Rv0057 Still unknown · low

Annotation: from legacy to revised

Curation note: 2026-10-02 (mtbc/Rv0057 project, back-propagation P52.2, destination C1, status: in preparation, not peer-reviewed). The dedicated project follows the DnaA-site argument outside M. tuberculosis. A scan anchored on the conserved position (Hamming distance of at most 2 from the published consensus TTATTCACA, both strands, in the window homologous to the Rv0057 site; 2 is the smallest threshold that recovers the known H37Rv site, whose best match lies at distance 2) finds a best hit within distance 2 in 47 of 48 non-tuberculous Mycobacterium genomes, with 172 hits observed against 24.7 expected by chance from the local GC content (ratio 7.0); the best hit is often the same sequence (TTGTCCACA or TTCTCCACA) at independent homologous positions. In 15 Corynebacteriales outside the genus the ratio is 4.3 and heterogeneous, in 4 other Actinobacteria 7.3 (small sample), and in the two controls outside the phylum (E. coli, B. subtilis) 0.7, which is chance level. The p values are Poisson and not corrected for phylogenetic structure. Rv0057 itself is absent from the 21 genomes outside the genus, so the anchor there is dnaB. Two tests of the idea that this conservation reflects a double constraint (DNA site plus protein sequence) did not support it: the number of quasi-invariant protein positions in the motif window is not higher than chance (hypergeometric p about 0.051), and conservation of synonymous third codon positions in that window across 45 non-tuberculous orthologues is not higher than in unrelated control windows (85th percentile, permutation p about 0.19, one control window scores higher than the test window), with low power (8 codons). A Boltz-2 and AF-Multimer model of the protein with a DNA duplex carrying the real site, against a scrambled paired control, shows no concentration of atomic contacts on the conserved block around residues 90 to 97; for a protein predicted fully disordered (mean pLDDT 32), such a model is weakly informative. This is conservation of a sequence position: it does not show that the site is functional in M. tuberculosis, nor that the protein binds it. 2026-10-02 (mtbc/Rv0057 project, back-propagation P52.2, destination C1, status: in preparation, not peer-reviewed). The presence count of this protein in non-MTBC Mycobacterium genomes served in this record (39 of 53, tblastn, identity of at least 30% and query coverage of at least 50%) is a strict-threshold count. The dedicated project searched 48 of the same 53 genomes with a search anchored on the gene order (ssb, rpsR1, rplI, Rv0057 and dnaB on one contig) and detects the locus in 45 of 48, between rplI and dnaB, with the five-gene order conserved in all 45. On these 48 genomes the strict atlas call gives 35 detections; the 10 genomes that differ (M. asiaticum, M. celatum, M. cookii, M. heckeshornense, M. kyorinense, M. leprae, M. lepromatosis, M. liflandii, M. shimoidei, M. xenopi) are detected in the project's search with 49 to 62% identity and 31 to 86% coverage. Seven of them are below the atlas coverage threshold (31 to 47%), which would account for the difference if the two searches measure coverage alike (not checked); for the other three (M. leprae, M. lepromatosis, M. liflandii, 79 to 86% coverage in the project's search) the difference is NOT explained here and has not been re-checked on the atlas side. The three genomes without the locus in the project's search (M. gallinarum, M. hackensackense, M. syngnathidarum) have the four flanking genes present at 70 to 100% coverage, which supports a real loss. Both counts are kept, and the classification 'genus-core' holds with either. The conserved gene order is not independent evidence for function: the flanking genes are core genes whose adjacency can be inherited. In the project's 63-sequence ColabFold alignment, 20 of 173 positions are at least 90% identical (columns covered by at least 20 sequences) although the protein is predicted fully disordered: sequence conservation without a fold, a property that says nothing about the molecular function. 