id: MODULE:bacterial_heptaprenylglyceryl_ether_lipid title: Bacterial heptaprenylglyceryl ether-lipid (Ac-HepG) pathway description: >- The heptaprenylglyceryl ether-lipid pathway characterized in Bacillus subtilis, with homologous PcrB chemistry demonstrated in other Caryophanales. It produces heptaprenylglycerol and its acetylated form (Ac-HepG). sn-Glycerol 1-phosphate (G1P) is condensed with all-trans-heptaprenyl diphosphate by the group I GGGPS-family enzyme PcrB to form an ether bond, giving 3-heptaprenyl-sn-glycero-1-phosphate (HepGP); HepGP is dephosphorylated by a phosphatase that has not been assigned to a gene, and the resulting heptaprenylglycerol (HepG) is O-acetylated from acetyl-CoA by YvoF. The product Ac-HepG is not elaborated into a bulk membrane lipid as in Archaea: it is a low-abundance lipophilic acetyl carrier that delivers acetyl groups to the extracytoplasmic face of the membrane, where LhaT (formerly YpjA) transfers them to the alpha-amino group of the N-terminal cysteine of mature diacylglyceryl lipoproteins. The module is of interest because a bacterium here uses three features of archaeal membrane chemistry at once - a G1P backbone, an ether linkage and an isoprenoid chain - to build a carrier cofactor rather than a membrane. Lipoprotein diacylglyceryl transfer and signal-peptide cleavage, which produce LhaT's acceptor, are modelled separately in bacterial_lipoprotein_maturation; isoprenoid (HepPP) supply and the downstream TLR2 immunology are outside the boundary. status: COMPLETE scope: CONCRETE references: - id: PMID:34046426 title: 'Mini Review: Bacterial Membrane Composition and Its Modulation in Response to Stress.' full_text_unavailable: false reference_review: review_notes: Full cached review read. Supports the upstream isoprenoid branch and processing sequence; the historical unknown-function statement is superseded by PMID40626731. relevance: HIGH correctness: VERIFIED findings: - statement: The 2021 review places HepST/HepPP supply upstream of several branches, and summarizes PcrB, an unassigned phosphatase and YvoF. Its unknown-function wording predates the 2025 lipoprotein-carrier finding; it does not refute that finding. supporting_text: Finally, heptaprenyl-PP can be coupled to glycerol-1-phosphate by PcrB, and then further processed by an unidentified phosphatase and the YvoF acetyltranferase to generate an ether linked lipid of unknown function - id: PMID:35976526 title: The catalytic and structural basis of archaeal glycerophospholipid biosynthesis. full_text_unavailable: false reference_review: review_notes: Full cached structural review read. Useful comparative synthesis of C35 PcrB and C20 GGGPS. The scope of each primary protein/structure is checked separately. relevance: HIGH correctness: VERIFIED findings: - statement: The structural review separates C35 PcrB group Ib from C20 GGGPS chemistry. Shared fold or G1P recognition alone cannot establish HepGP synthesis. supporting_text: Members of the PcrB sub-family catalyze the transfer of heptaprenyl diphosphate moieties (C35) to G1P forming heptaprenylglycerol phosphate in contrast to the shorter tetraprenyl moieties (C20) transferred by GGGPS - id: PMID:36941124 title: Four billion years of microbial terpenome evolution. full_text_unavailable: true reference_review: review_notes: Review located; only abstract cached and retrievable directly. PMID37455337 is its correction. Used solely for broad terpenoid context, not step activity, clade placement or native pathway sufficiency. relevance: LOW correctness: VERIFIED - id: PMID:37455337 title: 'Correction to: four billion years of microbial terpenome evolution.' full_text_unavailable: true reference_review: review_notes: Correction linked by PubMed to PMID36941124. Cached record contains metadata rather than the correction body; no corrected figure or evolutionary claim is relied on. relevance: LOW correctness: UNVERIFIED - id: PMID:40626731 title: 'Lipoprotein N-terminal modification in Bacillus: a new paradigm for extracellular acetylation and species-dependent Toll-like receptor 2 immunomodulation.' full_text_unavailable: false reference_review: review_notes: Full text read. Native B. subtilis genetics supports the carrier role; biochemical reconstitution uses a C20 analogue and heterologous LhaT membranes. G1P supply, native phosphatase identity, transport and donor regioisomer preference remain unresolved. relevance: HIGH correctness: VERIFIED findings: - statement: The reconstitution supplied a C20 surrogate donor, so evidence for the native C35 pathway also depends on genetics and earlier native lipid chemistry. supporting_text: We substituted the commercially available geranylgeranyl pyrophosphate (GGPP) (four isoprenyl repeats) for the native heptaprenyl pyrophosphate (seven isoprenyl repeats) carrier, as both PcrB and YvoF can accept the shorter substrate albeit with less efficiency (30). - id: PMID:24684232 title: A comprehensive analysis of the geranylgeranylglyceryl phosphate synthase enzyme family identifies novel members and reveals mechanisms of substrate specificity and quaternary structure organization. full_text_unavailable: false reference_review: review_notes: Cached abstract supplemented by independently accessed publisher full text (https://onlinelibrary.wiley.com/doi/full/10.1111/mmi.12596). Figure 2 assays long-chain substrate use in B. subtilis deletion-host complementation. Host-supplied processing does not establish complete native pathways in Geobacillus, Staphylococcus or Flavobacterium. Official PDB mappings independently establish the compared protein accessions. relevance: HIGH correctness: VERIFIED - id: PMID:23322418 title: 'Insights into TIM-barrel prenyl transferase mechanisms: crystal structures of PcrB from Bacillus subtilis and Staphylococcus aureus.' full_text_unavailable: false reference_review: review_notes: Full cached primary study read; B. subtilis and S. aureus Mu3 are source proteins, E. coli is the expression host. Provides comparative PcrB structural and enzymological evidence. relevance: HIGH correctness: VERIFIED - id: PMID:42286214 title: Bacterial lipid synthesizing enzymes PlsY and PlsC utilize both stereo-forms of glycerol-phosphate. full_text_unavailable: false reference_review: review_notes: Full cached primary paper distinguishes G1P-containing ester lipids from ether-lipid synthesis. Its discussion cites earlier engineered E. coli without AraM; the paper does not identify a new G1P-producing enzyme. relevance: HIGH correctness: VERIFIED - id: PMID:18558723 title: 'Identification and characterization of a bacterial glycerol-1-phosphate dehydrogenase: Ni(2+)-dependent AraM from Bacillus subtilis.' full_text_unavailable: true reference_review: relevance: HIGH correctness: VERIFIED review_notes: Cached primary abstract plus separately documented author-thesis evidence support purified Ni-dependent G1PDH. Neither substrate Km asymmetry nor exogenous G1P feeding establishes native EgsA-to-PcrB flux. - id: PMID:21761520 title: Functional assignment of an enzyme that catalyzes the synthesis of an archaea-type ether lipid in bacteria. full_text_unavailable: true reference_review: relevance: HIGH correctness: VERIFIED review_notes: Cached primary abstract establishes PcrB HepGP formation and subsequent processing. Cross-species enzyme-capacity experiments are distinguished from native complete pathways using the accessible 2014 follow-up. findings: - statement: PcrB is the Bacillales heptaprenylglyceryl phosphate synthase, and its product is dephosphorylated and acetylated in vivo; this establishes the ether-bond-forming step and the existence of the two processing steps. supporting_text: PcrB, the bacterial homologue of the archaea-specific geranylgeranylglyceryl phosphate synthase, produces heptaprenylglyceryl phosphate in bacillales. The product becomes dephosphorylated and acetylated in vivo. - id: PMID:27226549 title: Identification and Characterization of Heptaprenylglyceryl Phosphate Processing Enzymes in Bacillus subtilis. full_text_unavailable: true reference_review: relevance: HIGH correctness: VERIFIED review_notes: Cached primary abstract supports enriched but unassigned phosphatase, YvoF identification and failure of maltose acetyltransferase to complement. A separately documented indexed Results passage supports membrane-associated YvoF activity; complete full-text access remained unavailable. findings: - statement: 'Defines the module boundary downstream of PcrB: in Bacillales HepGP is dephosphorylated and acetylated rather than receiving a second polyprenyl chain as in archaeal di-ether lipids.' supporting_text: In Bacillales, heptaprenylglyceryl phosphate does not become linked to a second polyprenyl moiety like ether lipids in Archaea but is dephosphorylated and acetylated. - statement: The dephosphorylation step was enriched as an activity but never assigned to a gene product, which is why this module models it with an abstract ANY_WITH_FUNCTION participant. supporting_text: We enriched the phosphatase activity from a B. subtilis cell extract and suppose that dephosphorylation is catalyzed by the phosphatase PhoB or by any other phosphatase in an unspecific manner. - id: PMID:21214173 title: Two distinct mechanisms for TIM barrel prenyltransferases in bacteria. full_text_unavailable: false reference_review: relevance: HIGH correctness: VERIFIED review_notes: Cached full primary supports G1P acceptor specificity and in vitro use of shorter prenyl donors. Those assays are substrate-range evidence, not additional native pathway variants. - id: PMID:22614947 title: Dimerization determines substrate specificity of a bacterial prenyltransferase. full_text_unavailable: true reference_review: relevance: HIGH correctness: UNVERIFIED review_notes: Cached primary abstract supports a relationship between dimerization and long-chain substrate use. An erratum is indexed; its content is not yet verified, so no detailed residue or interface mapping is newly asserted from this source. evidence: - source_id: PMID:34046426 title: 'Mini Review: Bacterial Membrane Composition and Its Modulation in Response to Stress.' statement: The 2021 review places HepST/HepPP supply upstream of several branches, and summarizes PcrB, an unassigned phosphatase and YvoF. Its unknown-function wording predates the 2025 lipoprotein-carrier finding; it does not refute that finding. supporting_text: Finally, heptaprenyl-PP can be coupled to glycerol-1-phosphate by PcrB, and then further processed by an unidentified phosphatase and the YvoF acetyltranferase to generate an ether linked lipid of unknown function - source_id: PMID:35976526 title: The catalytic and structural basis of archaeal glycerophospholipid biosynthesis. statement: The structural review separates C35 PcrB group Ib from C20 GGGPS chemistry. Shared fold or G1P recognition alone cannot establish HepGP synthesis. supporting_text: Members of the PcrB sub-family catalyze the transfer of heptaprenyl diphosphate moieties (C35) to G1P forming heptaprenylglycerol phosphate in contrast to the shorter tetraprenyl moieties (C20) transferred by GGGPS - source_id: PMID:40626731 title: 'Lipoprotein N-terminal modification in Bacillus: a new paradigm for extracellular acetylation and species-dependent Toll-like receptor 2 immunomodulation.' statement: The reconstitution supplied a C20 surrogate donor, so evidence for the native C35 pathway also depends on genetics and earlier native lipid chemistry. supporting_text: We substituted the commercially available geranylgeranyl pyrophosphate (GGPP) (four isoprenyl repeats) for the native heptaprenyl pyrophosphate (seven isoprenyl repeats) carrier, as both PcrB and YvoF can accept the shorter substrate albeit with less efficiency (30). - source_id: PMID:21761520 title: >- Functional assignment of an enzyme that catalyzes the synthesis of an archaea-type ether lipid in bacteria. statement: >- PcrB is the Bacillales heptaprenylglyceryl phosphate synthase, and its product is dephosphorylated and acetylated in vivo; this establishes the ether-bond-forming step and the existence of the two processing steps. supporting_text: >- PcrB, the bacterial homologue of the archaea-specific geranylgeranylglyceryl phosphate synthase, produces heptaprenylglyceryl phosphate in bacillales. The product becomes dephosphorylated and acetylated in vivo. - source_id: PMID:27226549 title: >- Identification and Characterization of Heptaprenylglyceryl Phosphate Processing Enzymes in Bacillus subtilis. statement: >- Defines the module boundary downstream of PcrB: in Bacillales HepGP is dephosphorylated and acetylated rather than receiving a second polyprenyl chain as in archaeal di-ether lipids. supporting_text: >- In Bacillales, heptaprenylglyceryl phosphate does not become linked to a second polyprenyl moiety like ether lipids in Archaea but is dephosphorylated and acetylated. - source_id: PMID:27226549 title: >- Identification and Characterization of Heptaprenylglyceryl Phosphate Processing Enzymes in Bacillus subtilis. statement: >- The dephosphorylation step was enriched as an activity but never assigned to a gene product, which is why this module models it with an abstract ANY_WITH_FUNCTION participant. supporting_text: >- We enriched the phosphatase activity from a B. subtilis cell extract and suppose that dephosphorylation is catalyzed by the phosphatase PhoB or by any other phosphatase in an unspecific manner. - source_id: PMID:40626731 title: >- Lipoprotein N-terminal modification in Bacillus: a new paradigm for extracellular acetylation and species-dependent Toll-like receptor 2 immunomodulation. statement: >- Gives the four-step chain used as the module's parts, with the dephosphorylation step still unassigned to a gene. supporting_text: >- PcrB condenses sn-glycerol 1-phosphate (G1P) with heptaprenyl pyrophosphate (HepPP) to form heptaprenylglyceryl phosphate (28, 29), which is subsequently dephosphorylated by a non-specific phosphatase prior