Rv3196A Family assigned · low

Annotation: from legacy to revised

Curation note: 2026-09-08 (P45.5). First hand curation of this locus. Rv3196A is the gene that triggered the whole P45 full-text campaign and was the only one not to be instructed by it; its fiche still served the automatic sentence "Function unknown. Foldseek best (non-significant) hit: ... human Nup49 coiled-coil", a hit whose own probability (0.66) makes it noise. Three things were verified rather than repeated. Cronin et al. 2022, which eLife cites for the M. marinum ortholog, was identified (PNAS, ESX-1 proteo-genetics) and its Dataset S2 read: MMAR_1367 is in the secreted fraction of all 13 mutants and is not ESX-1-dependent -- a positive fact AND a negative one, both new to this fiche. Spectrum 2022 turned out not to be a passing table mention but a rank-2 induction (log2 FC 3.76) in an alkX mutant. Bell et al. 2012 stays cited-not-verified, and is written as such. HYPOTHESIS RAISED AND THEN REJECTED, recorded because the negative is informative. That alkX RNA-seq looks like a WhiB7 response: rank 1 is whiB7 (Rv3197A) and ranks 3, 5, 7 and 11 are erm(37), Rv1258c, eis and Rv0263c -- five members of the WhiB7 regulon -- with Rv3196A at rank 2. So: is Rv3196A a WhiB7-regulon gene? Three checks, all negative. (a) This atlas's own co-expression compendium (647 RNA-seq samples, iModulonDB) DOES contain a WhiB7 iModulon of 8 genes, and it recovers the canonical set -- whiB7, eis, erm(37), Rv1258c, Rv0263c. Rv3196A is not in it; its only iModulon is Unc_4, which contains none of those. The positive control shows the test could have said yes. (b) The chromosomal argument is weaker than proximity suggests: Rv3196A and whiB7 are ~1.5 kb apart on the same (minus) strand, but Rv3197 lies between them ON THE OPPOSITE strand (gaps 127 bp and 33 bp), so read-through from the whiB7 locus is not a simple explanation. (c) The atlas regulation layer lists only Rv0767c and CANNOT see WhiB7 (absent from the TFOE panel, 0 targets) -- an absence of assay, so it neither supports nor refutes. Conclusion: on the evidence available, Rv3196A is not a WhiB7-regulon member, and its rank-2 induction in the alkX mutant has no established explanation. Note that the primary Mtb whiB7-regulon papers (Morris 2005 PNAS; Burian 2012 and 2013 JBC) could not be checked directly: their supplementary tables are behind PMC's download gate, with no preprint. Verdict and confidence are unchanged (dark, low) on purpose: secretion and antigenicity are a status, not a molecular function, and this atlas does not requalify a gene on a status.

Annotated on the H37Rv protein: this gene has no 1:1 ancestral MTBC0 anchor (PE/PPE, paralogue, IS element, or otherwise unanchored CDS).

In the literature (TB corpus sweep) named only in full text

No TB publication mentions this locus tag in its title or abstract (sweep of a ~330k-abstract PubMed TB corpus). A full-text search does however find 4 paper(s) naming this gene in their body text — see the full-text field below. The gene remains dark on function, but it is not unmentioned, and this page no longer claims that it is.

Scope of the sweep: What was actually swept: the H37Rv locus tag and the ortholog locus tags (M. bovis Mb…, M. marinum MMAR_…, M. smegmatis MSMEG_…, M. leprae ML…, M. abscessus MAB_…), plus the gene name where one exists. NOT swept: protein and enzyme names, including family shorthands such as Ldt(Mt4) or VapC28, and their orthographic variants. An absent locus-tag mention is therefore evidence about the TAG, and only indirect evidence about the protein (P18.14).

An antigen status or a virulence phenotype is NOT a molecular function: the verdict stays unchanged. This layer adds the missing context, it does not requalify the gene. 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 of title+abstract: the 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, 2026-07-13.

Intrinsic disorder (sequence + structure) partially disordered

carries a substantial disordered region (17/66 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).

