Rv2569c Resolved · high

Annotation: from legacy to revised

Curation note: 2026-08-31 (mtbc/Rv2566 project, litterature_review --wide back-propagation, destination C1). Valas & Bourne 2008 (J Mol Evol 66(5):494-504) identify this gene's Pfam/COG1305 domain family as 'a transglutaminase-like protein' and show a genome-wide statistical association between COG1305 domain count and the presence of an alternative bacterial proteasome (Anbu or 20S; the MTBC has the 20S): p=2.27e-08 / p=3.42e-07, replicated in proteobacteria (p=0.0045). This is a FAMILY-LEVEL claim (applies to the shared Transglut_core/COG1305 fold) and is added here for that reason. Iyer et al. 2008's more specific architectural prediction (fusion to a C-terminal carboxylate-amine/ammonia ligase domain, GS-fold/ATP-grasp) is NOT carried over to this gene: cross-checked against this atlas's own Pfam calls (mtbc/Rv2566 project P9.3), only Rv2566 among the 5 H37Rv COG1305 paralogues carries the C-terminal DUF2126 extension Iyer's example describes -- this gene is Transglut_core-only. The proteostasis-coupling hypothesis (Anbu/proteasome) has only been network-tested for Rv2566 itself (mtbc/Rv2566 P5.2, negative at the local MTBC STRING level); it has NOT been tested for this paralogue and should not be assumed to transfer without a separate check. `verdict`/`confidence` left unchanged (family_assigned/medium). 2026-09-15 (P47.2 + P47.6, signalement inter-sessions du projet mtbc/Rv2569c, verifie ici avant ecriture, pas recopie). Barre de requalification franchie : deux appuis litterature independants (El-Etr et al. 2004, M. marinum ; Zang et al. 2024/2025, MEME groupe, compte pour un seul appui -- caveat du pair applique) PLUS une troisieme ligne de preuve, structurale, methodologiquement distincte : le Foldseek du pair vers le Facteur XIII humain (M-CSA 149, PDB 1ggt) et sa triade Cys196-His239-Asp259 mesuree sur le modele AlphaFold ont ete VERIFIES ici de maniere independante (distances recalculees sur data/af_models/Rv2569c.pdb, entree M-CSA 149 relue via l'API -- pas de chiffre recopie sans controle). Verdict family_assigned/medium -> requalified/high. Le produit propose par Zang et al. ('serine protease') est corrige en 'cysteine protease de la famille transglutaminase' : la triade catalytique verifiee est Cys-His-Asp (nucleophile cysteine), pas Ser-His-Asp -- une erreur de nomenclature mecanistique dans le titre de la publication primaire, sans consequence sur ses resultats experimentaux (clivage de l'E-cadherine, barriere epitheliale, transmission), qui restent la base du verdict fonctionnel. Le locus mel1 (P47.4) et le compte de litterature (P47.3) restent ouverts, non traites par cette note.

In the literature (TB corpus sweep) 3 publications

Found under: H37Rv (3).

3 TB publications mention this gene. 3 publication(s) mention this gene (title/abstract), verified against the text. The atlas states the function; these are the primary sources that discuss it.

This layer CITES the literature, it does not change the verdict or the function. A gene may be heavily studied (as an antigen, a resistance determinant, a drug target) while its molecular function is settled elsewhere in the fiche. This distinguishes a gene that is dark because nobody has looked from one that is dark despite having been studied. Source: PubMed (whole), MULTI-ALIAS sweep: H37Rv locus tag AND every ortholog identifier (M. bovis Mb…, M. marinum MMAR_…, M. smegmatis MSMEG_…, M. leprae ML…, M. abscessus MAB_…), each hit VERIFIED against the abstract text (word-boundary regex). phase73/phase75, 2026-07-13.

Intrinsic disorder (sequence + structure) partially disordered

carries a substantial disordered region (83/314 residues); disorder is a property, not a function

A property (biophysics), not a function. No LLPS/condensate claim is made from disorder alone. Verdict unchanged. Source: metapredict v3 (Emenecker/Holehouse) per-residue disorder + AlphaFold mean pLDDT (annotation_mtbc P16.13).