to acetylation by YvoF to form acetylated heptaprenylglyceryl (Ac-HepG) with acetyl-coenzyme A (Ac-CoA) as donor (27). - source_id: PMID:40626731 title: >- Lipoprotein N-terminal modification in Bacillus: a new paradigm for extracellular acetylation and species-dependent Toll-like receptor 2 immunomodulation. statement: >- The sequence from prenyl transfer through lipoprotein acetylation was reconstituted using PcrB, calf intestinal alkaline phosphatase, YvoF and LhaT-containing E. coli membranes. The assay supplied G1P and substituted a C20 geranylgeranyl donor for the native C35 donor; it neither tests EgsA supply nor establishes native C35 flux or membrane crossing. supporting_text: >- While the entire pathway could be reconstituted in vitro with just PcrB, YvoF, a non-specific phosphatase, and LhaT (YpjA) (Fig. 3), it is presently unclear how Ac-HepG/HepG transverses the membrane - source_id: PMID:40626731 title: >- Lipoprotein N-terminal modification in Bacillus: a new paradigm for extracellular acetylation and species-dependent Toll-like receptor 2 immunomodulation. statement: >- Genetic requirement in vivo: loss of pcrB, yvoF or ypjA (lhaT) abolishes lipoprotein N-acetylation, which is what licenses treating the carrier pathway and its consumer as one functional unit. supporting_text: >- Only pcrB, yvoF, and ypjA fully suppressed TLR2 activity, indicating these genes are strictly required for Ac-LP synthesis. - source_id: PMID:40626731 title: >- Lipoprotein N-terminal modification in Bacillus: a new paradigm for extracellular acetylation and species-dependent Toll-like receptor 2 immunomodulation. statement: >- The pathway's product is a catalytic acetyl carrier, not a structural membrane lipid; this is the reason the module is typed as a metabolic pathway for a cofactor rather than as membrane-lipid biosynthesis. supporting_text: >- A catalytic role as a transient carrier for acetyl groups, as opposed to being a structural membrane building block for an archeal-type lipid with ether-linked isoprenoid chains, is more consistent with the low overall abundance of HepG/Ac-HepG observed in B. subtilis membranes (28). - source_id: PMID:24684232 title: >- A comprehensive analysis of the geranylgeranylglyceryl phosphate synthase enzyme family identifies novel members and reveals mechanisms of substrate specificity and quaternary structure organization. statement: >- Family scoping for the ether-bond step: only group I bacterial members of the GGGPS family are heptaprenylglyceryl phosphate synthases, so the module's family grounding is the HepGP synthase subfamily, not GGGPS as a whole. supporting_text: >- the previously uncharacterized bacterial enzymes from group II have GGGPS activity like the archaeal enzymes and differ from the bacterial group I enzymes that are heptaprenylglyceryl phosphate synthases - source_id: file:modules/bacterial_lipoprotein_maturation.yaml title: Bacterial lipoprotein maturation module statement: >- Adjacent module, not duplicated here. It models Lgt diacylglyceryl transfer and LspA signal-peptide cleavage, which generate the diacylglyceryl lipoprotein (DA-LP) that LhaT acetylates, and it already records that the terminal modification of the mature lipoprotein is lineage-variable rather than always Lnt-type N-acylation. - source_id: file:genes/BACSU/pcrB/pcrB-notes.md title: BACSU pcrB curation notes statement: >- Worked evidence for the PcrB step, including the strict G1P acceptor specificity, the Mg2+ requirement, the decision to keep GO:0046474 and add GO:0008611 beside it rather than choose between them (GO:0008611 is not a descendant of GO:0046474, so replacing would discard a true claim), and the carrier (not membrane-lipid) reading of the pathway's purpose. - source_id: file:genes/BACSU/egsA/egsA-notes.md title: BACSU egsA curation notes statement: >- Worked evidence for the G1P-supply step, including purified AraM/EgsA activity and the unresolved assignment of native G1P supply to PcrB. Kinetic substrate preferences do not establish physiological direction or pathway flux. notes: >- See bacterial_heptaprenylglyceryl_ether_lipid-notes.md for the boundary argument, the GO term gaps found (no term for archaeal-type isoprenoid-ether lipid biosynthesis, for a prenyl-lipid acetyl-carrier cycle, or for heptaprenylglyceryl phosphate phosphatase activity), the reasons the PcrB and EgsA PANTHER/PAINT ancestral nodes were left unasserted, and the expected QC notices. Comparative reviews distinguish the BACSU carrier pathway from GEOKA and STAA1 PcrB step evidence and the FLAJ1 C20-GGGPS comparator. Carrier-pathway activity in another species requires evidence beyond a family match; native G1P supply and the phosphatase remain unassigned. module: id: bacterial_heptaprenylglyceryl_ether_lipid label: Bacterial heptaprenylglyceryl ether-lipid (Ac-HepG) pathway module_type: METABOLIC_PATHWAY description: >- Required G1P supply followed by three carrier-synthesis steps from G1P and heptaprenyl diphosphate to Ac-HepG, plus the optional terminal step in which the carried acetyl group is handed to a lipoprotein. The first four steps run on the cytoplasmic side; the last is catalysed by an integral membrane enzyme acting on an extracytoplasmic acceptor. concepts: - preferred_term: ether lipid biosynthetic process term: id: GO:0008611 label: ether lipid biosynthetic process description: >- The only GO process term that captures what this pathway does: it forms a single ether linkage between glycerol and a lipid alcohol (here an isoprenoid). GO has no term for archaeal-type (G1P, isoprenoid-ether) lipid biosynthesis, and none for a prenyl-lipid acetyl-carrier cycle. context: taxa: - preferred_term: Bacillales term: id: NCBITaxon:1385 label: Caryophanales description: >- Caryophanales is the comparative scope of the PcrB exemplars, not a claim that every member carries the complete pathway. Native carrier synthesis and lipoprotein acetylation are grounded in B. subtilis; Geobacillus kaustophilus and Staphylococcus aureus supply direct PcrB evidence only. C20-GGGPS in Flavobacterium johnsoniae lies outside this scope and is a specificity comparator. Broader G1PDH or LhaT family occurrence does not establish native pathway capacity. cellular_components: - preferred_term: cytoplasm term: id: GO:0005737 label: cytoplasm description: >- G1P formation, ether-bond formation, dephosphorylation and acetylation are cytoplasmic/cytoplasmic-face reactions. The membrane location of the terminal acetyl-transfer step is asserted on that annoton rather than here. conditions: - preferred_term: low-abundance carrier lipid pool description: >- HepG and Ac-HepG are minor membrane lipids in B. subtilis; the module describes a catalytic carrier cycle, not bulk membrane-lipid biosynthesis. parts: - order: 1 role: sn-glycerol 1-phosphate supply node: id: g1p_supply label: sn-Glycerol 1-phosphate supply module_type: REACTION description: >- Provision of the G1P acceptor. PcrB accepts G1P and not G3P, so a source