Doc ("death on curing") is the 126-residue toxin of the phd/doc type II toxin-antitoxin module carried by bacteriophage P1, which lysogenises Escherichia coli as a single-copy plasmid prophage. It consists of a single Fido (Fic/Doc) domain whose Fic motif is degenerate relative to the canonical AMPylating motif, and this degeneracy makes it a protein kinase rather than an adenylyltransferase: in the host cytoplasm Doc transfers the gamma-phosphate of ATP or GTP onto a single conserved threonine (Thr-382 of mature E. coli EF-Tu, Thr-383 in UniProt numbering) in domain 3 of the translation elongation factor EF-Tu. The modified EF-Tu can no longer form the ternary aminoacyl-tRNA:EF-Tu:GTP complex, so elongation halts, polysomes and mRNA are stabilised, and the bacterium arrests growth and dies. The same active site runs in reverse when ADP or GDP accumulates, removing the phosphate, which may allow return from dormancy. Doc acts once its cognate antitoxin Phd, which is labile and degraded by host ClpXP, disappears: loss of the P1 prophage therefore kills the plasmid-free daughter cell, the post-segregational killing that stabilises the prophage in the population. Doc has a second, mechanistically separate role in autoregulating its own operon. It does not bind DNA; instead monomeric Doc bridges two Phd dimers and orders Phd's intrinsically disordered C-terminal domain, which in turn structures Phd's N-terminal DNA-binding domain, deepening repression at low Doc:Phd ratios and derepressing the operon when Doc accumulates (conditional cooperativity).
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000043 | TOO GENERAL | Summary: Keyword-projected root-level binding term. True - Doc binds ATP or GTP as phosphate donor and ADP or GDP for the reverse reaction - but entirely subsumed by the ATP binding and GTP binding rows accepted in this review. Reason: The claim is correct and is not in doubt; the term simply carries no information once GO:0005524 ATP binding (accepted) and the proposed GO:0005525 GTP binding row are in place, both of which are is_a descendants of GO:0000166. Flagged as uninformative rather than wrong, per the TOO_GENERAL definition. Supporting Evidence: PMID:24141193 We found that Doc phosphorylated EF-Tu (using either ATP or GTP) at a relatively high rate of ~20 EF-Tu molecules per second by one molecule of Doc |
| GO:0003677 DNA binding | IEA GO_REF:0000043 | REMOVE | Summary: Projection of UniProt keyword KW-0238 (DNA-binding), which the Q06259 entry carries alongside the Repressor keyword because Doc autoregulates the phd/doc operon. The experimental literature shows the opposite: Doc regulates transcription without touching DNA, and the DNA-binding domain in the complex belongs to Phd. Reason: This is an electronic keyword mapping that the primary literature directly refutes, which is the sanctioned ground for REMOVE. Magnuson and Yarmolinsky tested DNA binding by the non-toxic but regulation-competent DocH66Y protein and found none, while the same protein strongly enhanced Phd's operator binding; Doc enters the protein-DNA complex (supershift) but contributes no DNA contact of its own. The structural work makes the division explicit: Doc binds the intrinsically disordered C-terminal domain of Phd and thereby orders Phd's N-terminal DNA-binding domain. Doc has only a Fido domain (PROSITE PS51459) and no recognisable DNA-binding motif. Comparator: in GOA, Phd (UniProtKB:Q06253) carries GO:0003677 by IDA from PMID:20603017, which is where the activity actually sits. The regulatory role is not lost by this removal - it is recaptured by the proposed GO:0003714 and GO:0045892 rows below. Supporting Evidence: PMID:9829946 DocH66Y failed to bind DNA in vitro yet enhanced the affinity, cooperativity, and specificity with which Phd bound the operator. PMID:20603017 The binding of Doc to the intrinsically disordered C-terminal domain of Phd structures its N-terminal DNA-binding domain file:BPP1/doc/doc-uniprot.txt DOMAIN 4..120 |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:24448800 Doc toxin is a kinase that inactivates elongation factor Tu. | ACCEPT | Summary: CACAO IDA row, and the central molecular function of this protein. Cruz et al. 2014 showed by direct in vitro assay that Doc transfers