Bacillus subtilis project
Bottom line: Bacillus subtilis is the model Gram-positive bacterium for
sporulation, competence, cell division and protein secretion. We reviewed every
existing GO annotation on 29 of its genes in three rounds: nine genes where
CACAO student curation had contributed heavily, ten key functional genes, and
ten more that complete the sporulation sigma cascade and the industrial
enzymes. That covers 343 annotation rows: 200 accepted, 28 kept as non-core,
45 modified, 14 removed, 10 marked over-annotated, 44 proposed as NEW, and 2 left
UNDECIDED (fliH, where the cited paper does not mention the gene). The
recurring corrections were removing RNA polymerase activity from sigma
factors, removing a fold-based acyltransferase call on spoVAD, and removing
the fliY and swrD rows that cite PMID:25313396, a paper that does not
examine those genes. All
29 reviews exist and render, and a sporulation cascade pathway summary is in
BACSU/. Most review files still carry status: DRAFT although the checklist
below says COMPLETE, and several UniProt IDs in the tables below differ from
the accessions the reviews use (the lipA review covers lipoyl synthase
O32129, not lipase A). Six further B. subtilis reviews made outside these
rounds (spoIIAA, spoIIAB, spoIIB, spoIIR, spoVD, yciC; 53 rows) are listed
in the frontmatter but not counted above.
Use uniprot code BACSU
Genes/proteins for which CACAO made major contributions
| Gene | UniProt ID | Protein |
|---|---|---|
| fliH | https://www.uniprot.org/uniprotkb/P23449 | Flagellar assembly protein FliH |
| fliK | https://www.uniprot.org/uniprotkb/P23451 | Flagellar hook-length control protein |
| fliW | https://www.uniprot.org/uniprotkb/P96503 | Flagellar assembly factor FliW |
| fliY | https://www.uniprot.org/uniprotkb/P24073 | Flagellar motor switch phosphatase FliY |
| gerD | https://www.uniprot.org/uniprotkb/P16450 | Spore germination protein GerD |
| spo0J | https://www.uniprot.org/uniprotkb/P26497 | Stage 0 sporulation protein J |
| spoVAD | https://www.uniprot.org/uniprotkb/P40869 | Stage V sporulation protein AD |
| swrD | https://www.uniprot.org/uniprotkb/C0H412 | Swarming motility protein SwrD |
| yddE | https://www.uniprot.org/uniprotkb/P96642 | ConE VirB4-like ATPase (ICEBs1 T4SS) |
Round 2 - Key functional genes
| Gene | UniProt ID | Protein |
|---|---|---|
| spo0A | https://www.uniprot.org/uniprotkb/P06534 | Sporulation master transcription factor |
| sigF | https://www.uniprot.org/uniprotkb/P07860 | RNA polymerase sigma-F factor |
| ftsZ | https://www.uniprot.org/uniprotkb/P17865 | Cell division protein FtsZ |
| divIVA | https://www.uniprot.org/uniprotkb/P71021 | Cell division initiation protein DivIVA |
| comK | https://www.uniprot.org/uniprotkb/P40396 | Competence transcription factor |
| aprE | https://www.uniprot.org/uniprotkb/P04189 | Subtilisin E (alkaline protease) |
| amyE | https://www.uniprot.org/uniprotkb/P00691 | Alpha-amylase |
| sacB | https://www.uniprot.org/uniprotkb/P05655 | Levansucrase |
| secA | https://www.uniprot.org/uniprotkb/P28366 | Protein translocase subunit SecA |
| minC | https://www.uniprot.org/uniprotkb/Q01463 | Division inhibitor MinC |
review these first
Round 3 - Sporulation cascade completion + biotechnology
| Gene | UniProt ID | Protein |
|---|---|---|
| sigE | https://www.uniprot.org/uniprotkb/P06222 | RNA polymerase sigma-E factor |
| sigG | https://www.uniprot.org/uniprotkb/P11469 | RNA polymerase sigma-G factor |
| sigK | https://www.uniprot.org/uniprotkb/P28014 | RNA polymerase sigma-K factor |
| spoIIE | https://www.uniprot.org/uniprotkb/P13801 | Stage II sporulation protein E |
| minD | https://www.uniprot.org/uniprotkb/P40770 | Septum site-determining protein MinD |
| nprE | https://www.uniprot.org/uniprotkb/P39899 | Extracellular neutral metalloprotease |
| lipA | https://www.uniprot.org/uniprotkb/P37957 | Lipase A |
| secY | https://www.uniprot.org/uniprotkb/P16336 | Protein translocase subunit SecY |
| comGA | https://www.uniprot.org/uniprotkb/P32390 | Competence protein ComGA |
| spoIIGA | https://www.uniprot.org/uniprotkb/P13800 | Stage II sporulation protein GA |
Why it's studied:
- Model Gram-positive bacterium - counterpart to E. coli (Gram-negative)