2026-10-02 (mtbc/Rv0057 project, back-propagation P52.2, destination C1, status: in preparation, not peer-reviewed). Resistance association of the lineage that carries the natural loss of this gene. Lineage 1.2.1 (carrier of the start-lost deletion described above; Coll classification) is not enriched in genotypic resistance. Over the complete rifampicin resistance determining region of rpoB (190 non-synonymous variants, codons 426 to 452; any non-synonymous variant of the window, not only the graded resistance mutations of the WHO catalogue, which were not cross-checked variant by variant), 6.62% of L1.2.1 strains (724 of 10,930) carry at least one, against 9.07% in the sister group 1.1 (1,246 of 13,741; odds ratio 0.711, 95% CI 0.647 to 0.782) and 21.26% in 1.2.2 (856 of 4,027; odds ratio 0.263, 0.236 to 0.292). The stratified Cochran-Mantel-Haenszel pooled odds ratio is 0.473 (0.440 to 0.508) with strong heterogeneity (Breslow-Day p close to 0), carried by 1.2.2 (2 to 3 times more carriers than the other two groups, cause not investigated) rather than by a uniform effect of L1.2.1; against the most comparable sister group (1.1) the effect is moderate. The same pattern holds for katG S315T (pooled odds ratio 0.564) and rpoB S450L (0.513). This is a lineage-level association on genotypic resistance. It does not attribute any resistance effect to the loss of Rv0057, and it neither supports nor contradicts the tolerance phenotype of the CRISPRi knockdown (Eldesouky et al. 2026), which is a different phenotype. 2026-09-29 (mtbc/Rv0057 project, back-propagation P52.2, destination C1, status: published). CRISPRi knockdown of Rv0057 lowers intracellular pH and ATP and increases tolerance to isoniazid and rifampicin, and Rv0057 is naturally repressed under proton-motive-force stress that promotes tolerance (Eldesouky et al., mBio 2026). This is a cellular phenotype of the knockdown: it does not establish a molecular function, and the mechanism linking the protein to pH homeostasis is not resolved. The article does not name the locus in its PubMed-indexed fields, so the atlas literature sweep cannot find it; the dedicated project found it in the full text. 2026-09-29 (mtbc/Rv0057 project, back-propagation P52.2, destination C1, status: unpublished, not peer-reviewed). The DnaA-binding site that Hicks et al. (2020) attribute to this locus is the most enriched peak of their ChIP-seq experiment in M. smegmatis (224.9-fold, against 44.1 for oriC). Re-analysis of their published supplementary tables (S8 to S10) by the dedicated project places it inside the coding sequence of Rv0057, not in an intergenic region upstream of it: the best match to the published DnaA consensus (TTATTCACA, Hamming distance 2) lies at codons ~90 to 97. This is an argument of position; the dedicated project does not read it as a function of the protein. 2026-09-29 (mtbc/Rv0057 project, back-propagation P52.2, destination C1, status: in preparation, not peer-reviewed). The dedicated Rv0057 project resolves the natural loss of this gene below lineage level. The deletion NC_000962.3:59895:AT:A (frameshift_variant and start_lost, p.Met1fs, destroying the initiator ATG) is carried by 8,634 of the 8,991 strains of Lineage 1.2.1 (96.0%) and by 99.8% of the 8,648 carriers worldwide, across 42 countries and 214 independent BioProjects (1993 to 2026); the call was checked against placement, cross-mapping and translation artefacts and against the raw reads of 10,548 strains. The loss of function is PREDICTED from the start_lost and frameshift annotation; no protein-level or phenotypic test was made on these isolates. The `lineage_relaxation` layer of this record independently reports 8,521 of 25,332 strains with a disrupted copy in L1 (33.6%) against 20 of 117,642 elsewhere (OR 2,908); it is kept as an independent measurement. The two counts use different denominators (sub-lineage carriers of one deletion versus disrupted copies in the whole lineage) and the gap between 8,521 and 8,634 is not reconciled.