of the archaeal-type glycerophosphate enantiomer is required. The only characterized bacterial route is NAD(P)H-dependent reduction of glycerone phosphate (DHAP) by a glycerol-1-phosphate dehydrogenase (EgsA/AraM). Whether that enzyme is in fact the in vivo source of the G1P used by PcrB has not been shown, so the step is modelled as a required activity with one known realizer rather than as a proven EgsA-to-PcrB flux. variant_sets: - id: g1p_supply_route label: Enzymes that can supply sn-glycerol 1-phosphate axis: G1P-supplying enzyme variant_kind: REALIZER selection: ONE_OR_MORE notes: >- Declared as a REALIZER set with a single modelled carrier on purpose: engineered E. coli produced correctly configured G1P-backbone archaeal lipids without AraM, so at least one further route to G1P exists and is not identifiable. A gap at this step should therefore be read as "activity required, carrier not identified", not as "no route exists". evidence: - source_id: PMID:42286214 title: >- Bacterial lipid synthesizing enzymes PlsY and PlsC utilize both stereo-forms of glycerol-phosphate. statement: >- This paper cites earlier engineered E. coli lipid production without AraM and infers an unidentified G1P-supply mechanism. It does not identify that mechanism, discover a second enzyme, or prove its use by native B. subtilis PcrB. supporting_text: >- other mechanism(s) of G1P biosynthesis must exist in E. coli and possibly other bacteria, as archaeal lipids with the correct chiral structure could be produced even in the absence of AraM variants: - id: g1pdh_route label: G1P dehydrogenase (EgsA/AraM) route module_type: REACTION annotons: - id: g1p_dehydrogenase label: "G1P dehydrogenase family: DHAP reduction to sn-glycerol 1-phosphate" participant: selector_type: FAMILY required_function: preferred_term: glycerol-1-phosphate dehydrogenase activity term: id: GO:0050492 label: glycerol-1-phosphate dehydrogenase [NAD(P)+] activity description: >- Required biochemical qualification: PTHR43622:SF1 also contains dehydroquinate and other sugar-phosphate enzymes. Membership alone does not establish G1PDH. family: preferred_term: >- bacterial glycerol-1-phosphate dehydrogenase (G1PDH, EgsA/AraM) family description: >- PANTHER places EgsA in a heterogeneous subfamily that also contains dehydroquinate and other sugar-phosphate enzymes; the official name is retained verbatim in `term.label` and the intended grouping is given in `preferred_term`. The curated family statement for the exemplar is UniProt's "glycerol-1-phosphate dehydrogenase family" (HAMAP-Rule MF_00497_B, InterPro IPR023003). term: id: PANTHER:PTHR43622:SF1 label: 3-DEHYDROQUINATE SYNTHASE representative_members: - preferred_term: egsA/araM (Bacillus subtilis exemplar) term: id: UniProtKB:P94527 label: Glycerol-1-phosphate dehydrogenase [NAD(P)+] description: >- Ni2+-dependent homodimeric G1PDH, EC 1.1.1.261, with measured reversible NADH-linked G1P/DHAP interconversion; native pathway flux is unresolved. function: preferred_term: glycerol-1-phosphate dehydrogenase [NAD(P)+] activity term: id: GO:0050492 label: glycerol-1-phosphate dehydrogenase [NAD(P)+] activity description: >- Purified AraM reduces DHAP using NADH and also accepts NADPH less efficiently. The reaction is drawn in the G1P-supplying direction needed by PcrB; this is a biochemical capability, not measured native flux. Cofactor-specific children are GO:0106357 (NAD+) and GO:0106358 (NADP+); native cofactor use is unresolved. substrates: - preferred_term: glycerone phosphate (dihydroxyacetone phosphate) term: id: CHEBI:57642 label: glycerone phosphate(2-) - preferred_term: NADH term: id: CHEBI:57945 label: NADH(2-) products: - preferred_term: sn-glycerol 1-phosphate term: id: CHEBI:57685 label: sn-glycerol 1-phosphate(2-) - preferred_term: NAD(+) term: id: CHEBI:57540 label: NAD(1-) cofactors: - preferred_term: nickel(2+) term: id: CHEBI:49786 label: nickel(2+) description: >- The B. subtilis enzyme is active with Ni2+ and not with other divalent cations tested. evidence: - source_id: RHEA:21412 title: RHEA:21412 statement: >- Balanced NAD+-linked reaction for G1P/glycerone phosphate interconversion, cross-referenced from the P94527 UniProt entry. - source_id: PMID:18558723 title: >- Identification and characterization of a bacterial glycerol-1-phosphate dehydrogenase: Ni(2+)-dependent AraM from Bacillus subtilis. statement: >- Direct enzymology for the reductive direction on the purified B. subtilis protein. supporting_text: >- the purified protein forms a homodimer that reversibly reduces dihydroxyacetone phosphate (DHAP) to glycerol-1-phosphate (G1P) in a NADH-dependent manner processes: - preferred_term: ether lipid biosynthetic process term: id: GO:0008611 label: ether lipid biosynthetic process description: >- Asserted as the module-level process context for this step rather than as a claim about the gene's own dedicated role: the same G1P pool may also feed ester-linked G1P-backbone phosphatidylglycerol, and no egsA-deletion lipidomics ties this enzyme's output to the HepG pool. locations: - preferred_term: cytoplasm term: id: GO:0005737 label: cytoplasm description: >- Soluble protein with no transmembrane segment; UniProt's cytoplasm assignment is a curator inference (ECO:0000305). role_description: >- Supplies the archaeal-type glycerophosphate enantiomer that the ether-bond-forming step requires. Only known bacterial enzyme for the reaction; not demonstrated to be the in vivo source for PcrB. knowledge_gaps: - gap_statement: >- It is not established which enzyme actually supplies the sn-glycerol 1-phosphate that PcrB uses in vivo. No egsA/araM-deletion lipidomics has been reported showing loss of HepG/Ac-HepG, egsA was not among the reported hits of the transposon screen for loss of lipoprotein N-acetylation, and at least one further bacterial route to G1P exists. boundary: >- Firmly established: purified AraM/EgsA reversibly reduces DHAP to G1P with NADH, without establishing physiological direction or flux from substrate Km values, and it is a characterized bacterial G1P-forming enzyme. Also established: B. subtilis contains G1P-backbone lipids, and PcrB accepts G1P exclusively. gap_kind: - BIOLOGY dark_aspect: BP_DARK status: OPEN significance: >- This is the join between the "archaeal" backbone chemistry and the ether-lipid pathway. Until it is closed, the pathway's first step is an activity requirement rather than an assigned gene, and a GO process annotation linking a G1P dehydrogenase to ether-lipid biosynthesis rests on inference from pathway logic. resolution: >- Lipidomics of an egsA/araM deletion (and of a complemented strain) measuring HepG, Ac-HepG and G1P-backbone phosphatidylglycerol; isotopic tracing from DHAP to the HepG pool. provenance: - reference_id: PMID:42286214 supporting_text: >- other mechanism(s) of G1P biosynthesis must exist in E. coli and possibly other bacteria, as archaeal lipids with the correct chiral structure could be produced even in the absence