- GRAS status (Generally Recognized As Safe) - safe for food/industrial use
- Natural competence - easily takes up DNA, great for genetic manipulation
- Sporulation - forms endospores; model for cell differentiation and stress survival
- Biofilm formation - forms complex multicellular communities
Key genes by function:
Sporulation (cell differentiation cascade):
- spo0A - master regulator, initiates sporulation
- sigF, sigE, sigG, sigK - compartment-specific sigma factors
- spoIIE, spoIIGA - signaling between mother cell and forespore
Cell division:
- ftsZ - tubulin homolog, forms Z-ring
- minC/minD - positioning of division septum
- divIVA - polar localization
Competence/DNA uptake:
- comK - master regulator of competence
- comGA, comGB - DNA uptake machinery
Biotechnology workhorses:
- aprE - subtilisin (alkaline protease) - detergent enzymes
- amyE - alpha-amylase - starch processing, commonly used integration locus
- sacB - levansucrase - counter-selection marker (sucrose sensitivity)
- nprE - neutral protease
- lipA - lipase
Secretion system:
- secA, secY - Sec pathway (major export route)
- B. subtilis is favored industrially because it secretes proteins directly into medium (no periplasm like E. coli)
Industrial applications:
- Enzyme production (proteases, amylases, lipases) - ~60% of commercial enzymes
- Vitamin B2 (riboflavin) production
- Poly-γ-glutamic acid production
- Probiotics (animal feed)
- Biocontrol agents in agriculture
STATUS
Round 1 - CACAO annotations review
- [x] fliW - COMPLETE (validated 2025-12-17)
- [~] fliH - DRAFT (2 annotations UNDECIDED - PMID:25313396 doesn't mention fliH)
- [x] fliK - COMPLETE (validated 2025-12-17, hook-length control protein)
- [x] fliY - COMPLETE (validated 2025-12-17, bifunctional CheY-P phosphatase + C-ring component)
- [x] gerD - COMPLETE (validated 2025-12-17, germinosome scaffold protein)
- [x] spo0J - COMPLETE (validated 2025-12-17, ParB/CTP-dependent chromosome partition clamp)
- [x] spoVAD - COMPLETE (validated 2025-12-17, SpoVA Ca-DPA channel plug; removed incorrect acyltransferase annotation)
- [x] swrD - COMPLETE (validated 2025-12-17, flagellar motor power enhancer; removed erroneous PMID:25313396)
- [x] yddE - COMPLETE (validated 2025-12-17, ConE VirB4-like conjugation ATPase; NOT uncharacterized!)
Round 2 - Key functional genes
- [x] spo0A - COMPLETE (validated 2025-12-18, master phosphorelay response regulator, 0A-box TF)
- [x] sigF - COMPLETE (validated 2025-12-18, forespore sigma factor, partner-switching regulation)
- [x] ftsZ - COMPLETE (validated 2025-12-18, tubulin-like GTPase, Z-ring scaffold for cytokinesis)
- [x] divIVA - COMPLETE (validated 2025-12-18, coiled-coil polar landmark protein)
- [x] comK - COMPLETE (validated 2025-12-18, competence master regulator, helix-turn-helix TF)
- [x] aprE - COMPLETE (validated 2025-12-18, subtilisin E serine protease, industrial enzyme)
- [x] amyE - COMPLETE (validated 2025-12-18, alpha-amylase, starch hydrolysis)
- [x] sacB - COMPLETE (validated 2025-12-18, levansucrase, counter-selection marker)
- [x] secA - COMPLETE (validated 2025-12-18, Sec translocase ATPase motor)
- [x] minC - COMPLETE (validated 2025-12-18, FtsZ polymerization inhibitor)
Round 3 - Sporulation cascade completion + biotechnology
- [x] sigE - COMPLETE (validated 2025-12-18, mother cell sigma-E factor, SpoIIGA-cleaved precursor)
- [x] sigG - COMPLETE (validated 2025-12-18, late forespore sigma-G factor, Gin-regulated)
- [x] sigK - COMPLETE (validated 2025-12-18, late mother cell sigma-K factor, SpoIVFB-processed)
- [x] spoIIE - COMPLETE (validated 2025-12-18, PP2C phosphatase + septum morphogenesis, activates SigF)
- [x] minD - COMPLETE (validated 2025-12-18, septum site-determining ATPase, MinC partner)
- [x] nprE - COMPLETE (validated 2025-12-18, bacillolysin zinc metalloprotease M4 family)
- [x] lipA - COMPLETE (validated 2025-12-18, lipoyl synthase, radical SAM enzyme)
- [x] secY - COMPLETE (validated 2025-12-18, SecYEG channel-forming subunit)
- [x] comGA - COMPLETE (validated 2025-12-18, competence pseudopilus ATPase)
- [x] spoIIGA - COMPLETE (validated 2025-12-18, pro-sigmaE processing aspartic protease)
NOTES
2025-12-18 (Session 2)
Round 3 complete! All 10 sporulation cascade + biotechnology genes validated.
