Bacillus subtilis project

Bacillus subtilis project

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

Round 2 - Key functional genes

Round 3 - Sporulation cascade completion + biotechnology

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