of AraM - reference_id: PMID:18558723 supporting_text: >- we propose that AraM generates G1P for the synthesis of phosphoglycerolipids in Gram-positive bacterial species full_text_unavailable: true - reference_id: file:genes/BACSU/egsA/egsA-notes.md - order: 2 role: Ether-bond formation between G1P and the isoprenoid chain node: id: ether_bond_formation label: Heptaprenylglyceryl phosphate synthesis (PcrB) module_type: REACTION description: >- PcrB, a homodimeric Mg2+-dependent group I GGGPS-family prenyltransferase, transfers the C35 heptaprenyl moiety of all-trans-heptaprenyl diphosphate to the C3 hydroxyl of sn-glycerol 1-phosphate, forming the ether bond and releasing diphosphate. This is the committed and name-giving step of the module. annotons: - id: hepgp_synthase label: "HepGP synthase subfamily: G1P heptaprenyltransferase" participant: selector_type: FAMILY family: preferred_term: >- bacterial group I GGGPS-family heptaprenylglyceryl phosphate synthase (PcrB) subfamily description: >- Group I bacterial members of the GGGPS family are HepGP synthases; group II bacterial members are GGGP synthases, so the subfamily rather than the family is the correct grounding for this activity. UniProt grounds the exemplar further with HAMAP-Rule MF_00112 and Pfam PF01884 (PcrB). term: id: PANTHER:PTHR40029:SF2 label: HEPTAPRENYLGLYCERYL PHOSPHATE SYNTHASE representative_members: - preferred_term: pcrB (Bacillus subtilis exemplar) term: id: UniProtKB:O34790 label: Heptaprenylglyceryl phosphate synthase description: >- EC 2.5.1.n9; homodimer, and dimerization is what allows the long C35 donor to be accepted. Crystal structures with bound G1P (PDB 3VZY, 3W00). - preferred_term: pcrB (Geobacillus kaustophilus HTA426) term: id: UniProtKB:Q5L3C1 label: Heptaprenylglyceryl phosphate synthase description: >- PcrB representative from another bacillaceous genus; G1P-bound structure 4NAE and companion structure 4NAF map to this accession (PMID:24684232). This is evidence for the prenyltransferase step, not native completion of the carrier or lipoprotein acetylation pathway. - preferred_term: pcrB (Staphylococcus aureus Mu3) term: id: UniProtKB:A7X435 label: Heptaprenylglyceryl phosphate synthase description: >- Staphylococcaceae exemplar from the comparative PcrB structural study (PMID:23322418; PDB 3W01/3W02). E. coli is the expression host, not the protein's source species. No complete native Ac-HepG/LhaT route is inferred from the purified PcrB experiments. function: preferred_term: heptaprenylglyceryl phosphate synthase activity term: id: GO:0120536 label: heptaprenylglyceryl phosphate synthase activity substrates: - preferred_term: sn-glycerol 1-phosphate term: id: CHEBI:57685 label: sn-glycerol 1-phosphate(2-) description: >- Strictly the sn-1 enantiomer; G3P and 3-phosphoglycerate are not accepted. - preferred_term: all-trans-heptaprenyl diphosphate term: id: CHEBI:58206 label: all-trans-heptaprenyl diphosphate(3-) description: >- The native C35 donor. Shorter C10/C20 donors are used in vitro with lower efficiency; that is in vitro substrate range, not a second physiological reaction, and it is not modelled as a variant. products: - preferred_term: 3-heptaprenyl-sn-glycero-1-phosphate (HepGP) term: id: CHEBI:64781 label: 3-heptaprenyl-sn-glycero-1-phosphate(2-) - preferred_term: diphosphate term: id: CHEBI:33019 label: diphosphate(3-) cofactors: - preferred_term: magnesium(2+) term: id: CHEBI:18420 label: magnesium(2+) description: Required for catalysis; no product forms with EDTA or without MgCl2. evidence: - source_id: PMID:35976526 title: The catalytic and structural basis of archaeal glycerophospholipid biosynthesis. statement: The structural review separates C35 PcrB group Ib from C20 GGGPS chemistry. Shared fold or G1P recognition alone cannot establish HepGP synthesis. supporting_text: Members of the PcrB sub-family catalyze the transfer of heptaprenyl diphosphate moieties (C35) to G1P forming heptaprenylglycerol phosphate in contrast to the shorter tetraprenyl moieties (C20) transferred by GGGPS - source_id: PMID:24684232 title: A comprehensive analysis of the geranylgeranylglyceryl phosphate synthase enzyme family identifies novel members and reveals mechanisms of substrate specificity and quaternary structure organization. statement: The comparative study tests long-chain use in a B. subtilis deletion host supplied with G1P. C35 products for group Ib enzymes establish PcrB capacity; host-provided donor synthesis, dephosphorylation and acetylation do not prove native complete pathways in the donor species. F. johnsoniae group IIb GGGPS is a C20 specificity comparator, not a representative of this C35 step. - source_id: RHEA:33495 title: RHEA:33495 statement: >- Balanced ether-bond-forming reaction, cross-referenced from the O34790 UniProt entry and from the GO:0120536 definition. - source_id: PMID:21761520 title: >- Functional assignment of an enzyme that catalyzes the synthesis of an archaea-type ether lipid in bacteria. statement: In vivo and in vitro assignment of the HepGP synthase activity to PcrB. supporting_text: >- PcrB, the bacterial homologue of the archaea-specific geranylgeranylglyceryl phosphate synthase, produces heptaprenylglyceryl phosphate in bacillales. - source_id: PMID:21214173 title: Two distinct mechanisms for TIM barrel prenyltransferases in bacteria. statement: "Acceptor specificity: G1P only." supporting_text: PcrB uses G1P (1) exclusively as an acceptor - source_id: PMID:23322418 title: >- Insights into TIM-barrel prenyl transferase mechanisms: crystal structures of PcrB from Bacillus subtilis and Staphylococcus aureus. statement: Mg2+ dependence of catalysis. supporting_text: >- No product was found, indicating that Mg2+ is likely essential for catalysis. - source_id: PMID:22614947 title: Dimerization determines substrate specificity of a bacterial prenyltransferase. statement: >- The homodimer is what permits the native seven-isoprene donor to be used, so quaternary structure is part of the chain-length specificity of this step. supporting_text: >- activity assays showed that monomerization limits the length of accepted polyprenyl pyrophosphates to three isoprene units, whereas the native PcrB substrate contains seven isoprene entities processes: - preferred_term: ether lipid biosynthetic process term: id: GO:0008611 label: ether lipid biosynthetic process description: >- This step forms the ether bond, so GO:0008611 is the apt process term. The existing GOA rows use GO:0046474 glycerophospholipid biosynthetic process, which is formally true of HepGP but describes a transient intermediate rather than the pathway's output. locations: - preferred_term: cytoplasm term: id: GO:0005737 label: cytoplasm role_description: >- Committed step of the module; creates the archaeal-type isoprenoid-ether linkage on a G1P backbone. - order: 3 role: Dephosphorylation of heptaprenylglyceryl phosphate node: id: hepgp_dephosphorylation label: HepGP dephosphorylation to heptaprenylglycerol (unassigned phosphatase) module_type: REACTION description: >- Hydrolysis of the phosphomonoester of HepGP to give the neutral ether lipid heptaprenylglycerol (HepG). The activity was enriched from a B. subtilis cell extract but never assigned to a gene product; PhoB was suspected and not demonstrated, and the authors explicitly allow that any phosphatase may do it unspecifically. The in vitro reconstitution of the whole pathway used calf intestinal alkaline phosphatase in this position. The step is therefore modelled as a required activity with no participant protein; no gene product is asserted. annotons: - id: hepgp_phosphatase label: Unassigned phosphatase acting on heptaprenylglyceryl phosphate participant: selector_type: ANY_WITH_FUNCTION required_function: preferred_term: >- phosphatase activity able to hydrolyse the phosphomonoester of heptaprenylglyceryl phosphate term: id: GO:0016791 label: phosphatase activity description: >- Deliberately broad. The responsible B. subtilis enzyme is unknown, the candidate named in the literature (PhoB) was not confirmed, and the activity may be contributed non-specifically by more than one phosphatase. No substrate-specific GO term (heptaprenylglyceryl phosphate phosphatase activity) exists. description: >- Any B. subtilis phosphatase that can dephosphorylate HepGP. This is an activity requirement, not a gene assignment: the module deliberately leaves the participant unidentified rather than naming PhoB, which the source only suspected. evidence: - source_id: PMID:27226549 title: >- Identification and Characterization of Heptaprenylglyceryl Phosphate Processing Enzymes in Bacillus subtilis. statement: >- The authors enriched the activity but did not assign it; the named candidate is explicitly a supposition. supporting_text: >- We enriched the phosphatase activity from a B. subtilis cell extract and suppose that dephosphorylation is catalyzed by the phosphatase PhoB or by any other phosphatase in an unspecific manner. function: preferred_term: phosphatase activity (on heptaprenylglyceryl phosphate) term: id: GO:0016791 label: phosphatase activity substrates: - preferred_term: 3-heptaprenyl-sn-glycero-1-phosphate (HepGP) term: id: CHEBI:64781 label: 3-heptaprenyl-sn-glycero-1-phosphate(2-) products: - preferred_term: heptaprenylglycerol (HepG) description: >- 3-heptaprenyl-sn-glycerol. No ChEBI entry was found for this compound or for its acetylated form, so these two intermediates carry no ontology grounding. - preferred_term: phosphate term: id: CHEBI:43474 label: hydrogenphosphate processes: - preferred_term: ether lipid biosynthetic process term: id: GO:0008611 label: ether lipid biosynthetic process locations: - preferred_term: cytoplasm term: id: GO:0005737 label: cytoplasm description: >- Inferred from the cytoplasmic placement of the flanking steps rather than measured for this activity. role_description: >- Converts the charged intermediate into the neutral ether lipid that YvoF acetylates. Required for the pathway; carried out by an enzyme that has not been identified. knowledge_gaps: - gap_statement: >- The gene product that dephosphorylates heptaprenylglyceryl phosphate in B. subtilis is unknown. PhoB was suspected on the basis of an enriched activity but not demonstrated, and whether one dedicated enzyme or several non-specific phosphatases carry the step is undetermined. boundary: >- Firmly established: HepGP is dephosphorylated in vivo, the activity is present in B. subtilis cell extract and can be enriched, and the step can be substituted in vitro by a heterologous alkaline phosphatase, so the chemistry and its place in the pathway are not in doubt. gap_kind: - BIOLOGY dark_aspect: MF_DARK status: OPEN significance: >- Without an assigned gene this step cannot be annotated to any protein, and the module has a step that no proteome scan can detect, which affects satisfiability calls in other Bacillales. resolution: >- Assay HepGP dephosphorylation in a phoB deletion and in multiple-phosphatase deletion backgrounds; purify the enriched activity to identity by mass spectrometry. provenance: - reference_id: PMID:27226549 supporting_text: >- We enriched the phosphatase activity from a B. subtilis cell extract and suppose that dephosphorylation is catalyzed by the phosphatase PhoB or by any other phosphatase in an unspecific manner. full_text_unavailable: true - order: 4 role: Acetylation of heptaprenylglycerol node: id: hepg_acetylation label: Heptaprenylglycerol O-acetylation (YvoF) module_type: REACTION description: >- YvoF transfers an acetyl group from acetyl-CoA to a glyceryl hydroxyl of HepG, giving the acetylated carrier Ac-HepG. YvoF was identified by screening a B. subtilis knockout library for loss of acetylation, and is a close relative of maltose O-acetyltransferase; the two overlap partly in vitro but maltose O-acetyltransferase does not complement a yvoF knockout in vivo, so the activity is YvoF's own physiological role. Mono-acetylated species on C1 or C2 and a diacetylated species can all be formed. annotons: - id: hepg_acetyltransferase label: "YvoF-related acetyltransferase subfamily: HepG O-acetyltransferase" participant: selector_type: FAMILY family: preferred_term: YvoF-related hexapeptide-repeat acetyltransferase subfamily description: >- Hexapeptide-repeat (LbetaH) acetyltransferase, Pfam PF00132, closely related to maltose O-acetyltransferase. The subfamily is named for YvoF itself, which is the appropriate grounding level: the parent family contains maltose O-acetyltransferases that do not substitute for YvoF in vivo. term: id: PANTHER:PTHR43300:SF6 label: ACETYLTRANSFERASE YVOF-RELATED representative_members: - preferred_term: yvoF (Bacillus subtilis exemplar) term: id: UniProtKB:O34993 label: Putative acetyltransferase YvoF description: >- EC 2.3.1.-; UniProt still names it "putative", although the acetyltransferase activity on HepG has been demonstrated genetically and biochemically. function: preferred_term: heptaprenylglycerol O-acetyltransferase activity (acetyl-CoA-dependent) term: id: GO:0016413 label: O-acetyltransferase activity description: >- The broadest apt GO term. No substrate-specific term exists for O-acetylation of a prenyl glycerol ether lipid, and the chemistry is transfer to a hydroxyl, not to a nitrogen. substrates: - preferred_term: heptaprenylglycerol (HepG) description: No ChEBI entry found for this compound. - preferred_term: acetyl-CoA term: id: CHEBI:57288 label: acetyl-CoA(4-) products: - preferred_term: acetylated heptaprenylglycerol (Ac-HepG) description: >- Mono-acetylated on C1 or C2 of the glyceryl unit, with a diacetylated species (Ac2-HepG) also observed. No ChEBI entry found. - preferred_term: coenzyme A term: id: CHEBI:57287 label: coenzyme A(4-) evidence: - source_id: PMID:34046426 title: 'Mini Review: Bacterial Membrane Composition and Its Modulation in Response to Stress.' statement: The 2021 review places HepST/HepPP supply upstream of several branches, and summarizes PcrB, an