Phenotype-driven functional lead (hypothesis) priority 8.0

disruption confers tolerance to Isoniazid; mixed in vivo signal: required in 1 of 9 significant measurements, disruption advantageous in 8 — the data supports no single direction; required under acid stress in phosphate-citrate buffer; altered fitness on cholesterol (lipid catabolism).

This locus is a "hypothetical" with a conditional Tn-seq phenotype. The statement above is a working hypothesis for its functional context, synthesised from the phenotype pattern and the corroborating layers on this page (conservation, STRING coupling, operon, regulon, localisation, structure) — a prioritised requalification candidate to validate, not an established function.

Genomic-neighbour overlap (structural caveat) co-directional · 1 % of gene

co-directional overlap: ordinary (e.g. shared stop/start codons in an operon), not the Rv2438A-type artefact P20.1, derived from GFF3 gene coordinates, 2026-08-03.

Post-translational modifications

1 reported modified residue(s): N-acetylserine @2.

Experimentally reported post-translational modification(s). A phosphosite indicates the protein is expressed and is a substrate of the M. tuberculosis Ser/Thr/Tyr kinase signalling network — a regulatory context, NOT a molecular function. Source: UniProt (Modified residue features; PTM sites curated from the M. tuberculosis literature).

CRISPRi vulnerability

Vulnerability index -0.32 (95% CI -2.56 to 2.73). 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, supplementary Table S2 'mmc3.xlsx', bulk import via Europe PMC -- supersedes the per-gene pebble.rockefeller.edu scrape of phase46_crispri_vulnerability.py).

Caveat on this estimate. Bosch et al. flag this gene's vulnerability estimate as NOT certain, and here that call has a mechanical cause visible in the library itself: every guide targeting this gene is >=21 nt (str_span=0.282 on a 0-1 scale, near the genome floor), and guide repression strength saturates from 21 nt on -- so the library never sampled a partial knockdown for this gene, only the near-maximal repression plateau. The dose-response fit (and this VI) is therefore essentially unconstrained by data, not merely uncertain -- a limitation of the CRISPRi library design for the non-essential class (Bosch et al. 2021), not a defect in this specific measurement. n_guides=12 is descriptive only and does not predict this. Do not cite this VI as a standalone vulnerability claim.

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)

As checked on 2026-09-23, UniProt listed this protein as Uncharacterized protein Rv2569c; the revised annotation above goes beyond that record. UniProt may have been recurated since: check the live entry.

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, 142 601 strains, snapshot of 2026-09-29)

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) Corynebacteriales

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.

Mutant phenotypes (conditional Tn-seq, MtbTnDB) in-vivo phenotype

Conditional fitness of transposon-disruption mutants across 16 significant condition(s) — the table lists the 12 strongest by |log2FC|, not all 16 (|log2FC|≥1, q≤0.05), from the standardized MtbTnDB compendium. A negative log2FC means the mutant is depleted — the gene contributes to fitness in that condition. An in-vivo defect for a "hypothetical" is strong evidence it matters for infection, even without a known molecular function. Disruption (Tn insertion), not a clean deletion; genetic-interaction screens excluded.

Provenance note on a condition above. MtbTnDB labels this condition in vivo (Collaborative Cross mouse panel) and attributes it to Smith et al. 2022, eLife 74419. Both are wrong and for the same reason: its left arm, `mbio_H37Rv`, is the saturated H37Rv reference of DeJesus et al. 2017 (mBio 8(1):e02133-16, fourteen independent Tn libraries grown in standard medium), not a mouse strain, and the upstream generator read the token before `_vs_` as a mouse-strain name. The same sample is the in vitro control of six knockout screens in the same file. Measured here on rank correlation over 3971 genes: this condition tracks the other 81 first-order in vivo conditions at rho = +0.03, where those conditions track each other at +0.270 -- it does not behave like an infection measurement. What the comparison actually is could not be established, so it is reported as undetermined rather than renamed, and it is excluded from the in vivo count of this fiche.

