identifier: METPO:1000699
label: gram negative
definition: A gram stain in which bacteria do not retain crystal violet dye and appear
  pink or red after staining, indicating a thin peptidoglycan layer and presence of
  an outer membrane.
definition_source: DOI:10.1038/s41579-019-0201-x
trait_category: MORPHOLOGY
term_kind: CLASS
mapping_status: REVIEWED
parent_traits:
- METPO:1000697
synonyms:
- synonym_text: G_negative
  synonym_type: RELATED_SYNONYM
  source: metpo.owl
- synonym_text: negative
  synonym_type: RELATED_SYNONYM
  source: metpo.owl
created_by: Luke Wang
evidence:
- reference: DOI:10.1038/s41579-019-0201-x
  snippet: defining feature of the Gram-negative cell envelope
  notes: Supports the outer membrane as a defining Gram-negative envelope feature.
- reference: PMID:27564552
  snippet: Gram-Negative Bacterium Escherichia coli
  notes: 'Organism example: Escherichia coli is described as Gram-negative.'
canonical_examples:
- taxon_id: NCBITaxon:562
  taxon_label: Escherichia coli
  note: "Model Gram-negative (outer membrane plus thin peptidoglycan)."
  reference: PMID:9278503
causal_graphs:
- graph_id: gram_negative_outer_membrane_dye_loss
  title: Gram-negative outer-membrane dye-loss mechanism
  description: Evidence-backed causal sketch linking Gram-negative staining to outer
    membrane architecture, lipopolysaccharide, thin peptidoglycan, decolorization,
    and counterstain uptake.
  nodes:
  - node_id: gram_negative_trait
    label: gram negative
    node_type: TRAIT
    grounding: METPO:1000699
    description: Gram stain phenotype in which cells lose crystal violet and appear
      pink or red after counterstaining.
  - node_id: outer_membrane
    label: outer membrane
    node_type: CELLULAR_LOCALIZATION
    description: Gram-negative outer membrane outside the peptidoglycan layer.
    grounding: GO:0019867
  - node_id: lipopolysaccharide
    label: lipopolysaccharide
    node_type: CHEMICAL
    description: Glycolipid found in the outer leaflet of Gram-negative outer membranes.
    grounding: CHEBI:16412
  - node_id: thin_peptidoglycan_layer
    label: thin peptidoglycan layer
    node_type: CELLULAR_LOCALIZATION
    description: Thin peptidoglycan layer in the Gram-negative periplasm.
  - node_id: periplasmic_space
    label: periplasmic space
    node_type: CELLULAR_LOCALIZATION
    description: Cell-envelope compartment between inner and outer membranes.
    grounding: GO:0042597
  - node_id: crystal_violet_iodine_complex
    label: crystal violet-iodine complex
    node_type: CHEMICAL
    description: Dye complex removed during decolorization in Gram-negative cells.
  - node_id: counterstain
    label: counterstain
    node_type: CHEMICAL
    description: Safranin or similar stain producing pink/red appearance.
  - node_id: outer_membrane_outer_leaflet
    label: outer membrane outer leaflet
    node_type: CELLULAR_LOCALIZATION
    description: Outer leaflet of the Gram-negative outer membrane, occupied by LPS.
  - node_id: divalent_cations
    label: divalent cations (Mg2+/Ca2+)
    node_type: CHEMICAL
    description: Magnesium and calcium ions that bridge and stabilize LPS packing
      in the OM.
  - node_id: outer_membrane_barrier_function
    label: outer membrane barrier function
    node_type: MOLECULAR_FUNCTION
    description: Low-permeability barrier property of the Gram-negative outer membrane.
  - node_id: lpt_pathway
    label: Lpt pathway
    node_type: PATHWAY
    description: Trans-envelope ATP-driven machinery transporting LPS from the inner
      to outer membrane.
  - node_id: bam_complex
    label: BAM complex
    node_type: GENE_OR_PROTEIN
    description: Beta-barrel assembly machinery (BamA-E) that folds and inserts OMPs
      into the outer membrane.
  - node_id: beta_barrel_omps
    label: beta-barrel outer membrane proteins
    node_type: GENE_OR_PROTEIN
    description: Beta-barrel outer membrane proteins folded and inserted by the BAM
      complex.