the gamma-phosphate of ATP to a single E. coli protein, identified as EF-Tu, at Thr-382. Reason: What the paper shows for this gene, assay by assay. In a defined coupled transcription/translation system Doc gave no signal with alpha-32P-ATP but a strong single band just above 40 kDa with gamma-32P-ATP (Fig. 2, A and B) - i.e. phosphate transfer, not adenylylation. The band was abolished by the Fic-motif mutant H66Y and by preincubation with the antitoxin Phd, excluding a contaminating kinase (Fig. 2B, Fig. 3A). The target was identified as recombinant EF-Tu (Fig. 3A), confirmed as the only Doc substrate in whole-cell lysate (Fig. 2C), and release of alpha-32P-ADP was shown by TLC (Fig. 3B). MS/MS at 100% coverage gave a single site, Thr-382, and the T382A mutant was not phosphorylated (Fig. 6). Castro-Roa et al. reached the same conclusion independently in the same year. The CACAO term matches this result: it is not too downstream (the assay is the catalytic activity itself), the evidence code fits a direct in vitro assay, and GO:0004674 is the most specific existing GO term available: GO created a threonine-specific term, GO:0106311 protein threonine kinase activity, and merged it into GO:0004674, so the serine/threonine parent is as precise as the ontology allows and is the canonical landing place for a threonine-only kinase. See the GO:0106310 row for the same point and the lookup that confirms the merge. Accepted as the core molecular function. Supporting Evidence: PMID:24448800 potent phosphorylation of a single product migrating just above 40 kDa was observed, indicating that Doc is in fact a kinase PMID:24448800 Sample analysis resulted in 100% protein coverage and yielded a single EF-Tu phosphorylation site in this Doc-treated sample at Thr-382 PMID:24448800 we demonstrated that Doc was unable to phosphorylate a T382A EF-Tu mutant PMID:24141193 Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering EF-Tu unable to bind aminoacylated tRNAs |
| GO:0004674 protein serine/threonine kinase activity | IEA GO_REF:0000003 | ACCEPT | Summary: EC-to-GO projection of EC 2.7.11.1 (non-specific serine/threonine protein kinase), which UniProt assigned to Q06259 on the strength of PubMed:24141193. It reinforces the CACAO IDA row for the same term. Reason: Unlike most EC projections on phage proteins this one is not a guess: the EC number on the Q06259 DE line carries ECO:0000269 with PubMed:24141193, i.e. it was assigned from the experimental demonstration of kinase activity. Rows sharing a term reinforce each other, so this is accepted in step with the IDA row above rather than treated as a redundant duplicate. The one caveat - that only a threonine acceptor has been demonstrated - applies to the ontology's granularity, not to this row, and is handled on the GO:0106310 row. Supporting Evidence: file:BPP1/doc/doc-uniprot.txt Phosphorylates EF-Tu on 'Thr-383' in vitro; can use ATP or GTP |
| GO:0005515 protein binding | IPI PMID:20603017 Allostery and intrinsic disorder mediate transcription regul... | MODIFY | Summary: IntAct row for the Doc-Phd toxin-antitoxin interaction. The interaction is real and extremely well characterised (four crystal structures of the complex), but bare protein binding says nothing about what the interaction does. Reason: Per the repository's protein-binding policy, a bare GO:0005515 row is replaced by a more informative molecular function where the cited paper supports one. Here it does: Garcia-Pino et al. show that monomeric Doc engages two Phd dimers and that binding to Phd's disordered C-terminal domain structures Phd's N-terminal DNA-binding domain, which is the mechanism by which Doc co-represses the operon. GO:0003714 transcription corepressor activity - an activity exerted by binding a DNA-binding transcription factor rather than DNA - is exactly this, and it is the role Magnuson and Yarmolinsky established genetically and by EMSA. Removal of the generic term does not mean the Doc-Phd interaction is doubted. Proposed replacements: transcription corepressor activity Supporting Evidence: PMID:20603017 Here we show that monomeric Doc engages two Phd dimers on two unrelated binding sites. PMID:20603017 The binding of Doc to the intrinsically disordered C-terminal domain of Phd structures its N-terminal DNA-binding domain |
