Rv2565 Family assigned · medium auto-curated

Annotation: from legacy to revised

Auto-curated: this verdict and function were generated by rules from PGAP + Pfam + Foldseek and have not been hand-reviewed.

In the literature (TB corpus sweep) 1 publication

1 TB publication mentions this gene. 1 publication(s) discuss this gene (1 in a M. tuberculosis context).

This layer CITES the literature and adds context; it does not change the verdict or the function stated elsewhere in this fiche. This distinguishes a gene that is dark because nobody has looked from one that is dark despite having been studied. Source: PubMed (whole): H37Rv locus tag + GENE NAME + ortholog identifiers (Mb…, MMAR_…, MSMEG_…, ML…, MAB_…), under a mycobacterial context filter; hits verified against the abstract text. Species-context counts distinguish M. tuberculosis literature from literature on other mycobacteria. phase76/phase77, 2026-07-13.

CRISPRi vulnerability

Vulnerability index 1.71 (95% CI 0.14 to 4.33). A more negative index = more vulnerable to knockdown (better drug-target quality); indicative threshold VI ≤ -6 = highly vulnerable.

Quantitative CRISPRi knockdown, graded (finer than binary Tn-seq essentiality). Source: CRISPRi vulnerability index (Bosch 2021, pebble.rockefeller.edu).

Legacy record & comparison (Mycobrowser) ahead of Mycobrowser

Mycobrowser classes this locus among conserved hypotheticals; the atlas now assigns a functional handle (COG category). Mycobrowser is no longer maintained, so its EC numbers predate recent nomenclature revisions (e.g. the 2018 EC 7 "translocase" class) — most EC differences are re-numberings of the same enzyme, not conflicts.

Orthologues (reciprocal best hits across mycobacteria)

Reciprocal-best-hit orthologues (DIAMOND) against the Mycobrowser reference proteomes. A missing species is informative: e.g. a gene absent from M. leprae was likely lost in its reductive genome evolution. Locus tags link to Mycobrowser.

Curated reference (UniProt)

UniProt still lists this protein as Uncharacterized NTE family protein Rv2565; the revised annotation above is ahead of the current UniProt record.

Functional vocabulary (eggNOG-mapper, orthology transfer)

Orthology-based transfer (eggNOG 5.0.2, diamond). EC/KO/GO/CAZy are computed annotations, not manual curation; cross-check against the primary literature before treating a specific reaction as established.

Conservation & selection (intra-MTBC, 145 209 strains)

pN/pS from segregating SNPs (singletons removed) normalised by possible sites. Low pN/pS = purifying selection (a strong signal that a "hypothetical" is a real, constrained gene). A high pN/pS is ambiguous: relaxed constraint or positive selection (drug resistance, antigenic variation) inflate it; e.g. rpoB/katG/pncA score high here for resistance, not loss of function. A clonal disruption (one allele over a clade) suggests lineage pseudogenisation; a convergent one (many independent alleles) is typical of resistance loss-of-function.

Outgroup conservation (beyond the MTBC) Mycobacterium

Two orthogonal outgroup signals. M. canettii (the immediate outgroup) gives a deep-divergence dN/dS (a low value confirms a constrained, real gene; shown as confident only at ≥8 substitutions, else flagged low-power). Genus-wide presence/absence (tblastn vs assembled non-MTBC genomes) places the gene on the ancient-core ↔ MTBC-specific axis: a gene absent even from the closest MTBAP relatives is a candidate MTBC-specific innovation (possible host-adaptation factor, to confirm by synteny). The phylostratum extends that axis outside the genus (tblastn vs 13 reference genomes spanning Mycobacteriaceae → Corynebacteriales → Actinomycetia → outside the phylum): it is the deepest clade in which a homolog is still detected, i.e. a proxy for gene age. Read it with the null model in mind: a shallow (young) stratum can also reflect homology-detection failure for short or fast-evolving ORFs, so it is a descriptive axis, not a proof of novelty.

Essentiality (transposon mutagenesis)

Genome-wide Himar1 transposon essentiality in H37Rv (DeJesus 2017). An essential call (ES/ESD/GD) is strong, independent evidence that a "hypothetical" locus encodes a functional, selectively required gene — orthogonal to intra-species conservation.

Enzyme activity (activity-based protein profiling) active serine hydrolase confirms atlas call

Directly labelled by a fluorophosphonate activity-based probe in live M. tuberculosis: biochemical proof that this protein is a catalytically active serine hydrolase, not merely a predicted fold. This experimentally corroborates the atlas assignment (Patatin-like phospholipase family protein. Pfam: cNMP_binding (PF00027.36), Pata), which was derived independently from structure and orthology.

experimental (activity-based) confirmation of the atlas serine-hydrolase family assignment; proves the enzyme is catalytically active in live M. tuberculosis. It never changes the verdict here. Source: Li M, Patel HV, ..., Canaan S, Aldridge BB et al., Cell Chem Biol 2021 (PMC8964833; doi:10.1016/j.chembiol.2021.09.002); competitive activity-based protein profiling of FP-reactive serine hydrolases.