  - node_id: mla_pathway
    label: Mla pathway
    node_type: PATHWAY
    description: Retrograde phospholipid transport system maintaining outer membrane
      lipid asymmetry.
  - node_id: outer_membrane_lipid_asymmetry
    label: outer membrane lipid asymmetry
    node_type: QUALITY
    description: Asymmetric lipid distribution (LPS outer / phospholipid inner) of
      the OM.
  - node_id: tol_pal_system
    label: Tol-Pal system
    node_type: GENE_OR_PROTEIN
    description: Envelope-spanning complex safeguarding connectivity of the Gram-negative
      cell envelope.
  - node_id: cell_envelope_connectivity
    label: cell envelope connectivity
    node_type: QUALITY
    description: Maintained connectivity between outer membrane, peptidoglycan, and
      inner membrane.
  edges:
  - subject: outer_membrane
    predicate: defines
    object: gram_negative_trait
    description: The outer membrane is a defining feature of the Gram-negative cell
      envelope.
    evidence:
    - reference: DOI:10.1038/s41579-019-0201-x
      snippet: defining feature of the Gram-negative cell envelope
      notes: Supports outer membrane as the key Gram-negative envelope feature.
    predicate_id: METPO:2007500
  - subject: lipopolysaccharide
    predicate: localized in
    object: outer_membrane
    description: LPS is found on the outer surface of the Gram-negative outer membrane.
    evidence:
    - reference: DOI:10.1038/s41579-019-0201-x
      snippet: lipopolysaccharide exclusively found on the surface
      notes: Supports LPS as a surface-exposed outer-membrane component.
    predicate_id: biolink:located_in
  - subject: thin_peptidoglycan_layer
    predicate: localized in
    object: periplasmic_space
    description: Gram-negative peptidoglycan is a thin layer in the periplasm.
    evidence:
    - reference: DOI:10.1016/j.bbalip.2016.10.010
      snippet: periplasm ... containing a thin layer of peptidoglycan
      notes: Supports thin periplasmic peptidoglycan in Gram-negative envelopes.
    predicate_id: biolink:located_in
  - subject: thin_peptidoglycan_layer
    predicate: fails to retain
    object: crystal_violet_iodine_complex
    description: Thin peptidoglycan and outer-membrane disruption allow dye complex
      loss during decolorization.
    evidence:
    - reference: DOI:10.3109/10520299609117151
      snippet: cell wall ... responsible for retention
      notes: Inference from Gram-positive retention mechanism; thinner Gram-negative
        peptidoglycan contributes to non-retention.
  - subject: counterstain
    predicate: produces
    object: gram_negative_trait
    description: Counterstain gives decolorized Gram-negative cells their pink/red
      appearance.
    evidence:
    - reference: DOI:10.3109/10520299609117151
      snippet: mechanism of the Gram stain
      notes: Supports Gram stain procedure context; counterstain appearance follows
        dye-complex loss.
    predicate_id: METPO:2000202
  - subject: lipopolysaccharide
    predicate: occupies
    object: outer_membrane_outer_leaflet
    description: LPS occupies the outer leaflet of the Gram-negative outer membrane
      (OM asymmetry).
    evidence:
    - reference: DOI:10.1146/annurev-micro-032521-014507
      notes: LPS occupies the outer leaflet while phospholipids occupy the inner leaflet.
  - subject: divalent_cations
    predicate: stabilizes
    object: lipopolysaccharide
    description: Divalent cations (Mg2+/Ca2+) stabilize LPS packing in the OM outer
      leaflet.
    evidence:
    - reference: DOI:10.3390/pathogens13100889
      notes: Divalent cations (Mg2+/Ca2+) stabilize LPS packing.
  - subject: lipopolysaccharide
    predicate: contributes to
    object: outer_membrane_barrier_function
    description: Tight LPS packing with OM proteins creates the impermeable OM barrier.
    evidence:
    - reference: DOI:10.1146/annurev-micro-032521-014507
      notes: The tight packing of LPS and OM proteins creates an impermeable barrier.
    predicate_id: RO:0002326
  - subject: lpt_pathway
    predicate: transports
    object: lipopolysaccharide
    description: The Lpt machinery transports LPS across the periplasm for insertion
      into the OM.
    evidence:
    - reference: DOI:10.1007/s12275-024-00137-w
      notes: LPS transported to the OM by the ATP-driven Lpt machinery; moves LPS
        across periplasm for OM insertion.
    predicate_id: METPO:2000207
  - subject: bam_complex
    predicate: inserts
    object: beta_barrel_omps
    description: The BAM complex folds and inserts beta-barrel OMPs into the outer
      membrane.
    evidence:
    - reference: DOI:10.3390/pathogens13100889
      notes: The Bam complex (BamA-E) folds uOMPs into beta-barrels.