| GO:0005515 protein binding | IPI PMID:24141193 The Fic protein Doc uses an inverted substrate to phosphoryl... | MODIFY | Summary: IntAct row for the Doc-EF-Tu interaction (P0CE47 is E. coli tufA). The interaction is the enzyme-substrate encounter of a kinase, which the generic term fails to convey. Reason: Castro-Roa et al. did not merely detect binding; they showed Doc phosphorylates the bound EF-Tu on Thr382 and that the R64G mutant loses binding to EF-Tu-GDP. The informative molecular function is therefore the kinase activity for which EF-Tu is the substrate, already asserted by IDA elsewhere in this review, and the bare binding term is replaced by it rather than kept. Removing the generic term does not dispute the IntAct interaction. Proposed replacements: protein serine/threonine kinase activity Supporting Evidence: PMID:24141193 Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu file:BPP1/doc/doc-uniprot.txt R->G: No binding to EF-Tu-GDP. |
| GO:0005524 ATP binding | IEA GO_REF:0000043 | ACCEPT | Summary: Keyword-projected ATP binding. Directly supported: ATP is the phosphate donor in the kinase reaction, and the Fido active site binds it in an inverted orientation that presents the gamma- rather than the alpha-phosphate to the acceptor. Reason: Doc phosphorylates EF-Tu using ATP (or GTP) and releases ADP, shown by gamma-32P transfer and by TLC detection of alpha-32P-ADP; the N78W mutant shows a 100-fold decrease in affinity of the Doc-EF-Tu-GTP complex for the ATP analogue AMP-PNP, confirming a genuine nucleotide site rather than keyword inference. Kept as a core part of the catalytic mechanism rather than marked general, because the nucleotide identity is mechanistically informative here (the ATP/GTP versus ADP/GDP distinction switches the enzyme between kinase and phosphatase directions). Supporting Evidence: PMID:24141193 We found that Doc phosphorylated EF-Tu (using either ATP or GTP) at a relatively high rate of ~20 EF-Tu molecules per second by one molecule of Doc file:BPP1/doc/doc-uniprot.txt N->W: No change in binding to EF-Tu, 100-fold decrease of affinity of doc-EF-Tu-GTP complex for AMP-PNP |
| GO:0016301 kinase activity | IEA GO_REF:0000120 | TOO GENERAL | Summary: Combined IEA projection from the Doc InterPro family (IPR006440) and the Kinase keyword. True but subsumed by the accepted protein serine/threonine kinase activity row. Reason: GO:0016301 is an ancestor of GO:0004674, which is accepted here on direct experimental evidence, so the row adds nothing. The underlying family assignment is sound - Doc is the eponymous member of IPR006440 and the prediction that all Doc-family toxins with the degenerate Fic motif are kinases comes from this work - so this is a granularity judgement on the term, not a doubt about the claim. Supporting Evidence: PMID:24448800 By extension, all Doc toxins with this Fic domain are predicted to function as kinases as well. |
| GO:0016740 transferase activity | IEA GO_REF:0000043 | TOO GENERAL | Summary: Keyword-projected root transferase term, two levels above the accepted kinase activity. Reason: Correct (phosphate group transfer) but uninformative: GO:0016740 is an ancestor of GO:0016301 and GO:0004674, the latter accepted on direct evidence in this review. Judged on the term, not the evidence; the claim is not in question. Supporting Evidence: PMID:24448800 Doc is a protein kinase that blocks translation elongation through phosphorylation of Thr-382 of the essential elongation factor EF-Tu |
| GO:0039604 symbiont-mediated suppression of host translation | IDA PMID:18398006 Bacterial addiction module toxin Doc inhibits translation el... | ACCEPT | Summary: CACAO IDA row. Liu et al. 2008 induced Doc in E. coli and measured a rapid arrest of growth with marked inhibition of translation, no perturbation of transcription or replication, stabilised polysomes and a large increase in mRNA half-life. The term matches that outcome exactly. Reason: What the paper shows for this gene: after Doc induction protein synthesis shuts down while transcription and replication continue, so the block is specific to translation; polysomes are stabilised rather than running off, placing the block at elongation; mRNA half-life