Rv0007 Resolved · medium

Annotation: from legacy to revised

Curation note: 2026-09-05 (P2.19, external report from the companion project mtbc/Rv0007, closed 2026-09-03 on a do-not-disseminate verdict -- this fiche is the only public output of that work). Corrects an inversion: the fiche stated that full-length MtbCofA interacts with BOTH aPBPs, whereas the primary source reports a weak signal with PonA2 and explicitly none with PonA1. Partner reported as PonA2 only, with the source's own internal inconsistency stated rather than silently resolved. Confidence lowered high -> medium: one heterologous assay (POLAR in E. coli, isolated TM domains), called weak by its authors, with no trace in curated interaction databases (IntAct 0/3 proteins, STRING experimental 0/15 partners). Adds as a positive result the residue-level alignment of the C-terminal DUF3566 module against the characterised C. glutamicum ortholog, which is not published in this form. The requalified verdict is unchanged: family assignment does not depend on the partner debate.

In the literature (TB corpus sweep) characterised phenotype

2 TB publications mention this gene. Named **CofA** by Sher, Lim & Bernhardt (eLife 2020): a conserved actinobacterial cofactor for the bifunctional class-A PBP cell wall synthases (aPBPs). In a heterologous POLAR two-hybrid assay in E. coli, full-length Mtb CofA gives a weak interaction signal with the transmembrane domain of **PonA2** (Rv3682, the Mtb ortholog of Cglu PBP1a) and explicitly none with that of PonA1 (Rv0050, the PBP1b ortholog); deleting the Mtb-specific N-terminal extension strengthens it. Genetic support is indirect and concerns the OTHER paralog: Tn-seq shows Mtb CofA becomes less dispensable when the non-cognate aPBP PonA1 is inactivated, which the authors read -- through the known PonA1-PonA2 synthetic lethality -- as consistent with CofA being required for PonA2 function. Protein-fusion/localisation experiments place CofA at the division septum alongside its aPBP partner, conserved across the Corynebacterineae. A secondary 2025 report (Sci Rep, Tn-seq screen for rifampicin/streptomycin tolerance) independently lists Rv0007 as peptidoglycan synthesis and describes a genetic interaction with **ponA2** specifically -- consistent with the primary source, now verified against its full text. Consistent with the existing transmembrane domain (DUF3566) and TnSeq phenotypes (required in vivo, in vitro passage, altered under meropenem). A phenotype/genetic-interaction is not itself a solved molecular mechanism: treat CofA as a strong, named lead for a dedicated deep-dive, not a settled function. reformulation, not a quotation

Source sentence, verbatim: « Using the full-length CofA from Mtb (MtbCofA-FL) as the prey, a weak interaction signal was detected with the transmembrane domain of Mtb PonA2, the paralog of Cglu PBP1a, but not the transmembrane domain of Mtb PonA1, the paralog of Cglu PBP1b. »
Corrected 2026-09-05 (P2.19, external report from the companion project mtbc/Rv0007). The previous summary left the physical partner undetermined and flagged the 2025 ponA2 claim as not verified against the primary source, on the grounds that the eLife epistasis data concern PonA1. The full text has now been re-read: the epistasis data do concern PonA1, but the INTERACTION data name PonA2 and explicitly exclude PonA1 -- two different experiments about different things, which the fiche header had conflated into interacts with both. Verbatim source sentence now shown above. One sentence of that paper's Results attributes the CofA-DeltaN gain to PonA1; its Discussion, its figure-supplement title and its orthology notes all say PonA2, and this atlas follows the latter.

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).

CRISPRi vulnerability

Vulnerability index 1.00 (95% CI -2.03 to 4.64). 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=0.230 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=5 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 Rv0007; 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) 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.

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

Conditional fitness of transposon-disruption mutants across 7 significant condition(s) (|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.

Predicted localisation (DeepTMHMM + lipobox)

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 59.7, 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)

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.

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: gyrA (DNA gyrase subunit A), high confidence (score 943, all STRING channels except text-mining; the partner is chosen on its genomic-context evidence).

Showing the 15 strongest of this gene's 58 partners at medium confidence (12 at high confidence). The table is an extract, not a neighbourhood: across the proteome the real median degree is 34 while the served median is 15, so no hub, degree distribution or centrality may be read on this table. None of the 58 partners 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

• POLAR two-hybrid assay (heterologous, in E. coli; bait = isolated aPBP transmembrane domain): full-length MtbCofA gives a weak interaction signal with the TM domain of PonA2 (Rv3682, residues 1-41) but NOT with that of PonA1 (Rv0050, residues 120-167); deleting the Mtb-specific N-terminal extension (1-190) strengthens the signal (Sher et al. 2020, eLife, PMID 32167475, Figure 6D and Figure 6-figure supplement 1).
• Internal inconsistency of the primary source, resolved in favour of PonA2: one Results sentence names PonA1 for the CofA-DeltaN gain, while the Discussion, the figure-supplement title ("Interaction of full length MtbCofA with PonA2") and the orthology notes in the Figure 6D legend all name PonA2 (full text re-read 2026-09-05).
• No curated physical-interaction trace: IntAct queried directly returns 0 interactions for each of CofA (P9WMA7), PonA1 (P71707) and PonA2 (I6YGX2); in this atlas's own STRING layer, all 15 partners of Rv0007 have experimental=0, and neither PonA1 nor PonA2 appears among them (companion project mtbc/Rv0007, 2026-09-03).
• Family assignment is independent of that debate: Pfam DUF3566 (PF12089) at i-E 1.7e-41 over residues 187-302, plus residue-level alignment of the C-terminal module against the characterised C. glutamicum ortholog CofA/cgp_0016 (UniProt Q8NUC5, 114 aa, DUF3566): 34.3% identity over 105 aligned positions (BLOSUM62 local), the aligned window covering both DeepTMHMM helices (TM1 206-226, TM2 260-282) and 93% of the C. glutamicum protein. The N-terminal 1-186 extension is disordered and specific to Mtb, absent from the ortholog (companion project mtbc/Rv0007, 2026-09-03; not published in this form elsewhere).
• Independent Tn-seq genetic interaction: MtbCofA (Rv0007) becomes less dispensable upon inactivation of the non-cognate aPBP PonA1, read by the primary source as consistent with a requirement for PonA2 function via the known PonA1-PonA2 synthetic lethality (Kieser et al. 2015, PNAS, PMID 26438867).
• Consistent with this atlas's own Tn-seq phenotype data (present before this connection was made): altered fitness under meropenem and in mouse infection.
• Found via full-text-only literature sweep (P16.2a-septies, phase138, 2026-08-17) -- invisible to the title+abstract PubMed sweep because the locus tag appears only in a figure legend.

Ancestral MTBC0 protein sequence

>mtbc0_000007|Rv0007|
MTAPNEPGALSKGDGPNADGLVDRGGAHRAATGPGRIPDAGDPPPWQRAATRQSQAGHRQPPPVSHPEGRPTNPPAAADARLNRFISGASAPVTGPAAAVRTPQPDPDASLGCGDGSPAEAYASELPDLSGPTPRAPQRNPAPARPAEGGAGSRGDSAAGSSGGRSITAESRDARVQLSARRSRGPVRASMQIRRIDPWSTLKVSLLLSVALFFVWMITVAFLYLVLGGMGVWAKLNSNVGDLLNNASGSSAELVSSGTIFGGAFLIGLVNIVLMTALATIGAFVYNLITDLIGGIEVTLADRD