  - subject: mla_pathway
    predicate: maintains
    object: outer_membrane_lipid_asymmetry
    description: The Mla pathway counteracts phospholipid flipping to maintain OM
      lipid asymmetry.
    evidence:
    - reference: DOI:10.3390/pathogens13100889
      notes: Mla system performs retrograde transport to counteract PL flipping to
        the outer leaflet.
  - subject: tol_pal_system
    predicate: safeguards
    object: cell_envelope_connectivity
    description: Tol-Pal safeguards connectivity between the three layers of the Gram-negative
      envelope.
    evidence:
    - reference: DOI:10.1038/s44259-024-00065-0
      notes: Tol-Pal safeguards connectivity between the three layers of the Gram-negative
        cell envelope.
curation_history:
- timestamp: '2026-05-05T01:35:46.903067+00:00'
  curator: seed_from_metpo
  action: SEEDED_FROM_METPO
  changes: imported from data/raw/metpo.owl (CLASS)
  llm_assisted: false
- timestamp: '2026-05-06T14:40:41-07:00'
  curator: codex
  action: CURATED_WITH_ORGANISM_EXAMPLE
  changes: Added Escherichia coli organism example with PMID-backed evidence.
  llm_assisted: true
- timestamp: '2026-05-11T00:00:00-07:00'
  curator: codex
  action: CURATED_WITH_LITERATURE
  changes: Replaced PMID definition source with DOI-backed Gram-negative envelope
    source and added causal graph for outer membrane, lipopolysaccharide, periplasmic
    thin peptidoglycan, dye loss, and counterstaining.
  llm_assisted: true
- timestamp: '2026-05-20T03:35:31Z'
  curator: claude
  action: GROUND_CAUSAL_PREDICATES
  changes: Grounded 1 causal-edge predicate_id field(s) via mappings/predicate_grounding.tsv
    (METPO:2000202×1).
  llm_assisted: true
- timestamp: '2026-05-23T08:21:50Z'
  curator: claude
  action: GROUND_CAUSAL_PREDICATES
  changes: Grounded 2 causal-edge predicate_id field(s) via mappings/predicate_grounding.tsv
    (biolink:located_in×2).
  llm_assisted: true
- timestamp: '2026-05-24T04:38:00Z'
  curator: claude
  action: GROUND_CAUSAL_NODES
  changes: Grounded 2 causal-node grounding field(s) via mappings/node_grounding.tsv
    (GO:0019867×1, GO:0042597×1).
  llm_assisted: true
- timestamp: '2026-05-24T07:37:40Z'
  curator: claude
  action: GROUND_CAUSAL_PREDICATES
  changes: Grounded 1 causal-edge predicate_id field(s) via mappings/predicate_grounding.tsv
    (METPO:2007500×1).
  llm_assisted: true
- timestamp: '2026-06-14T05:51:46Z'
  curator: claude
  action: GROUND_CAUSAL_NODES
  changes: Grounded 1 causal-node grounding field(s) via mappings/node_grounding.tsv
    (CHEBI:16412×1).
  llm_assisted: true
- timestamp: '2026-06-23T00:00:00Z'
  curator: claude
  action: ENRICH_CAUSAL_GRAPH
  changes: Added 7 evidence-backed generic edges (10 new nodes) from the deep-research
    report.
  llm_assisted: true
- timestamp: '2026-06-24T17:21:13Z'
  curator: claude
  action: GROUND_CAUSAL_PREDICATES
  changes: Grounded 1 causal-edge predicate_id field(s) via mappings/predicate_grounding.tsv
    (RO:0002326×1).
  llm_assisted: true
- timestamp: '2026-06-24T17:23:39Z'
  curator: claude
  action: GROUND_CAUSAL_PREDICATES
  changes: Grounded 1 causal-edge predicate_id field(s) via mappings/predicate_grounding.tsv
    (METPO:2000207×1).
  llm_assisted: true