Sporulation sigma factors:
- sigE: 8 annotations reviewed. Mother cell sigma-E factor (sigma-29) cleaved from pro-sigmaE (P31) by SpoIIGA. MODIFY: GO:0003700 (TF activity) → GO:0016987 (sigma factor activity). ACCEPT sigma factor activity, sporulation.
- sigG: 14 annotations reviewed. Late forespore sigma-G factor regulated by anti-sigma Gin (CsfB). REMOVE GO:0003899 (RNAP activity) - sigma factors are NOT catalytic. REMOVE GO:0000976 (cis-regulatory binding) - sigma factors require core RNAP. NEW: GO:0045152 (antisigma factor binding), GO:0042601 (forespore localization).
- sigK: Validated with clean pass. Late mother cell sigma-K factor processed by SpoIVFB.
Sporulation signaling:
- spoIIE: 8 annotations reviewed. PP2C-family phosphatase that dephosphorylates SpoIIAA-P to activate SigF. ACCEPT phosphatase activities. Added supporting_text for PMID:25374563 findings (Y2H with RacA/RecA, in vitro dephosphorylation).
- spoIIGA: 13 annotations reviewed. Membrane-embedded aspartic protease that cleaves pro-sigmaE. MODIFY: GO:0030435/GO:0030436 (sporulation) → GO:0034301 (endospore formation) for specificity. MARK_AS_OVER_ANNOTATED: GO:0016787 (hydrolase) too general.
Cell division:
- minD: Validated with clean pass. Septum site-determining ATPase, MinC partner.
Industrial enzymes:
- nprE: 8 annotations reviewed. Bacillolysin (neutral protease), peptidase M4 family zinc metalloendopeptidase. ACCEPT metalloendopeptidase activity (GO:0004222). MODIFY: GO:0046872 (metal ion binding) → GO:0008270 (zinc ion binding) + GO:0005509 (calcium ion binding). MARK_AS_OVER_ANNOTATED: GO:0016787 (hydrolase) too general.
- lipA: 13 annotations reviewed. Lipoyl synthase, radical SAM enzyme inserting sulfur into octanoyl groups. MODIFY: GO:0003824 (catalytic activity) → GO:0016992 (lipoate synthase activity). ACCEPT all Fe-S cluster binding terms.
Protein secretion:
- secY: 12 annotations reviewed. SecYEG channel-forming subunit (10 TM helices). ACCEPT protein transmembrane transporter activity, signal sequence binding. KEEP_AS_NON_CORE: generic protein transport/targeting terms.
Competence:
- comGA: 6 annotations reviewed. AAA+ ATPase powering competence pseudopilus assembly. NEW: GO:0060187 (cell pole localization), GO:0009297 (pilus assembly). ACCEPT ATP hydrolysis activity.
Summary of key decisions:
- Sigma factors consistently: REMOVE RNAP catalytic activity, MODIFY TF activity → sigma factor activity
- Metal-binding terms: MODIFY generic metal ion binding → specific zinc/calcium binding where applicable
- Over-general terms (hydrolase activity): MARK_AS_OVER_ANNOTATED when more specific terms exist
- Sporulation terms: Use GO:0034301 (endospore formation) for B. subtilis specificity
2025-12-18
Round 2 complete! All 10 key functional genes validated. Summary of findings:
Transcriptional regulators:
- spo0A: 19 annotations reviewed. Master sporulation phosphorelay response regulator. Key actions: ACCEPT for phosphorelay signal transduction (GO:0000160), ACCEPT for DNA-binding TF activity (GO:0003700). Added supporting_text from publications. REMOVE calcium ion binding (not Ca²⁺-dependent).