Binding-pocket screen (P2Rank, geometric prediction) detector blind at this length

Read with care. This protein (66 aa) is below the size where this detector has meaningful power: on proven enzymes, only 3.8% (1/26) under 200 aa reach this layer's P2Rank confidence threshold of best_probability >= 0.70, versus 60.5% (75/124) above it (P16.3b calibration, negative control EsxA/EsxB-scale panel). Both figures are recall AT 0.70, which is also the best separation this detector reaches on that panel (Youden J = 0.416) and whose false-positive rate above 200 aa is 18.9% (81.1% specificity); the looser 0.50 that appears elsewhere in this repository would raise recall above 200 aa to 74.2% and drop specificity to 64.9%. A negative or weak pocket result here should NOT be read as evidence against a ligand-binding role -- the test essentially has no power at this length, not that the protein lacks a site. The same holds in the other direction, and that half was missing here: a STRONG score at this length is not a result either. The same calibration panel does carry that other arm below 200 aa, and the two do not separate: the 3.8% above is matched by 6.6% (5/76) of proteins annotated as non-catalytic clearing 0.70 (Fisher p = 1.00; 15.4% against 11.8% at 0.50, p = 0.73). At this length the score therefore carries no measured information in either direction -- which is a stronger statement than untested power, and the reason a strong score here is not a result. P16.3e, calibration phase72b_pocket_calibration.py, 2026-08-03.

Conditional expression context (iModulons)

Member of 1 independently-modulated gene set(s): Unc_4.

iModulon membership (independently-modulated gene sets from a 647-sample RNA-seq compendium): the conditional co-expression context. Co-expression is a regulatory context, NOT a molecular function. Source: iModulonDB / modulome_mtb (Yoo 2022).

CRISPRi vulnerability

Vulnerability index -2.76 (95% CI -13.91 to 8.33). 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, 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=0.002 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=2 is descriptive only and does not predict this. Do not cite this VI as a standalone vulnerability claim.

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; 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.

Lineage-specific relaxation (candidate, one evidence handle) 1 lineage(s)

Candidate lineage-specific loss-of-function signal: rare/absent in the other well-sampled lineages (<5%) but risen in the listed lineage(s). ONE handle, not a verdict change (P8 two-handle policy) -- the general phenomenon of lineage-specific gene loss reflecting ecotype-specific selective pressure is already established at genome scale (Silva-Pereira et al. 2024); this signal on an individual dark gene needs an independent second handle before any requalification.

Caveat: Sample-size heterogeneity across MTBC lineages (up to 4 orders of magnitude, 16 smaller lineages excluded for n<500); essentiality is NOT resolvable per lineage (TnSeq data is H37Rv-only). For the two smallest adequate lineages (L5 n=630, L6 n=1364) the frequency floor bottoms out at the absolute minimum of 3 carrier strains -- a handful of strains from a SINGLE transmission cluster (clonal outbreak) can pass this floor without representing independent evolutionary origins of the disrupting allele; no phylogenetic correction for clonal sampling was applied. Treat low-count hits in these two lineages with extra caution.

Reference: Silva-Pereira TT, Soler-Camargo NC, Sá Guimarães AM 2024, Microbiology Spectrum, doi:10.1128/spectrum.02289-23

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.

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.

Predicted localisation (DeepTMHMM + lipobox) no export signal, no TM helix

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)

No Pfam-A domain above the gathering threshold (hmmscan --cut_ga scan completed).

HHpred profile-profile (no confident hit): No confident hit (best E=8.4, non-significant); biologically incoherent cohort (pilus regulator, archaeal virus coat protein, human hemoglobin). Remains dark. top hits 2IP6_A PapB pilus regulatory protein (84.7%, E=8.4, 52 cols), 3F2E_A SIRV archaeal virus coat protein (77.3%, E=11), 2R80_A hemoglobin subunit alpha (65.9%, E=28). Remote homology search on the deep UniRef30 profile (MPI Toolkit), run for the residual dark set.

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

No experimental structure is deposited for this gene: showing the AlphaFold DB predicted model (mean pLDDT 73.6, confident), 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 neighbours (Foldseek on the ESMFold model, exploratory)

ESMFold model confidence: mean pLDDT 66.4 (low). Low-confidence model: the fold may be unreliable, so treat these structural hits with caution.

Best matches against the PDB, ranked by Foldseek homology probability. A high probability / TM-score suggests a shared fold; unless flagged sig (E < 0.01) these are fold hypotheses, not assignments.

Genomic context (neighbours & predicted operon)

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.

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: Rv3197 (ABC transporter ATP-binding protein), medium confidence (score 546, all STRING channels except text-mining; the partner is chosen on its genomic-context evidence).