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. Read the dataset count as abundance, not as replication. Measured across this atlas (3,491 detected proteins), the number of datasets a protein appears in tracks its abundance almost monotonically: median 1.2 ppm at 1 dataset of 16, 27 ppm at 10, 392 ppm at 15, 1,325 ppm at 16 — three orders of magnitude. A high count therefore says the protein is abundant more than it says several independent experiments confirmed it. What the count does establish, at any value, is that the product exists.

Localisation (measured + predicted) experimentally localised

A verified absence of signals, which is not a compartment. No Sec/Tat signal peptide, no transmembrane helix and no lipobox were found on this sequence. The predictor reports topology and export signals; it does not measure where the protein ends up, so this page draws no cytoplasmic conclusion from it. In M. tuberculosis that reservation is not theoretical: the 12 Esx paralogues of this atlas — EsxA/ESAT-6 and EsxB/CFP-10 among them, the best-established secreted proteins of the species — carry this very call, because substrates of the type VII (ESX) secretion system leave no recognisable signal. « Secreted » and « no signal peptide » are true together here.

Transmembrane topology and signal peptide from DeepTMHMM (deep-learning reference predictor); lipoproteins from a (myco)bacterial lipobox motif. A sequence-based prediction of subcellular context.

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)

3D structure (interactive — Mol*/pdbe-molstar)

No experimental structure is deposited for this gene: showing the AlphaFold DB predicted model (mean pLDDT 91.3, very high), coloured by per-residue confidence (dark blue = very high, orange/red = low — treat low-confidence regions as unreliable, especially termini and loops). Drag to rotate, scroll to zoom.

Structural search (AlphaFold DB model, Foldseek vs PDB — genome-wide) pLDDT 91.3

Foldseek search of the AlphaFold DB model (mean pLDDT 91.3, 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 2

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).

Published operon evidence (two independent sources)

Two independent published operon maps, cited alongside the distance heuristic above: an association corroborating or contradicting the heuristic, never proof of shared function. REMap's RNA-seq coverage continuity is the most direct of the three signals; Roback's logistic regression and the intergenic-distance heuristic are statistical proxies.

Named locus (published gene set, not an operon) mel1

El-Etr, Subbian, Cirillo & Cirillo 2004 (Infect Immun 72(12):6902-6913, PMID 15557611, PMC529147) identified the mel1 cosmid region in M. marinum by a gain-of-function screen (cosmid library transformed into M. smegmatis, screened for enhanced macrophage association) and then pinpointed melE by transposon mutagenesis (Tn22) and allelic exchange: the M. marinum melE mutant is defective for cell association with the murine (J774A.1) and fish (CLC) macrophage cell lines, and this defect is complemented by expressing the M. tuberculosis Rv2569c gene itself from the hsp60 promoter (construct pJDC77). melA, melB, melC and melD were annotated ONLY by sequence homology to their M. tuberculosis counterparts (Table 1 of the paper) and were NEVER individually mutated or tested in this study -- the direct functional evidence in mel1 concerns melE/Rv2569c alone.

'mel1' names a conserved gene SET (near-identical order across M. marinum and the MTBC, per the source paper), not a demonstrated operon: El-Etr et al. 2004 measured neither a shared transcript nor co-expression. A single five-gene transcription unit is not even geometrically possible in H37Rv: melC (Rv2567) sits on the '+' strand while melD (Rv2568c) and melE (Rv2569c, this gene) sit on the '-' strand. Only melD-melE are adjacent and co-directional (8 bp gap), which the co-directional heuristic above already captures as a 2-gene predicted operon -- and Roback et al. 2007 (published_operon_evidence, above) assigns that specific pair a LOW probability (0.011) and flags disagreement with the heuristic. melA (Rv2560) is not even adjacent to melB (Rv2566) in H37Rv: five unrelated ORFs intervene (Rv2561-Rv2565, including a glutamine ABC transporter).

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 physiological-context lead, not a function.

Condition: Condition-blind by construction. These edges come from transcription-factor OVEREXPRESSION in a single laboratory condition, so a listed regulator is not a claim that it governs this gene in every circumstance. Measured counter-examples exist within this atlas (see Rv2516c, where the DosR regulon collapses under moxifloxacin while the gene itself rises). Read a regulator as 'capable of shifting this gene', and look for the condition before building a hypothesis on it.

