id: A2AXI2 gene_symbol: cfr product_type: PROTEIN status: DRAFT taxon: id: NCBITaxon:1292 label: Staphylococcus warneri description: >- cfr encodes a radical SAM 23S rRNA methyltransferase that methylates carbon 8 of adenine 2503 in bacterial 23S rRNA. This A2503 C8 modification confers cross-resistance to multiple antibiotic classes that bind near the peptidyl transferase center. Cfr is related to RlmN, but the clinically relevant Cfr activity is C8 methylation rather than the housekeeping C2 methyltransferase activity represented by existing GO specificity terms. reference_proteome_assessments: - reference_proteomes: - UP000814367 status: NO_SUPPORTED_COUNTERPART reviewed_on: '2026-10-19' reason: Retain A2AXI2 as the primary Swiss-Prot accession. The only hit is the related RlmN methyltransferase A0A2T4Q1F7 (33.69% global identity), not an established Cfr counterpart. Shared radical-SAM/RNA-methyltransferase ancestry does not establish the Cfr resistance activity or gene identity. The source is strain 11562A; no supported reference-strain counterpart was identified. evidence: - type: PROTEOME_SEARCH description: MMseqs2 searched 2,456 sequences across UP000814367 at E <= 1e-5 and sensitivity 7.5, returning 1 hits. Every returned sequence was globally aligned using BLOSUM62 with gap-open -10 and extension -0.5. Scores and sequences for all 1 returned candidates are retained, with 1 detailed alignment files. The heuristic search does not establish genomic absence or unique locus identity. supported_by: - reference_id: file:STAWA/cfr/cfr-record-evidence.yaml supporting_text: 'target_sequence_count: 2456' - reference_id: file:STAWA/cfr/cfr-record-evidence.yaml supporting_text: 'candidate_count: 1' - type: PROTEOME_MEMBERSHIP description: The primary accession is outside every searched exact-taxon reference proteome. supported_by: - reference_id: file:STAWA/cfr/cfr-record-evidence.yaml supporting_text: 'primary_in_any_reference_proteome: false' - type: RECORD_HISTORY description: 'Sampled UniSave observations: 11-NOV-2015, entry version 43.: no Proteomes cross-reference; 02-DEC-2020, entry version 58.: no Proteomes cross-reference; 27-NOV-2024, entry version 68.: no Proteomes cross-reference. These dated observations do not establish uninterrupted membership/absence, the exact change date, or UniProt''s motivation.' supported_by: - reference_id: file:STAWA/cfr/cfr-record-evidence.yaml supporting_text: |- entry_date: 11-NOV-2015, entry version 43. proteomes: [] - reference_id: file:STAWA/cfr/cfr-record-evidence.yaml supporting_text: |- entry_date: 02-DEC-2020, entry version 58. proteomes: [] - reference_id: file:STAWA/cfr/cfr-record-evidence.yaml supporting_text: |- entry_date: 27-NOV-2024, entry version 68. proteomes: [] - type: CURATION_CONTEXT description: The primary UniProt entry records plasmid origin. Reference-proteome membership at species or strain level does not ensure coverage of every accessory plasmid. supported_by: - reference_id: file:STAWA/cfr/cfr-record-evidence.yaml supporting_text: 'geneEncodingType: Plasmid' existing_annotations: - term: id: GO:0003824 label: catalytic activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: Technically true but too broad to be useful for Cfr. action: MODIFY reason: >- Following the RADICAL_SAM project rule for the generic domain-derived rows (projects/RADICAL_SAM.md): the row is true, so it is refined rather than flagged as an over-annotation. Cfr has a defined reaction, methylation of C8 of adenine 2503 in 23S rRNA, and GO:0016433 is the term this entry already carries for it. supported_by: - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["CC:FUNCTION"] supporting_text: "Specifically methylates position 8 of adenine 2503 in 23S" proposed_replacement_terms: - id: GO:0016433 label: rRNA (adenine) methyltransferase activity - term: id: GO:0005737 label: cytoplasm evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: located_in review: summary: Correct location for a bacterial rRNA-modifying enzyme. action: ACCEPT reason: The UniProt record carries cytoplasm as the subcellular-location-derived GO annotation. supported_by: - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["DR:GO"] supporting_text: "DR GO; GO:0005737; C:cytoplasm" - term: id: GO:0006364 label: rRNA processing evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: involved_in review: summary: Broad process annotation that should be replaced by rRNA base methylation for Cfr. action: MODIFY reason: >- Cfr performs a base methylation