All 1 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

• MHC class I presentation (primary, full text read): Sher et al., eLife 2023 (10.7554/elife.84070), Discussion -- "we also identified peptides derived from a Sec pathway substrate (TB8.4), and from uncharacterized hypothetical proteins (Rv1211, Rv3196A)", and "suggesting that either or both of these proteins could be secreted despite lacking readily identifiable secretion signals".
• Secreted fraction of M. marinum, VERIFIED IN THE PRIMARY TABLE (this is new; eLife only cited it): Cronin et al., PNAS 2022 (10.1073/pnas.2123100119), Dataset S2, accession B2HFI1 = MMAR_1367. The protein is quantified in the secreted fraction of all 13 deletion strains versus wild type -- deltaeccCb1 (log2 FC 0.0003, P 0.78), deltaesxA (0.494, 0.57), deltaesxB (-0.032, 0.86), deltaespA (0.505, 0.63), deltaespB (0.038, 0.94), deltaespC (0.222, 0.82), deltaespE (-0.065, 0.87), deltaespF (-0.108, 0.76), deltaespJ (0.451, 0.46), deltaespK (0.034, 0.56), deltape35 (0.547, 0.54), deltappe68 (0.804, 0.49), deltaMMAR_2894 (-0.094, 0.79). None passes the authors' threshold (P < 0.05 and |log2 FC| >= 1): MMAR_1367 is present in the culture filtrate and is NOT an ESX-1-dependent substrate.
• CAVEAT on the point above, and it is now MEASURED rather than merely stated (this atlas, P5.1c, 2026-09-08). Mapping the whole Cronin secreted-fraction inventory onto the atlas shows that detection there tracks ABUNDANCE, not export: predicted-cytoplasmic globular proteins are detected at 42.1%, predicted signal-peptide proteins at 42.0% (Fisher P = 0.07, i.e. no enrichment at all), and in the most abundant PaxDb quartile 80.6% of globular proteins are detected. RpoB and GroEL2, both strictly cytoplasmic, are in the table. Rv3196A sits in that top abundance quartile, so its presence in the M. marinum culture filtrate is what its abundance alone predicts and carries LITTLE independent weight for export. What the Cronin data do establish for this gene is the negative: whatever puts MMAR_1367 in that fraction, it is not ESX-1. The case for export therefore rests on the MHC class I result, which comes from a different group and a different method, not on the culture-filtrate detections.
• NOT verifiable, reported as cited: Bell et al. 2012 (J Proteome Res, 10.1021/pr2007939, PMID 22053987), MS detection of Rv3196A in M. tuberculosis culture filtrates. The reference itself was confirmed independently (tbmonitor), but the article is closed access with no preprint and its supplementary tables could not be retrieved, so the Rv3196A row was never seen here.
• Transcription, alkX mutant: Rv3196A is the second most upregulated gene (log2 FC 3.76, adjusted P 1.77e-30) of the 41 upregulated in an alkX (rv3249c) deletion mutant versus wild-type H37Rv (Microbiol Spectrum 2022, 10.1128/spectrum.01969-22, Table 1). The authors themselves caution that their top hit, whiB7, did not reproduce by qRT-PCR and shows no ChIP-seq binding: this table is an RNA-seq screen, and Rv3196A specifically received no orthogonal confirmation there.
• Transcription, CO2: Rv3196A is listed among genes induced at 5% versus 0.5% CO2 (>1.5-fold, q < 0.05) at both pH values, 1.51-fold at pH 5.7 and 1.57-fold at pH 7.0 (Infect Immun 2025, 10.1128/iai.00568-24, Table 1). Both values sit at the authors' own cutoff: a real but weak effect.
• Fourth full-text hit, no functional content: Rv3196A is named only inside a list of genes with an elevated abundance of secondary-structure-forming CGGC motifs (Sci Rep 2026, 10.1038/s41598-026-47170-w). A sequence-composition observation, no claim about the protein.
• This atlas, converging independently: no signal peptide, no transmembrane helix, predicted globular (DeepTMHMM); abundance 733 ppm, rank 301 of 3519, 14 of 16 PaxDb datasets; genus-core conservation (present in 31 of 53 non-tuberculous mycobacteria, mean 76.0% identity); non-essential (DeJesus 2017, 2 TA sites only -- weak power); singleton operon on the minus strand.

Ancestral MTBC0 protein sequence

>H37Rv|Rv3196A|
MQEGGPQETMSARSTQHDAADALFRAIIETLDKHRNERTLTEDVLDTLARAYASISTNVPEQGRLG