Proteomics (mass spectrometry) detected

Detection by mass spectrometry is direct, experimental evidence that the protein product exists — orthogonal to sequence conservation and to Tn-seq essentiality, and especially decisive for a "hypothetical" locus. Reproducible detection across several independent datasets (PaxDb) makes the existence claim robust; the integrated abundance places the protein in the proteome's dynamic range.

Physico-chemical properties (computed, ProtParam)

Computed from the ancestral MTBC0 sequence with the ExPASy ProtParam method (Biopython). Descriptive biophysical context: a positive GRAVY flags a hydrophobic (often membrane) protein, a high instability index (>40) predicts a short in-vitro half-life, an extreme pI hints at compartment or binding partner.

Domains (Pfam, hmmscan --cut_ga)

Structural search (AlphaFold DB model, Foldseek vs PDB — genome-wide) pLDDT 84.8

Foldseek search of the AlphaFold DB model (mean pLDDT 84.8, gated at 70) against the PDB — a genome-wide extension of the ESMFold dark-gene search that also covers proteins beyond the single-sequence length limit. Confident structural neighbours (E < 0.01) shown.

Genomic context (neighbours & predicted operon) operon of 3

Neighbours from the H37Rv annotation (+ strand). The operon is predicted by co-directional intergenic distance (same strand, gaps ≤50 bp) — a transcription-unit hypothesis, not a mapped TSS. For a "hypothetical", co-transcription with a characterised operon is a concrete functional lead (complements the STRING neighborhood channel below).

Transcriptional regulation (signed TRN: ChIP-seq + TFOE)

Regulatory edges from the ISB signed transcriptional regulatory network (TF ChIP-seq binding, Minch 2015 + TF-overexpression response, Rustad 2014). An edge is regulatory evidence (binding and/or expression change), not necessarily direct. For a "hypothetical", membership in a known regulon (e.g. DosR dormancy, PhoP virulence) is a strong physiological-context lead.

Node colour = verdict, dashed = hypothetical; edge colour = evidence (green experimental, orange genomic-context, grey co-expression), width ∝ score. Click a partner to open its page; "Explore full network" to walk the graph.

STRING combines evidence channels (neighborhood, fusion, cooccurrence, coexpression, experimental, database, text-mining) into a 0–1000 score. The ctx badge marks edges carried by the genomic-context channels (conserved neighborhood, fusion, phylogenetic co-occurrence), which are independent of orthology and structure and the strongest signal for an unknown gene. The exp badge marks an experimentally-supported partner (measured interaction, experimental/database channel ≥400) as opposed to a purely predicted one — but note that the M. tuberculosis experimental interactome is dominated by a noisy bacterial-two-hybrid screen, so a strong measured link that contradicts the operon/localisation context is likely a false positive. The no text-mining column recomputes the score from data alone, so a link that does not depend on the literature is visible. Association is a function hypothesis, not proof: corroborate with the operon context and the primary literature before assigning a function.

Evidence

• Legacy H37Rv annotation: NTE family protein
• MTBC0 PGAP product: patatin-like phospholipase family protein
• Pfam (hmmscan --cut_ga): cNMP_binding PF00027.36 (E=7e-17), Patatin PF01734.28 (E=4e-27)
• (auto-curated by rules from PGAP + Pfam + Foldseek; not hand-reviewed)

Ancestral MTBC0 protein sequence

>mtbc0_002732|Rv2565|
MTTARRRPKRRGTDARTALRNVPILADIDDEQLERLATTVERRHVPANQWLFHAGEPADSIYIVDSGRFVAVAPEGHVFAEMASGDSIGDLGVIAGAARSAGVRALRDGVVWRIAAETFTDMLEATPLLQSAMLRAMARMLRQSRPAKTARRPRVIGVVSNGDTAAAPMVDAIATSLDSHGRTAVIAPPVETTSAVQEYDELVEAFSETLDRAERSNDWVLVVADRGAGDLWRHYVSAQSDRLVVLVDQRYPPDAVDSLATQRPVHLITCLAEPDPSWWDRLAPVSHHPANSDGFGALARRIAGRSLGLVMAGGGARGLAHFGVYQELTEAGVVIDRFGGTSSGAIASAAFALGMDAGDAIAAAREFIAGSDPLGDYTIPISALTRGGRVDRLVQGFFGNTLIEHLPRGFFSVSADMITGDQIIHRRGSVSGAVRASISIPGLIPPVHNGEQLLVDGGLLNNLPANVMCADTDGEVICVDLRRTFVPSKGFGLLPPIVTPPGLLRRLLTGTDNALPPLQETLLRAFDLAASTANLRELPRVAAIIEPDVSKIGVLNFKQIDAALEAGRMAARAALQAQPDLVR