on mature 23S rRNA. GO:0070475 is the better process-level representation than generic rRNA processing. proposed_replacement_terms: - id: GO:0070475 label: rRNA base methylation supported_by: - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["CC:FUNCTION"] supporting_text: "Specifically methylates position 8 of adenine 2503 in 23S" - term: id: GO:0006400 label: tRNA modification evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: involved_in review: summary: Incorrect electronic carryover from broad methylthiotransferase signatures. action: REMOVE reason: >- The Cfr record and Cfr mechanism literature identify 23S rRNA A2503 as the substrate. This review found no support that Cfr modifies tRNA. supported_by: - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["CC:FUNCTION"] supporting_text: "Specifically methylates position 8 of adenine 2503 in 23S" - term: id: GO:0008173 label: RNA methyltransferase activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: Correct but broad; replace with rRNA (adenine) methyltransferase activity. action: MODIFY reason: >- Cfr is an rRNA adenine methyltransferase, not a generic RNA methyltransferase. GO still lacks the exact C8/A2503 child term. proposed_replacement_terms: - id: GO:0016433 label: rRNA (adenine) methyltransferase activity supported_by: - reference_id: PMID:19144912 supporting_text: "Cfr catalyzes formation of 8-methyladenosine." - term: id: GO:0016433 label: rRNA (adenine) methyltransferase activity evidence_type: IEA original_reference_id: GO_REF:0000104 qualifier: enables review: summary: Correct core molecular-function parent for Cfr, but missing the C8/A2503 specificity. action: ACCEPT reason: >- Cfr methylates adenine in 23S rRNA. The existing term is the best current GO parent; a Cfr-specific A2503 C8 term is proposed below. supported_by: - reference_id: PMID:19144912 supporting_text: "Cfr catalyzes formation of 8-methyladenosine." - term: id: GO:0016740 label: transferase activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: Correct but too broad; replace with rRNA (adenine) methyltransferase activity. action: MODIFY reason: Generic transferase activity is a weak representation of Cfr compared with GO:0016433. proposed_replacement_terms: - id: GO:0016433 label: rRNA (adenine) methyltransferase activity supported_by: - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt supporting_text: "Ribosomal RNA large subunit methyltransferase Cfr" - term: id: GO:0019843 label: rRNA binding evidence_type: IEA original_reference_id: GO_REF:0000104 qualifier: enables review: summary: Plausible substrate-binding annotation, but secondary to catalytic methyltransferase activity. action: KEEP_AS_NON_CORE reason: Cfr acts on 23S rRNA, but substrate binding is not the core evolved function. supported_by: - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["CC:CATALYTIC ACTIVITY"] supporting_text: "adenosine(2503) in 23S rRNA" - term: id: GO:0030488 label: tRNA methylation evidence_type: IEA original_reference_id: GO_REF:0000118 qualifier: involved_in review: summary: Incorrect TreeGrafter propagation for Cfr. action: REMOVE reason: | Retained. Cfr's characterized substrate is 23S rRNA: it methylates C8 of adenosine 2503 (PMID:19144912), and the record's rRNA methyltransferase, rRNA base methylation and rRNA binding rows are accepted. The tRNA specificity in this family belongs to the paralogous radical-SAM enzyme RlmN, which methylates C2 of A2503 in 23S rRNA and of A37 in tRNA; Cfr lacks the tRNA activity, so this row is a substrate-class error propagated from the shared RlmN/Cfr PANTHER family rather than a merely coarse term. No tRNA substrate is reported for Cfr in the UniProt record or the cited literature. supported_by: - reference_id: PMID:19144912 supporting_text: "The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA" - term: id: GO:0051536 label: iron-sulfur cluster binding evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables review: summary: Broad cofactor-binding term; the 4Fe-4S child is more informative and already annotated. action: MODIFY reason: Replace broad iron-sulfur cluster binding with the specific 4Fe-4S cluster binding annotation for Cfr. proposed_replacement_terms: - id: GO:0051539 label: 4 iron, 4 sulfur cluster binding supported_by: - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["CC:COFACTOR"] supporting_text: "Name=[4Fe-4S] cluster" - term: id: GO:0051539 label: 4 iron, 4 sulfur cluster binding evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables review: summary: Correct cofactor-binding annotation for a radical SAM Cfr enzyme. action: ACCEPT reason: >- Core cofactor binding under the project rule. The [4Fe-4S] cluster is not merely required for the mechanism: it generates the 5'-deoxyadenosyl radical that abstracts the C8 hydrogen, which is the step that distinguishes Cfr from the conventional SAM methyltransferases. Catalysis is established, so ACCEPT rather than KEEP_AS_NON_CORE. supported_by: - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["CC:COFACTOR"] supporting_text: Binds 1 [4Fe-4S] cluster. - term: id: GO:0070475 label: rRNA base methylation evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: involved_in review: summary: Correct process annotation for the Cfr-catalyzed rRNA A2503 methylation. action: ACCEPT reason: Cfr directly methylates a base in 23S rRNA; the process term is appropriate. supported_by: - reference_id: PMID:19144912 supporting_text: "Antibiotic susceptibility data confirm that the antibiotic resistance conferred by Cfr is provided by methylation at the 8 position" - term: id: GO:0046677 label: response to antibiotic evidence_type: RCA original_reference_id: PMID:19144912 qualifier: involved_in review: summary: NEW high-level process annotation for Cfr-mediated multidrug resistance. action: NEW reason: >- Cfr-mediated A2503 C8 methylation provides resistance to multiple peptidyl-transferase-center antibiotic classes. This is a high-level process annotation, while the core curation need remains the C8-specific molecular-function term. additional_reference_ids: - PMID:17145796 - file:genes/STAWA/cfr/cfr-uniprot.txt supported_by: - reference_id: PMID:19144912 supporting_text: "The Cfr methyltransferase confers combined resistance to five different classes" - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["CC:FUNCTION"] supporting_text: "Confers resistance to some classes of antibiotics." - term: id: GO:1904047 label: S-adenosyl-L-methionine binding evidence_type: IC original_reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt qualifier: enables review: summary: >- cfr binds S-adenosyl-L-methionine at the unique iron of its radical SAM [4Fe-4S] cluster and cleaves it to the 5'-deoxyadenosyl radical; two SAM are consumed per methyl transferred, one as the methyl donor and one as the radical source. action: NEW reason: >- Not in GOA. Added under the RADICAL_SAM treatment rule, which treats GO:1904047 as a core cosubstrate-binding function of every radical SAM enzyme whose catalysis is established. This is a molecular function the protein itself performs, so the participation test is met directly: cfr binds SAM and cleaves it, rather than merely being required for a process that consumes it. UniProt's curated cofactor note records the exchangeable S-adenosyl-L-methionine on the radical SAM cluster and the entry's binding features name the 4Fe-4S-S-AdoMet ligand, so the claim rests on curated record content rather than on an electronic GO line. Coded IC because the inference is the reviewer's, drawn from that curated content and the established rRNA (adenine) methyltransferase activity rather than from a SAM-binding assay on this protein. Comparator check, against GOA rather than against this project's own proposals: GOA does annotate GO:1904047 to radical SAM enzymes in exactly this role, by UniRule -- ECOLI/queE (UR000100349), ECOLI/moaA and PSEPK/moaA (UR000101305), METEA/pqqE and PSEPK/pqqE (UR000098303). So the absence here is patchy UniRule coverage, which rules happen to carry the term, not a GO convention against annotating a radical SAM enzyme's own cosubstrate. That is also why those five genes have an existing row to ACCEPT or MODIFY while this one needs a NEW row. supported_by: - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["CC:COFACTOR"] supporting_text: Note=Binds 1 [4Fe-4S] cluster. The cluster is coordinated with 3 - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["CC:COFACTOR"] supporting_text: cysteines and an exchangeable S-adenosyl-L-methionine - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["FT:BINDING"] supporting_text: /ligand_note="4Fe-4S-S-AdoMet" references: - id: file:STAWA/cfr/cfr-record-evidence.yaml title: Compact archived record and sequence evidence for A2AXI2 - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: [] - id: GO_REF:0000104 title: Electronic Gene Ontology annotations created by transferring manual GO annotations between related proteins based on shared sequence features findings: [] - id: GO_REF:0000118 title: TreeGrafter-generated GO annotations findings: [] - id: GO_REF:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: [] - id: file:genes/STAWA/cfr/cfr-uniprot.txt title: UniProt flat file for cfr (A2AXI2) findings: - statement: UniProt names Cfr as a 23S rRNA A2503 C8 methyltransferase. record_section: [DE] supporting_text: "23S rRNA (adenine(2503)-C(8))-methyltransferase" - id: PMID:17145796 title: IS21-558 insertion sequences are involved in the mobility of the multiresistance gene cfr. findings: - statement: The source-species cfr paper supports Cfr as a multiresistance gene in Staphylococcus warneri genetic context. supporting_text: "gene cfr for combined resistance to phenicols, lincosamides, oxazolidinones," - id: PMID:19144912 title: Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria. findings: - statement: Cfr catalyzes 8-methyladenosine formation at A2503, and resistance is provided by the C8 methylation. supporting_text: "Cfr catalyzes formation of 8-methyladenosine." - id: file:projects/ANTIMICROBIAL_RESISTANCE/aro2go.sssom.yaml title: Curated ARO to GO mapping set for AMR gene families findings: - statement: The AMR mapping records the Cfr C8/A2503 methyltransferase as a GO molecular-function gap. supporting_text: "Cfr methylates C8 of A2503." core_functions: - description: >- Radical-SAM 23S rRNA A2503 C8 methyltransferase activity that produces 8-methyladenosine in 23S rRNA and confers resistance to several peptidyl-transferase-center antibiotic classes. molecular_function: id: GO:0016433 label: rRNA (adenine) methyltransferase activity directly_involved_in: - id: GO:0070475 label: rRNA base methylation - id: GO:0046677 label: response to antibiotic locations: - id: GO:0005737 label: cytoplasm supported_by: - reference_id: PMID:19144912 supporting_text: "Cfr catalyzes formation of 8-methyladenosine." - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["CC:FUNCTION"] supporting_text: "Specifically methylates position 8 of adenine 2503 in 23S" - description: >- Binds the catalytic radical SAM [4Fe-4S] cluster through the CxxxCxxC motif; its unique iron coordinates the S-adenosyl-L-methionine that is reductively cleaved to the 5'-deoxyadenosyl radical starting every turnover of rRNA (adenine) methyltransferase activity. molecular_function: id: GO:0051539 label: 4 iron, 4 sulfur cluster binding supported_by: - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["CC:COFACTOR"] supporting_text: Note=Binds 1 [4Fe-4S] cluster. The cluster is coordinated with 3 - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["CC:COFACTOR"] supporting_text: cysteines and an exchangeable S-adenosyl-L-methionine - description: >- Binds S-adenosyl-L-methionine as the radical-generating cosubstrate at the unique iron of the [4Fe-4S] cluster: two SAM are consumed per methyl transferred, one as the methyl donor and one as the radical source. molecular_function: id: GO:1904047 label: S-adenosyl-L-methionine binding supported_by: - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["CC:COFACTOR"] supporting_text: cysteines and an exchangeable S-adenosyl-L-methionine - reference_id: file:genes/STAWA/cfr/cfr-uniprot.txt record_section: ["FT:BINDING"] supporting_text: /ligand_note="4Fe-4S-S-AdoMet" proposed_new_terms: - proposed_name: 23S rRNA A2503 C8 methyltransferase activity proposed_definition: >- Catalysis of methyl transfer to carbon 8 of adenine 2503 in bacterial 23S rRNA, forming 8-methyladenosine at A2503. justification: >- Existing GO specificity for this enzyme family includes the RlmN/C2 methyltransferase activity, but Cfr's resistance-conferring activity is C8 methylation at A2503. The AMR mapping records this as a NoTermFound gap. proposed_parent: id: GO:0016433 label: rRNA (adenine) methyltransferase activity proposed_mappings: - predicate: skos:exactMatch target_term: id: ARO:3000202 label: Cfr 23S ribosomal RNA methyltransferase supported_by: - reference_id: PMID:19144912 supporting_text: "Cfr catalyzes formation of 8-methyladenosine." - reference_id: file:projects/ANTIMICROBIAL_RESISTANCE/aro2go.sssom.yaml supporting_text: "Cfr methylates C8 of A2503." suggested_questions: - question: Should GO add a Cfr-specific C8 term as a sibling of the existing RlmN/C2 23S rRNA methyltransferase term? experts: [] suggested_experiments: - description: >- Compare Cfr and RlmN activity on the same 23S rRNA substrate by MS mapping of A2503 products to support separate C8 and C2 GO molecular functions. experiment_type: in vitro enzyme assay