Node colour = verdict, dashed = still uncharacterised; 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.

Closest characterised functional partner: Rv2568c (hypothetical protein), high confidence (score 952, all STRING channels except text-mining; the partner is chosen on its genomic-context evidence).

All 7 partner(s) at medium confidence are shown. None rests on an interaction measured in M. tuberculosis itself.

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 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.

Where the experimental evidence comes from, and why it matters here. STRING's experimental channel fuses interactions measured in M. tuberculosis with interactions measured in another organism and transferred to this pair by orthology. The two are not equivalent for this atlas, whose standing rule is that better studied elsewhere is not a function established in M. tuberculosis. Measured on the served network: of the 38 229 partner pairs shown across the atlas, 13 483 partner rows carry a non-zero experimental channel, and only 106 pairs rest on an interaction measured in M. tuberculosis itself — 0.3 % of the network. Those 106 are long-established complexes (ESX-1/ESAT-6, RNA polymerase with CarD and RbpA, the Pup–Mpa proteasome, gyrase, Emb, GarA with PknB and PknG, the Rel modules); no still-uncharacterised gene is among them. The reason is not a gap in this atlas but in the literature: there is almost no experimental interactome of this bacterium, IntAct holding 134 curated protein pairs for a ~4 000-gene proteome. A exp (transferred) badge is therefore a lead, never a measurement, and an absence of badge is not evidence that the pair does not interact.

Evidence

• Legacy H37Rv annotation: hypothetical protein
• MTBC0 PGAP product: transglutaminase family protein
• Pfam (hmmscan --cut_ga): Bact_transglu_N PF08379.16 (E=2e-25), Transglut_core PF01841.26 (E=3e-14)
• (initial automatic assignment: auto-curated by rules from PGAP + Pfam + Foldseek; superseded by hand review)
• P9.3 back-propagation (mtbc/Rv2566 project, litterature_review --wide, 2026-08-31): Valas & Bourne 2008 (J Mol Evol) identify the COG1305 domain family shared by this gene as 'a transglutaminase-like protein' and show a genome-wide statistical association between COG1305 domain count and the presence of an alternative bacterial proteasome (Anbu or 20S; p=2.27e-08 / p=3.42e-07, replicated in proteobacteria p=0.0045). Family-level claim; this gene is Transglut_core-only (no C-terminal DUF2126 ligase extension, unlike Rv2566), so Iyer et al. 2008's more specific fusion-architecture prediction is deliberately NOT extended to it here.
• P47.6 (2026-09-15, signalement inter-sessions du projet mtbc/Rv2569c) : catalytic triad Cys196-His239-Asp259 verified independently on this atlas's own AlphaFold model (data/af_models/Rv2569c.pdb) -- Cys196 SG-His239 ND1 3.69 A, His239 NE2-Asp259 OD2 2.74 A, pLDDT 95.3-97.9 -- matching the pair's report exactly. M-CSA 149 (EC 2.3.2.13, PDB 1ggt, human Factor XIII) independently confirms a Cys315-His374-Asp397 catalytic triad, ruling out the 'serine protease' label of Zang et al. 2024/2025 (same group, one support) for this cysteine-nucleophile transglutaminase-family enzyme. El-Etr et al. 2004 (M. marinum ortholog locus, macrophage-interaction phenotype) added as the second, independent literature support.

Ancestral MTBC0 protein sequence

>mtbc0_002736|Rv2569c|
MSADSSLSLPLSGTHRYRVTHRTEYRYSDVVTSSYGRGFLTPRNSLRQRCVAHRLTIDPAPADRSTSRDGYGNISSYFHVTEPHRTLTITSDSIVDVSPPPPGLYTSGPALQPWEAARPAGLPGSLATEFTLDLNPPEITDAVREYAAPSFLPKRPLVEVLRDLASRIYTDFTYRSGSTTISTGVNEVLLAREGVCQDFARLAIACLRANGLAACYVSGYLATDPPPGKDRMIGIDATHAWASVWTPQQPGRFEWLGLDPTNDQLVDQRYIVVGRGRDYADVPPLRGIIYTNSENSVIDVSVDVVPFEGDALHA