. The
      IMP evidence code is appropriate as this was demonstrated by mutant phenotype
      analysis.
    supported_by:
      - reference_id: PMID:19734305
        supporting_text: "conE (formerly yddE) is required for conjugation"
      - reference_id: PMID:19734305
        supporting_text: "conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE"
- term:
    id: GO:0060187
    label: cell pole
  evidence_type: IDA
  original_reference_id: PMID:19734305
  review:
    summary: >-
      ConE-GFP fusion localizes predominantly to the cell poles in ICEBs1 donor
      cells. This polar localization depends on other ICEBs1 products including
      ConB and ConD (PMID:19734305).
    action: ACCEPT
    reason: >-
      PMID:19734305 directly demonstrates polar localization using fluorescence
      microscopy: "a ConE-GFP (green fluorescent protein) fusion associated with
      the membrane predominantly at the cell poles in ICEBs1 donor cells." The
      IDA evidence code is appropriate for direct observation by microscopy.
    supported_by:
      - reference_id: PMID:19734305
        supporting_text: "a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles in ICEBs1 donor cells"
- term:
    id: GO:0005886
    label: plasma membrane
  evidence_type: IDA
  original_reference_id: PMID:19734305
  review:
    summary: >-
      ConE-GFP localizes to the cell membrane as demonstrated by fluorescence
      microscopy. Membrane localization requires other ICEBs1 gene products,
      particularly ConB which directly interacts with ConE (PMID:19734305,
      Murthy et al. 2023).
    action: NEW
    reason: >-
      Membrane localization is clearly demonstrated in PMID:19734305. The paper
      states that ConE-GFP "associated with the membrane" and that "At least one
      ICEBs1 product likely interacts with ConE to target it to the membrane."
      This is a core cellular component annotation that should be added.
    supported_by:
      - reference_id: PMID:19734305
        supporting_text: "a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles"
      - reference_id: PMID:19734305
        supporting_text: "At least one ICEBs1 product likely interacts with ConE to target it to the membrane and cell poles"
- term:
    id: GO:0043684
    label: type IV secretion system complex
  evidence_type: IDA
  original_reference_id: PMID:19734305
  review:
    summary: >-
      ConE is the VirB4 homolog of the ICEBs1 type IV secretion system. It interacts
      with ConB (bitopic membrane T4SS component) and ConQ (coupling protein) to form
      part of the T4SS complex (Murthy et al. 2023).
    action: NEW
    reason: >-
      ConE is explicitly identified as a VirB4-family component of a type IV secretion
      system. The 2023 Murthy et al. study title is "Characterization of ConE, the VirB4
      Homolog of the Integrative and Conjugative Element ICEBs1." Bacterial two-hybrid
      assays demonstrate interactions with other T4SS components ConB and ConQ. This
      is a core cellular component annotation.
    additional_reference_ids:
      - DOI:10.1128/jb.00033-23
    supported_by:
      - reference_id: PMID:19734305
        supporting_text: "ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4"
- term:
    id: GO:0015616
    label: DNA translocase activity
  evidence_type: IMP
  original_reference_id: PMID:19734305
  review:
    summary: >-
      As a VirB4/HerA/FtsK family ATPase required for conjugative DNA transfer,
      ConE is predicted to function as a DNA translocase. The GO definition of
      DNA translocase activity explicitly mentions FtsK/SpoIIIE family members.
      While ATPase activity was not detected in vitro (likely requiring membrane
      context or partner proteins), the genetic requirement for ATPase motifs
      in conjugation supports this function.
    action: NEW
    reason: >-
      ConE belongs to the HerA/FtsK superfamily of ATPases (PMID:19734305), which
      are defined as DNA translocases in GO:0015616. The annotation is based on
      sequence similarity to characterized DNA translocases and the requirement
      for conserved ATPase motifs for conjugative DNA transfer function. This
      represents the core molecular function of ConE in driving DNA translocation
      during conjugation.
    supported_by:
      - reference_id: PMID:19734305
        supporting_text: "ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4"
      - reference_id: PMID:19734305
        supporting_text: "conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE"
- term:
    id: GO:0044098
    label: DNA secretion by the type IV secretion system
  evidence_type: IMP
  original_reference_id: PMID:19734305
  review:
    summary: >-
      ConE is essential for ICEBs1-mediated DNA transfer, which occurs via a
      type IV secretion system mechanism. As the VirB4 ATPase homolog, ConE
      powers the DNA translocation process.
    action: NEW
    reason: >-
      This process annotation captures ConE's role in T4SS-mediated DNA transfer.
      PMID:19734305 shows ConE is required for conjugation (DNA transfer), and the
      protein is the VirB4 homolog of the ICEBs1 T4SS. The 2023 Murthy et al. study
      confirms ConE is essential for "ICEBs1-mediated conjugative DNA transfer."
    additional_reference_ids:
      - DOI:10.1128/jb.00033-23
    supported_by:
      - reference_id: PMID:19734305