rises substantially; and no mRNA cleavage occurs, distinguishing Doc from the RNase toxins. E. coli is the host of this phage and Doc is the phage-encoded symbiont protein, so GO:0039604 (a symbiont inhibiting or disrupting the translation of host mRNA into protein) is the right branch and the right granularity - neither a downstream phenotype read as a process nor a parent-level restatement. One caveat does not change the action: this paper's mechanistic model, direct occlusion of the 30S subunit at the hygromycin B site, was overturned in 2013-2014 (recorded as a finding_review on this reference). GO:0039604 asserts the outcome and not the mechanism, so the term stands while the explanation has changed. The cached record is abstract-only and the GONUTS page that would show the student's reading is offline, so figure-level checking was not possible; the abstract states the annotated claim directly. Accepted as a core process. Supporting Evidence: PMID:18398006 Doc expression resulted in rapid cell growth arrest and marked inhibition of translation without significant perturbation of transcription or replication. PMID:18398006 However, Doc did not cleave mRNA as do other addiction-module toxins whose activities result in translation inhibition. PMID:24141193 Inhibition of translation by Doc is the direct result of phosphorylation of the translation elongation factor EF-Tu without the involvement of the ribosome itself or of any other player of the translation machinery. |
| GO:0039604 symbiont-mediated suppression of host translation | IMP PMID:11222604 Postsegregational killing mediated by the P1 phage "addictio... | ACCEPT | Summary: CACAO IMP row. Hazan et al. 2001 report that host protein synthesis is inhibited under phd-doc post-segregational killing conditions - the same claim as the IDA row above, reached genetically in the natural prophage-loss setting rather than by overexpression. Reason: The cached publication is abstract-only (full_text_available false), and the GONUTS wiki page that would record the student's reading of the figures is offline ("This site is temporarily offline while we perform upgrades"), so the figure-level detail cannot be checked. The abstract nonetheless states the annotated claim directly, for this module in its native context, and the evidence code (IMP, from comparison of strains retaining versus losing the prophage and of mazEF-plus versus mazEF-minus hosts) fits that design. Under the curator-deference rule an experimental row whose full text cannot be read is not removed on abstract-level doubt; here there is no doubt to begin with, because the claim is independently established by the IDA row and by the EF-Tu kinase mechanism. Rows sharing a term reinforce each other, so this is accepted in step with the IDA row. Note that what this paper adds beyond the translation block - the mazEF dependence, and the RelE induction reported elsewhere - are host responses downstream of Doc, not activities of Doc, and were correctly not annotated to it. Supporting Evidence: PMID:11222604 we demonstrate that under conditions of postsegregational killing, mediated by phd-doc, protein synthesis of E. coli is inhibited |
| GO:0044696 killing by virus of host cell by post-segregational killing | IMP PMID:11222604 Postsegregational killing mediated by the P1 phage "addictio... | ACCEPT | Summary: CACAO IMP row for the defining phenotype of this gene. GO:0044696 was written for exactly this phenomenon - its definition describes a two-gene extrachromosomal module whose long-lived toxic product kills daughter cells that lose the viral DNA - and Doc is the toxic product in question. Reason: What the paper shows for this gene: Hazan et al. work with the P1 phd-doc module and its post-segregational killing of plasmid-free cells, and show that this killing requires the host mazEF system, so the phenotype itself is assayed with doc present and the readout is cell death on prophage loss. The term is not a downstream phenotype misread as a process: post-segregational killing is the biological process Doc evolved to execute, and Doc performs the committed step in it by phosphorylating EF-Tu and shutting down host translation, which satisfies the participation test. It is also at the right granularity - more specific than its parent killing by virus of host cell, and the most specific term GO offers. The cached record is abstract-only and GONUTS is offline, so figure-level checking was impossible, but the claim is independently established in the founding paper for this gene, which named doc for death on curing. GO:0044696 is a host-directed term, so the "own-process term for a host-directed activity" failure shape catalogued for phage proteins does not apply. Accepted as a core process. Supporting Evidence: PMID:11222604 The extrachromosomal addiction module phd-doc, located on the P1 prophage, is responsible for the postsegregational killing effect (death of plasmid-free cells). PMID:8411153 The plasmid withdrawal response depends on a gene named doc (death on curing), encoding a 126 amino acid protein. PMID:8411153 In cells that retain P1 the killing is prevented by the product of a gene named phd (prevent host death), encoding a 73 amino acid protein. |
| GO:0106310 protein serine kinase activity | IEA GO_REF:0000116 | MODIFY | Summary: Rhea-to-GO projection of RHEA:17989, the generic seryl half of EC 2.7.11.1 that UniProt lists under CATALYTIC ACTIVITY beside the threonyl reaction RHEA:46608. No serine phosphorylation by Doc has ever been observed; the single demonstrated acceptor is a threonine. Reason: This is a lateral correction, not a refinement: the right acceptor residue is threonine, not serine. Cruz et al. searched the EF-Tu phosphopeptides for differential modifications of phosphotyrosine, phosphoserine and phosphothreonine at 100% sequence coverage and recovered one site, Thr-382, with the T382A mutant no longer a substrate; Castro-Roa et al. independently identify Thr382. The two UniProt CATALYTIC ACTIVITY lines are the generic pair that accompanies EC 2.7.11.1, and only RHEA:46608 (threonyl) is backed by data, so the Rhea mapping has projected the unsupported half. There is no threonine-specific sibling of GO:0106310 to point at, and that is a resolved GO decision rather than a gap: GO:0106311 protein threonine kinase activity was created and then merged into GO:0004674, which a QuickGO lookup of GO:0106311 confirms by resolving to GO:0004674. So the canonical landing place for a kinase whose only demonstrated acceptor is a threonine is the serine/threonine parent, which this gene already carries by CACAO IDA and which core function 1 uses. The same conclusion is reached for this shape in the AAK1, AKT1 and CSNK2A1 reviews in this repository. Proposed replacements: protein serine/threonine kinase activity Supporting Evidence: PMID:24448800 re-searched with SEQUEST against the EF-Tu sequence without taking into account enzyme specificity and with differential modifications of phosphorylated tyrosine, serine, and threonine residues PMID:24448800 Sample analysis resulted in 100% protein coverage and yielded a single EF-Tu phosphorylation site in this Doc-treated sample at Thr-382 PMID:24448800 Doc is a kinase that inactivates EF-Tu at a highly conserved threonine at the C-terminal end |
| GO:0005525 GTP binding | IDA PMID:24141193 The Fic protein Doc uses an inverted substrate to phosphoryl... | NEW | Summary: Proposed new annotation. Doc uses GTP as phosphate donor as readily as ATP, and GDP (like ADP) drives the reverse, dephosphorylating reaction - so GTP binding is a demonstrated, mechanistically meaningful capability that GOA does not record. Reason: Castro-Roa et al. state directly that Doc phosphorylated EF-Tu using either ATP or GTP, and UniProt's curated FUNCTION line records the same with ECO:0000269. The nucleotide promiscuity is not incidental: the triphosphate forms make Doc a kinase while the diphosphate forms make it run backwards, so the GTP/GDP side of the site is part of the proposed switch between toxicity and recovery. Doc itself is not a GTPase - it binds GTP as a cosubstrate - which the term does not imply. ATP binding is already annotated; this row completes the pair at the same granularity rather than restating it. Supporting Evidence: PMID:24141193 We found that Doc phosphorylated EF-Tu (using either ATP or GTP) at a relatively high rate of ~20 EF-Tu molecules per second by one molecule of Doc file:BPP1/doc/doc-uniprot.txt Phosphorylates EF-Tu on 'Thr-383' in vitro; can use ATP or GTP |