- sigF: 13 annotations reviewed. Forespore-specific sigma-70 factor. Key actions: ACCEPT sigma factor activity (GO:0016987), REMOVE DNA-directed RNAP activity (sigma factors don't catalyze RNA synthesis - they confer promoter specificity), MODIFY general TF term to sigma factor activity.
- comK: 11 annotations reviewed. Competence master regulator. All IEA annotations validated.
Cell division proteins:
- ftsZ: 27 annotations reviewed. Tubulin-like GTPase. Key actions: ACCEPT GTPase activity (IDA from PMID:23577149), ACCEPT cell division (EXP), MARK_AS_OVER_ANNOTATED generic protein binding from high-throughput Y2H.
- divIVA: 18 annotations reviewed. Polar landmark/scaffold protein. Key actions: ACCEPT cytoskeletal protein binding, ACCEPT cell pole localization.
- minC: 7 annotations reviewed. FtsZ polymerization inhibitor. All annotations validated.
Industrial enzymes:
- aprE: 14 annotations reviewed. Subtilisin E serine endopeptidase. ACCEPT serine-type endopeptidase activity (GO:0004252).
- amyE: 12 annotations reviewed. Alpha-amylase. ACCEPT alpha-amylase activity (GO:0004556), starch metabolic process.
- sacB: 8 annotations reviewed. Levansucrase with dual activity (sucrose hydrolysis + fructan polymerization). ACCEPT both activities.
Protein secretion:
- secA: 18 annotations reviewed. Sec translocase ATPase. ACCEPT ATPase activity (GO:0016887), protein secretion (GO:0009306).
Minor issues noted:
- Some reviews have warnings for missing supporting_text in reference findings (sacB has 14 warnings, secA has 6 warnings)
- comK has warning about 0% supporting_text in existing annotations
2025-12-17
Starting BACSU project review. fliW was already reviewed and validated - it has comprehensive coverage of the flagellar assembly factor role and partner-switching mechanism with CsrA.
Session progress:
- fliW: Pre-existing complete review
- fliH: Reviewed but has UNDECIDED annotations - PMID:25313396 doesn't mention fliH in abstract/minimal components. Proposed GO:0042030 (ATPase inhibitor activity) based on Salmonella FliH studies.
- fliK: Complete - hook-length control protein. PMID:22730131 directly demonstrates "FliK regulated hook length". Fixed incorrect GO:0009424 (hook CC) - FliK is NOT a structural component.
- fliY: Complete - bifunctional protein with 17 annotations. CheY-P phosphatase activity (IDA from PMID:12920116) well-supported. Removed incorrect GO:0003774 (motor activity) and unsupported PMID:25313396 annotations.
Key finding about PMID:25313396: This paper lists minimal components for FlgM secretion (FliO, FliP, FliQ, FliR, FlhA, FlhB, FliF, FliG, FliK) - notably MISSING fliH, fliY, fliW, swrD. CACAO annotations citing this paper for genes not in the minimal list need scrutiny.
Session 2 progress (completed 2025-12-17):
- gerD: Germinosome scaffold protein for spore germination. Proposed new GO term for "germinosome".
- spo0J: ParB/CTP-dependent DNA-sliding clamp for chromosome segregation. Proposed 6 new annotations.
- spoVAD: SpoVA Ca-DPA channel plug. REMOVED incorrect GO:0016746 (acyltransferase) annotation - fold misprediction.
- swrD: Flagellar motor power enhancer via MotAB stator modulation. REMOVED erroneous PMID:25313396 annotation.
- yddE: NOT uncharacterized! ConE VirB4-like ATPase for ICEBs1 conjugation. Updated to reflect known function.
Key issues identified:
1. PMID:25313396 erroneously cited for swrD (and fliH, fliY, fliW) - paper doesn't mention these genes
2. spoVAD incorrectly annotated as acyltransferase based on thiolase-like fold
3. yddE is well-characterized as ConE but labeled "uncharacterized" in UniProt
Slides
- Slides (Marp source: BACSU-slides.md) — AI generated