unassigned phosphatase and YvoF. Its unknown-function wording predates the 2025 lipoprotein-carrier finding; it does not refute that finding. supporting_text: Finally, heptaprenyl-PP can be coupled to glycerol-1-phosphate by PcrB, and then further processed by an unidentified phosphatase and the YvoF acetyltranferase to generate an ether linked lipid of unknown function - source_id: PMID:27226549 title: >- Identification and Characterization of Heptaprenylglyceryl Phosphate Processing Enzymes in Bacillus subtilis. statement: >- Genetic identification of YvoF as the acetyltransferase of this pathway, and its acetyl-CoA dependence. supporting_text: >- By screening a B. subtilis knock-out library for deficiency in acetylation, the yvoF gene product was identified to be the acetyltransferase. The acetyl-CoA-dependent enzyme YvoF is a close relative of maltose O-acetyltransferase (MAT). - source_id: PMID:40626731 title: >- Lipoprotein N-terminal modification in Bacillus: a new paradigm for extracellular acetylation and species-dependent Toll-like receptor 2 immunomodulation. statement: >- Recombinant YvoF acetylates the dephosphorylated prenyl glycerol with acetyl-CoA in the reconstituted pathway, using a C20 geranylgeranyl analogue of native C35 HepG. supporting_text: >- which is subsequently dephosphorylated by a non-specific phosphatase prior to acetylation by YvoF to form acetylated heptaprenylglyceryl (Ac-HepG) with acetyl-coenzyme A (Ac-CoA) as donor (27). processes: - preferred_term: ether lipid biosynthetic process term: id: GO:0008611 label: ether lipid biosynthetic process locations: - preferred_term: plasma membrane term: id: GO:0005886 label: plasma membrane description: >- Native activity fractionation supports membrane association (PMID:27226549; indexed Results, documented in the YvoF gene source analysis). Cytoplasmic-side loading follows acetyl-CoA accessibility and the pathway model. The physical attachment is unresolved; no integral-membrane topology is asserted. role_description: >- Loads the carrier. Produces the module's output, the high-energy acetyl donor Ac-HepG. - order: 5 optional: true role: Delivery of the carried acetyl group to a lipoprotein notes: >- Marked optional because PcrB has a broader strain distribution than LhaT, and some species encoding both do not make acetylated lipoprotein. Inactive LhaT alleles are demonstrated in B. anthracis, whereas the precise defect in other B. cereus-group members is unresolved. The carrier steps can therefore run in genomes where this consumer is absent or inactive, and Ac-HepG may have further uses. Keeping the step inside the module (rather than treating it purely as downstream biology) is what makes the pathway's purpose explicit: the module's real product is a transferred acetyl group, and the carrier is regenerated. node: id: lipoprotein_acetyl_transfer label: Lipoprotein N-acetylation from Ac-HepG (LhaT/YpjA) module_type: REACTION description: >- LhaT, a polytopic membrane protein formerly annotated only as uncharacterized YpjA, transfers the acetyl group from Ac-HepG to the alpha-amino group of the N-terminal cysteine of a mature diacylglyceryl lipoprotein, giving acetylated lipoprotein (Ac-LP) and releasing HepG for reloading. The acceptor is produced by Lgt diacylglyceryl transfer and LspA signal-peptide cleavage, which are modelled in bacterial_lipoprotein_maturation and are not repeated here. How Ac-HepG reaches the outer leaflet is unknown. annotons: - id: lipoprotein_n_acetyltransferase label: "LhaT (DUF1405) family: lipoprotein heptaprenylglyceryl N-acetyltransferase" participant: selector_type: FAMILY required_function: preferred_term: lipid-donor-dependent lipoprotein N-acetyltransferase term: id: GO:0008080 label: N-acetyltransferase activity description: >- Requires demonstrated transfer from an acetylated prenyl glycerol lipid to a lipoprotein N terminus. A DUF1405 family match or generic acetyltransferase activity is insufficient. family: preferred_term: >- lipoprotein heptaprenylglyceryl N-acetyltransferase (LhaT/YpjA, DUF1405) family description: >- Pfam PF07187 (DUF1405) integral membrane proteins, predicted to have six transmembrane helices. The N-acetyltransferase function is established for the B. subtilis member; family membership alone does not imply the activity, since LhaT is pseudogenized by poly-tyrosine insertion in Bacillus anthracis isolates and some orthologue-bearing species do not make acetylated lipoprotein. term: id: PANTHER:PTHR40042:SF1 label: DUF1405 DOMAIN-CONTAINING PROTEIN representative_members: - preferred_term: lhaT/ypjA (Bacillus subtilis exemplar) term: id: UniProtKB:P54392 label: Uncharacterized protein YpjA description: >- UniProt still names P54392 "Uncharacterized protein YpjA"; the renaming to LhaT and the assignment of lipoprotein N-acetyltransferase activity are from the 2025 genetic and reconstitution work and have not yet reached the record. function: preferred_term: >- lipoprotein N-terminal N-acetyltransferase activity, using acetylated heptaprenylglycerol as donor term: id: GO:0008080 label: N-acetyltransferase activity description: >- GO:0004596 protein-N-terminal amino-acid acetyltransferase activity was checked and rejected: its definition fixes acetyl-CoA as the donor, whereas LhaT uses the lipid Ac-HepG. GO:0008080 is the nearest term that does not misstate the donor. substrates: - preferred_term: acetylated heptaprenylglycerol (Ac-HepG) description: The acetyl donor. No ChEBI entry found. - preferred_term: diacylglyceryl lipoprotein (DA-LP) description: >- Mature lipoprotein with a free alpha-amino group on the N-terminal cysteine, produced by the Lgt and LspA steps of bacterial_lipoprotein_maturation. products: - preferred_term: N-acetylated lipoprotein (Ac-LP) - preferred_term: heptaprenylglycerol (HepG) description: The discharged carrier, available for reloading by YvoF. targets: - preferred_term: bacterial lipoprotein N-terminal cysteine alpha-amino group evidence: - source_id: PMID:40626731 title: >- Lipoprotein N-terminal modification in Bacillus: a new paradigm for extracellular acetylation and species-dependent Toll-like receptor 2 immunomodulation. statement: >- Native genetic requirement of ypjA. Separate biochemical reconstitution uses an acetylated C20 geranylgeranyl analogue and a model lipopeptide, not purified native C35 donor with a resolved acetylation regioisomer. supporting_text: >- Only pcrB, yvoF, and ypjA fully suppressed TLR2 activity, indicating these genes are strictly required for Ac-LP synthesis. - source_id: PMID:40626731 title: >- Lipoprotein N-terminal modification in Bacillus: a new paradigm for extracellular acetylation and species-dependent Toll-like receptor 2 immunomodulation. statement: >- Topology of the step: the donor is made in the cytoplasm and used by a membrane enzyme acting on an extracytoplasmic acceptor. supporting_text: >- The Ac-HepG extracellular acetyl donor is then shuttled across the membrane by an unidentified process