| GO:0003714 transcription corepressor activity | IDA PMID:9829946 Corepression of the P1 addiction operon by Phd and Doc. | NEW | Summary: Proposed new annotation capturing Doc's second, non-catalytic function: it represses the phd/doc operon by binding the DNA-binding repressor Phd and increasing Phd's affinity, cooperativity and specificity for the operator, without binding DNA itself. Reason: Participation test: Doc does the work. The conformational ordering it imposes on Phd's disordered C-terminal domain is what structures Phd's DNA-binding domain and what bridges two Phd dimers across the two adjacent operator sites, and Doc remains in the resulting protein-DNA complex (further mobility retardation). Repression is weaker when Doc is absent and is restored by the catalytically dead DocH66Y, so the activity is separable from the kinase activity and is not a knock-on of toxicity. GO:0003714 is defined as a coregulator activity exerted by binding a DNA-binding transcription factor rather than DNA, which is precisely this arrangement. Comparator: GOA gives Phd the DNA-side terms (GO:0003677, GO:0001217 and GO:0045892 on UniProtKB:Q06253) but gives Doc only a bare protein-binding row for the same interaction, so the coregulator half of a two-protein repressor is simply unannotated. This row and the MODIFY of the Doc-Phd GO:0005515 row state the same claim, once as a replacement term and once as a proposable annotation. Supporting Evidence: PMID:9829946 DocH66Y failed to bind DNA in vitro yet enhanced the affinity, cooperativity, and specificity with which Phd bound the operator. PMID:9829946 Mobility of the protein-DNA complex is further retarded in the presence of Doc, indicating that Doc may directly participate in the protein-DNA complex PMID:20603017 The binding of Doc to the intrinsically disordered C-terminal domain of Phd structures its N-terminal DNA-binding domain |
| GO:0045892 negative regulation of DNA-templated transcription | IDA PMID:9829946 Corepression of the P1 addiction operon by Phd and Doc. | NEW | Summary: Proposed new annotation for the process side of the corepressor activity: Doc deepens repression of the phd/doc promoter about fourfold in vivo in the presence of Phd, and point mutations that abolish the regulatory function map to the Doc protein itself. Reason: In vivo reporter assays show transcription repressed 40-fold with Phd plus Doc versus 10-fold with Phd alone, and DocH66Y - which has no toxicity - reproduces the effect, so the readout is regulation rather than a consequence of growth arrest. Point mutants L12P, L82P, L84P and L118P lose transcriptional regulatory activity, placing the function on Doc. The term is deliberately the negative-regulation term and not a bidirectional one even though high Doc:Phd ratios derepress the operon (conditional cooperativity): the derepression arises when antitoxin capacity is exceeded and the operon has left its homeostatic set point, whereas corepression is the regulatory behaviour in the physiological regime where Phd is in excess. The bidirectional case is flagged in suggested_questions rather than annotated as a second, opposing arm. Supporting Evidence: PMID:9829946 transcription was repressed 40-fold, while in the presence of Phd, transcription was repressed only 10-fold, indicating that under these conditions, Doc enhanced repression about fourfold. PMID:9829946 In addition to acting as antidote and toxin, Phd and Doc collaborate to autoregulate transcription of the addiction promoter. file:BPP1/doc/doc-uniprot.txt L->P: Loss of toxin and transcriptional regulatory activity. |
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Download this section (compressed HTML)Q: Should GO:0106310 protein serine kinase activity carry a usage comment directing threonine-specific kinases to GO:0004674, or be merged into it as GO:0106311 protein threonine kinase activity already was? The asymmetry of a serine child with no threonine sibling invites exactly the annotation this review corrects: GO_REF:0000116 projects the seryl half of EC 2.7.11.1 onto an enzyme whose only demonstrated acceptor is a threonine. The CSNK2A1 review in this repository raises the same question from the opposite direction, so it is not specific to Doc.