In the literature (TB corpus sweep) antigen / vaccine

1 TB publication mentions this gene. Studied as an immunogen: recombinant Rv0057-Rv1352 fusion protein elicits immune responses. reformulation, not a quotation

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) dark by construction (IDR-orphan)

both sequence (metapredict) and structure (low AlphaFold pLDDT) indicate little stable globular structure: this gene is dark BY CONSTRUCTION — there is no confident fold for structure/homology search to match, so its obscurity is expected, not a pipeline failure

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).

How to read this gene's constraint (qualifying attributes, not exclusions) HIGH

Genomic-neighbour overlap (structural caveat) co-directional · 4 % 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.

Conditional expression context (iModulons)

Member of 1 independently-modulated gene set(s): WhiB4 (whiB4).

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 -6.98 (95% CI -13.61 to 0.98). 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=0.435 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=8 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 Rv0057; the revised annotation above goes beyond that record. UniProt may have been recurated since: check the live entry.

Conservation & selection (intra-MTBC, 142 601 strains, snapshot of 2026-09-29) pseudogene candidate

Read the disruption percentage against its ceiling, not against 100%. The strain collection is deeply imbalanced by clade, so a loss fixed in an entire lineage still shows a small overall fraction: a gene disrupted in every L6 strain caps at 0.96% of the collection, in every M. caprae strain at 0.20%, in every M. microti strain at 0.02%. Of the 705 clades in this collection, 689 have a ceiling below 1%. A figure of "0.15% of strains" is therefore uninterpretable on its own — it can be noise or a complete, lineage-defining loss. Use the clade attribution row above, which gives the sub-clade sizes, and never read the percentage alone.

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.

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) not detected

Not detected in the mass-spectrometry datasets integrated by PaxDb. This is not evidence of absence, and this atlas can say which bias dominates, because it measured it on its own 482 undetected proteins. Size is the main factor: undetected proteins have a median length of 145 aa against 295 aa for detected ones, and 25.3 % of them are shorter than 100 aa against 6.0 %. Predicted localisation matters far less than usually assumed: detection rates are 89.3 % for globular, 84.9 % for transmembrane, 88.0 % for lipoproteins and 77.8 % for signal-peptide proteins — so a short protein is under-sampled whatever its predicted compartment, and hydrophobicity alone is a weak excuse. Condition-specific expression and few tryptic peptides (PE/PPE repeats) add to this.

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; near-orphan. Remains dark. top hits Neff=1.2 (near-orphan), single trivial hit (30.1%, E=220). 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 32.0, very low), 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.

Genomic context (neighbours & predicted operon) operon of 5

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: dnaB (replicative DNA helicase), high confidence (score 889, all STRING channels except text-mining; the partner is chosen on its genomic-context evidence). Read this as a lead toward a shared pathway or operon, not as evidence of a physical complex: when this atlas co-folded 98 such context-only pairs, their predicted interfaces were no more confident than those of length-matched control pairs.

All 12 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

• MTBC0 PGAP product: 'hypothetical protein' (PGAP itself leaves it unannotated)
• Pfam (hmmscan --cut_ga): no domain above threshold
• Foldseek on the ESMFold model: no structural hit -- one of the few truly dark genes of the set

ESM Atlas signal (exploratory)

Ancestral protein hash be9ce41de1497f9ff42715afc4b5a900 · 10 ESM-space neighbours (max similarity 0.607). SAE features are orienting indices, not validated domains.

Ancestral MTBC0 protein sequence

>mtbc0_000062|Rv0057|
MPVVTAVGRRRGFAMPWVSTARSGAVMLANYSAGVCGRVSSPGLNVRKMCLKANTPGAVTWLDTPKRFLSTQTASRCMAVNSSDVVTGRIDPQVLHTPLNTDVDGYAHAMHSSINSGPLEYLPATFSVFPALGDVGDLGGGVGAATYALDRLSNMRSGACVGGGESPWRSLMT