where it is utilized by LhaT (YpjA) processes: - preferred_term: N-terminal protein amino acid acetylation term: id: GO:0006474 label: N-terminal protein amino acid acetylation - preferred_term: lipoprotein biosynthetic process term: id: GO:0042158 label: lipoprotein biosynthetic process description: >- The terminal N-terminal modification of a mature bacterial lipoprotein; the shared process term with the Lgt/LspA steps in bacterial_lipoprotein_maturation. locations: - preferred_term: plasma membrane term: id: GO:0005886 label: plasma membrane description: >- Polytopic membrane protein (six predicted transmembrane helices) acting on an acceptor on the outer leaflet. role_description: >- Consumes the carrier's acetyl group and regenerates HepG, which is what makes the upstream steps a catalytic cycle rather than a lipid-accumulating pathway. knowledge_gaps: - gap_statement: >- How Ac-HepG (and the discharged HepG) crosses the membrane between the cytoplasmic loading steps and the extracytoplasmic transfer step is unknown; no flippase, transporter or spontaneous-flip mechanism has been identified. boundary: >- Firmly established: Ac-HepG is made on the cytoplasmic side from cytosolic acetyl-CoA, LhaT is an integral membrane protein, the acceptor lipoprotein is extracytoplasmic, and the downstream chain can be reconstituted using a C20 donor analogue in detergent, where compartmentation does not apply; native membrane crossing is not demonstrated by that assay. gap_kind: - BIOLOGY dark_aspect: BP_DARK status: OPEN significance: >- A transport step with no assigned gene is a missing part of this module, and determines whether a further protein should be a sixth part of it. resolution: >- Screen for mutants that accumulate cytoplasmic Ac-HepG while retaining PcrB/YvoF/LhaT; test candidate flippases; measure spontaneous flip rates of Ac-HepG in defined liposomes. provenance: - reference_id: PMID:40626731 supporting_text: >- it is presently unclear how Ac-HepG/HepG transverses the membrane - gap_statement: >- Whether Ac-HepG has acetyl-donor roles beyond lipoprotein N-acetylation is unknown. PcrB occurs in strains without LhaT, and species encoding both PcrB and LhaT orthologues do not always make acetylated lipoprotein. boundary: >- Firmly established: in B. subtilis, Ac-HepG is required for and sufficient as the donor for lipoprotein N-acetylation, and loss of pcrB, yvoF or ypjA abolishes the modification. gap_kind: - BIOLOGY dark_aspect: BP_DARK status: OPEN significance: >- It decides whether this module's terminal step is its only consumer, which is why that step is modelled as optional rather than required. resolution: >- Look for other extracellular acetylated metabolites or macromolecules lost in a pcrB or yvoF deletion in a species that lacks LhaT. provenance: - reference_id: PMID:40626731 supporting_text: >- The co-occurrence of PcrB with YpjA is not absolute, however, with PcrB enjoying a broader strain distribution than LhaT. Ac-HepG may therefore have cellular roles beyond just supplying acetyl units for lipoproteins. connections: - source: g1p_supply target: ether_bond_formation connection_type: PROVIDES_INPUT_FOR description: >- Supplies the sn-glycerol 1-phosphate acceptor for ether-bond formation. chaining_status: KNOWLEDGE_GAP chaining_note: >- The connecting metabolite is unambiguous (G1P, CHEBI:57685, is the product of the upstream reaction and the obligatory acceptor of the downstream one), but the enzyme that supplies it to PcrB in vivo is not established: there is no egsA-deletion lipidomics, and a further bacterial route to G1P exists. Recorded as a knowledge gap rather than VERIFIED so the edge is not read as a demonstrated metabolic flux. - source: ether_bond_formation target: hepgp_dephosphorylation connection_type: PRECEDES description: HepGP is the substrate of the dephosphorylation step. chaining_status: VERIFIED chaining_note: >- Continuity is on HepGP (CHEBI:64781), not a CoA thioester, so the automated CoA-carrier check cannot see it. The identity of the intermediate is established; only the enzyme of the downstream step is unassigned. - source: hepgp_dephosphorylation target: hepg_acetylation connection_type: PRECEDES description: >- HepG produced by dephosphorylation is the acetyl acceptor for YvoF; the order was established in vitro, where acetylation follows phosphatase treatment. chaining_status: VERIFIED chaining_note: >- Continuity is on heptaprenylglycerol, which has no ChEBI identifier, so it cannot be checked automatically; it is supported by the stepwise in vitro reconstitution. - source: hepg_acetylation target: lipoprotein_acetyl_transfer connection_type: PROVIDES_INPUT_FOR description: >- Ac-HepG made by YvoF is the acetyl donor used by LhaT; the transfer regenerates HepG for reloading, closing a carrier cycle. chaining_status: VERIFIED chaining_note: >- Continuity is on Ac-HepG, which has no ChEBI identifier; it is supported jointly by native genetics and an in vitro reconstitution with a C20 geranylgeranyl analogue in place of C35 HepG. The fluorescent lipopeptide acquired +42 Da with acetyl-CoA, YvoF and YpjA-containing membranes. The assay does not resolve the native donor regioisomer preference or prove native transbilayer transport. knowledge_gaps: - gap_statement: >- Why Bacillales retain an archaeal-type G1P, isoprenoid-ether lipid at all, and whether HepG/Ac-HepG has any structural membrane role alongside its carrier role, is not resolved. The pathway makes a lipid with three archaeal features but uses it as a cofactor. boundary: >- Firmly established: the lipid is made, it is a low-abundance membrane species, it is not extended with a second polyprenyl chain as archaeal di-ether lipids are, and its acetylated form is required as the acetyl donor for lipoprotein N-acetylation. gap_kind: - BIOLOGY dark_aspect: BP_DARK status: NARROWING significance: >- This is the question that makes the module interesting for the lipid divide: a bacterium using archaeal membrane chemistry for something other than a membrane. It also determines whether GO should treat the pathway as membrane-lipid biosynthesis or as cofactor biosynthesis. resolution: >- Quantitative lipidomics across growth conditions and stresses in wild type versus pcrB and yvoF deletions, including any G1P-backbone ester lipids, and biophysical measurement of HepG effects on membrane properties at native abundance. provenance: - reference_id: PMID:40626731 supporting_text: >- A catalytic role as a transient carrier for acetyl groups, as opposed to being a structural membrane building block for an archeal-type lipid with ether-linked isoprenoid chains, is more consistent with the low overall abundance of HepG/Ac-HepG observed in B. subtilis membranes (28). - reference_id: PMID:27226549 supporting_text: >- In Bacillales, heptaprenylglyceryl phosphate does not become linked to a second polyprenyl moiety like ether lipids in Archaea but is dephosphorylated and acetylated. full_text_unavailable: true