Q: Does a dedicated phosphatase dephosphorylate EF-Tu Thr-382 in vivo, and is Doc-induced dormancy therefore reversible, or is the Doc-catalysed ADP/GDP-dependent reverse reaction the only route back?
Suggested experts: Nikolay Zenkin, Abel Garcia-Pino
Q: At high Doc:Phd ratios the operon is derepressed rather than repressed. Which protein-DNA species is responsible, and does Doc leave the operator complex or remain in an altered one?
Suggested experts: Remy Loris, Roy D. Magnuson
Q: Do Doc-family toxins from bacterial genomes outside P1 also target EF-Tu Thr-382, or has the family diversified its substrates while keeping the degenerate Fic motif?
Suggested experts: Nancy A. Woychik, Jonathan W. Cruz
Q: Is the mazEF dependence of phd/doc post-segregational killing a downstream host death pathway triggered by translation arrest, or does Doc act on mazEF more directly? No activity of Doc on MazE or MazF has been reported.
Suggested experts: Hanna Engelberg-Kulka
Experiment: Screen the E. coli phosphatase complement (and candidate PPM/PPP-family enzymes) for activity against purified phospho-Thr-382 EF-Tu in vitro, then test the hits in vivo: induce Doc transiently in wild-type and phosphatase-deletion backgrounds and follow phospho-EF-Tu by Phos-tag gel or targeted mass spectrometry alongside regrowth kinetics after Doc shut-off. A phosphatase mutant that retains phospho-EF-Tu and fails to resume growth would establish the recovery route.
Hypothesis: A host serine/threonine phosphatase reverses EF-Tu Thr-382 phosphorylation and allows recovery from Doc-induced dormancy.
Type: biochemical screen plus genetic epistasis
Experiment: Titrate purified DocH66Y against fixed Phd on single-site and double-site operator DNA and resolve the complexes by native mass spectrometry and mass photometry alongside EMSA, assigning each electrophoretic species a Doc:Phd:DNA stoichiometry; pair this with SAXS or cryo-EM of the repressing and the derepressing species.
Hypothesis: The species that derepresses the phd/doc operon at high Doc:Phd ratio is a distinct Doc-Phd-operator complex of defined stoichiometry.
Type: structural and biophysical stoichiometry
Experiment: Express and purify Doc-family toxins (with their cognate antitoxins for neutralisation) from a phylogenetically spread panel of bacterial genomes, assay each against its own host EF-Tu and against whole-cell lysate with gamma-32P-ATP, and map any phosphosites by tandem mass spectrometry. A toxin that labels lysate but not EF-Tu would identify a substrate switch within the family.
Hypothesis: EF-Tu Thr-382 is the conserved target of the whole Doc subfamily of Fic proteins.
Type: comparative enzymology
Experiment: Test purified Doc for phosphorylation of MazE and MazF with gamma-32P-ATP and for binding by pull-down or isothermal titration calorimetry; in parallel, ask whether an unrelated translation-arresting toxin substitutes for Doc in a mazEF-dependent killing assay, and whether cells expressing EF-Tu T382A are protected from Doc-induced, mazEF-dependent death.
Hypothesis: The mazEF requirement for phd/doc post-segregational killing is downstream of translation arrest and not a direct Doc-MazEF interaction.
Type: biochemistry plus genetic complementation
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