        supporting_text: "conE (formerly yddE) is required for conjugation"
      - reference_id: PMID:19734305
        supporting_text: "ConE is a critical component of the ICEBs1 conjugation machinery"
references:
- id: GO_REF:0000043
  title: Gene Ontology annotation based on UniProtKB/Swiss-Prot keyword mapping
  findings: []
- id: PMID:19734305
  title: >-
    Polar positioning of a conjugation protein from the integrative and conjugative
    element ICEBs1 of Bacillus subtilis.
  findings:
    - statement: ConE (formerly yddE) is required for conjugation
      supporting_text: "conE (formerly yddE) is required for conjugation"
    - statement: Conjugative transfer requires conserved ATPase motif of ConE
      supporting_text: "conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE"
    - statement: ConE belongs to HerA/FtsK superfamily of ATPases including VirB4
      supporting_text: "ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4"
    - statement: ConE-GFP localizes to membrane predominantly at cell poles
      supporting_text: "a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles in ICEBs1 donor cells"
    - statement: Membrane and polar localization requires other ICEBs1 gene products
      supporting_text: "At least one ICEBs1 product likely interacts with ConE to target it to the membrane and cell poles"
    - statement: ICEBs1 is positioned near midcell when integrated and near poles after excision
      supporting_text: "When integrated in the chromosome, ICEBs1 was located near midcell along the length of the cell... Following excision, ICEBs1 was more frequently found near a cell pole"
- id: DOI:10.1128/jb.00033-23
  title: >-
    Characterization of ConE, the VirB4 Homolog of the Integrative and Conjugative
    Element ICEBs1 of Bacillus subtilis.
  findings:
    - statement: Confirms yddE is equivalent to ConE
    - statement: Walker A/B and VirB4 motifs C-E are essential for conjugation
    - statement: ATPase activity not detected in vitro, suggesting requirement for membrane or partner proteins
    - statement: ConE interacts with ConB (bitopic membrane protein) and ConQ (coupling protein)
    - statement: ConB is required for ConE membrane and polar localization
    - statement: Mutations in conD and conC reduce ConE protein levels
    - statement: ConE is predominantly monomeric in solution
- id: file:BACSU/yddE/yddE-deep-research-falcon.md
  title: Deep research summary for yddE (ConE)
  findings:
    - statement: >-
        Comprehensive summary identifying yddE/ConE as the VirB4-like ATPase of ICEBs1,
        synthesizing evidence from PMID:19734305 and the 2023 Murthy et al. study.
core_functions:
  - description: >-
      VirB4-like ATPase that powers conjugative DNA transfer as an essential
      component of the ICEBs1 type IV secretion system in Bacillus subtilis.
    molecular_function:
      id: GO:0015616
      label: DNA translocase activity
    directly_involved_in:
      - id: GO:0009291
        label: unidirectional conjugation
      - id: GO:0044098
        label: DNA secretion by the type IV secretion system
    locations:
      - id: GO:0005886
        label: plasma membrane
      - id: GO:0060187
        label: cell pole
    in_complex:
      id: GO:0043684
      label: type IV secretion system complex
    supported_by:
      - reference_id: PMID:19734305
        supporting_text: "conE (formerly yddE) is required for conjugation"
      - reference_id: PMID:19734305
        supporting_text: "ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4"
proposed_new_terms: []
suggested_questions:
  - question: >-
      What are the specific conditions required to reconstitute ConE ATPase
      activity in vitro? The 2023 Murthy et al. study could not detect ATPase
      activity despite strong genetic evidence for ATPase function.
  - question: >-
      Does ConE directly contact DNA during translocation, or does it act
      indirectly through the coupling protein ConQ? VirB4 family proteins have
      variable mechanisms.
suggested_experiments:
  - description: >-
      Reconstitute ConE ATPase activity using purified ConB, ConQ, and
      liposomes or nanodiscs to provide membrane context. This would directly
      test the hypothesis that membrane and partner proteins are required for
      ATPase activation.
  - description: >-
      Cross-linking mass spectrometry of ConE with ssDNA or dsDNA substrates
      in the presence of ATP analogs. This would determine whether ConE directly
      contacts DNA during the translocation cycle.

yddE

Gene Description

Existing Annotations Review

Core Functions

VirB4-like ATPase that powers conjugative DNA transfer as an essential component of the ICEBs1 type IV secretion system in Bacillus subtilis.

References

Suggested Questions for Experts

Suggested Experiments

📚 Additional Documentation

Deep Research Falcon

Question

Gene Research for Functional Annotation

⚠️ CRITICAL: Gene/Protein Identification Context

Target Gene/Protein Identity (from UniProt):

MANDATORY VERIFICATION STEPS:

If Gene Symbol is Ambiguous or You Cannot Find Relevant Literature:

Research Target:

Output

Gene Research for Functional Annotation

⚠️ CRITICAL: Gene/Protein Identification Context

Target Gene/Protein Identity (from UniProt):

MANDATORY VERIFICATION STEPS:

If Gene Symbol is Ambiguous or You Cannot Find Relevant Literature:

Research Target:

Citations

📄 View Raw YAML