YbjN is a small (158 aa) cytoplasmic protein of Escherichia coli K-12 whose orthologs are confined to the Enterobacteriaceae and whose biochemistry is unknown. It carries no catalytic motif, no transmembrane segment and no signal peptide, and is classified only as a putative sensory transduction regulator (Pfam PF10722 YbjN, InterPro IPR019660). The one solved structure of the family, the Erwinia amylovora ortholog AmyR, is a homodimer with a class I type III secretion chaperone-like fold and no evidence of a secretion or catalytic role, suggesting that YbjN proteins act through specific protein-protein interactions. Functionally, YbjN behaves as a dosage-sensitive negative regulator of the Escherichia coli multicellular and surface-appendage programme: loss of ybjN raises type 1 fimbriation, swimming motility, flagellar gene expression (fliA and the FliA-dependent flgB, flgD, flgE, fliL, fliM), colanic acid capsule production and biofilm formation on abiotic surfaces, while extra copies of ybjN suppress all of these. The gene was originally isolated as a multicopy suppressor of the temperature-sensitive coaA (pantothenate kinase) allele in the ts9 strain, which led to the proposal that it stabilizes unstable proteins, and its expression is growth-phase- and temperature-dependent and induced under many stresses. Deletion of ybjN also causes a modest loss of survival after high-dose ionizing radiation. Whether YbjN works as a chaperone, as an adaptor feeding into the Rcs phosphorelay, or by some other route is not established.
Definition: Any process that stops, prevents or reduces the frequency, rate or extent of pilus assembly, the assembly of a pilus or fimbria on the surface of a bacterial cell.
Justification: GO has `GO:0009297 pilus assembly` and `GO:0140623 type I pilus assembly`, but regulation terms exist only for type IV pili (`GO:1903656 regulation of type IV pilus biogenesis` and its positive and negative children). There is therefore no term for a protein that suppresses chaperone-usher (type 1) fimbriation, which is what E. coli YbjN does: deleting ybjN gives about three times more fimbriae by TEM and raises fimC, fimD and fimG, while multicopy ybjN abolishes pili. The absence of such a term is why the existing annotation landed on the unregulated parent `GO:0009297`, asserting the opposite of the observed phenotype. A term at the general `pilus assembly` level is proposed rather than a type-1-specific one so that it can also serve the regulators of other chaperone-usher and curli-type appendages; if GO prefers the specific level, a sibling "negative regulation of type I pilus assembly" under `GO:0140623` would also fit this gene.
Parent term: negative regulation of cellular component organization
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005515 protein binding | IPI PMID:24561554 The binary protein-protein interaction landscape of Escheric... | REMOVE | Summary: Bare protein binding from the Rajagopala et al. Escherichia coli binary yeast two-hybrid interactome, reporting a YbjN-YpjF (Q46953) pair. Reason: GO:0005515 is uninformative: it records that an interaction was detected without saying what YbjN does, and the repository's protein-binding policy removes such rows. Removal does not dispute the interaction, which UniProt records independently as three experiments. The pair is in fact interesting - multicopy ybjN strongly induces the mqsR/ygiT and relE/relB toxin-antitoxin modules (PMID:21980417), and YpjF is a YpjF/YfjZ-type toxin - but a two-hybrid pair does not license any specific molecular function term, and nothing in the cited paper's text or in any other source characterizes a YbjN activity, so no replacement term is proposed. Supporting Evidence: file:ECOLI/ybjN/ybjN-uniprot.txt P0AAY6; Q46953: ypjF; NbExp=3; IntAct=EBI-9138440, EBI-9134503. |
| GO:0009297 pilus assembly | IMP PMID:21980417 The orphan gene ybjN conveys pleiotropic effects on multicel... | MODIFY | Summary: CACAO IMP. The cited paper shows the opposite of what this term asserts: the ybjN deletion makes about three times MORE type 1 fimbriae than wild type by transmission electron microscopy (Fig. 3C), with fimD up 8-fold (Fig. 3D), while multicopy ybjN abolishes pili entirely. YbjN suppresses pilus formation; it is not part of the assembly machinery. Reason: CACAO row, assessed against the paper. The assay is the right one for an IMP and is unambiguously about this gene: Fig. 3C is TEM of BW25113, the ybjN mutant and the overexpression strain, Fig. 3D the matching fimD qRT-PCR, and the microarray (Table 1) adds fimC, fimD and fimG. But the direction is inverted. `involved_in pilus assembly` places YbjN among the proteins that build the pilus - the chaperone-usher components FimC and FimD - whereas the paper's own conclusion is that YbjN negatively regulates fimbriae formation by suppressing fimbrial gene expression. This is the taxonomy's "sign inverted" shape, and it cannot be repaired by substituting an existing term: GO has `GO:0009297 pilus assembly` and `GO:0140623 type I pilus assembly`, and regulation terms only for type IV pili (`GO:1903656`/`GO:1903657`), with no `regulation of pilus assembly` anywhere. The replacement is therefore proposed as a new term (see proposed_new_terms). `GO:0010629 negative regulation of gene expression` would be literally true of the fimD/fimC/fimG result but loses the pilus, and YbjN has never been shown to act on transcription directly, so it is not proposed. Proposed replacements: negative regulation of pilus assembly Supporting Evidence: PMID:21980417 Deletion of the ybjN gene led to over-production of fimbriae, and the ybjN mutant produced about three times more fimbriae than that of the BW PMID:21980417 In contrast, no pili were observed on cells over-expressing ybjN. PMID:21980417 The fimD gene was up-regulated 8-fold in the ybjN mutant, but it was down-regulated 12-fold in strain over-expressing ybjN as compared to that of the BW PMID:21980417 These results indicate that YbjN negatively regulated fimbriae formation by suppressing fimbrial gene expression PMID:21980417 Genes encoding major components of bacterial pili including fimC, fimD and fimG were significantly up-regulated in the ybjN mutant, but down-regulated in the ybjN over-expression strain |
| GO:0010212 response to ionizing radiation | IMP PMID:25049088 Escherichia coli genes and pathways involved in surviving ex... | KEEP AS NON CORE | Summary: EcoCyc IMP. Byrne et al. identified ybjN by transposon-directed insertion sequencing as required for survival of high-dose ionizing radiation and validated it with a clean deletion and survival curves, where it falls in the mildest validated class (group 1, under ~1 log loss at 2,000 Gy). Reason: This is genuine loss-of-function evidence from a professional curation source: the gene was both screened and individually validated (Fig. 2), and the paper explicitly flags it as one of the uncharacterized genes for which this is the first phenotype. `acts_upstream_of_or_within` is the right relation for a survival phenotype whose mechanism is unknown, and the term is at the granularity the data support. It is kept as non-core rather than accepted because the effect is at the bottom of the validated range and is one of several stress-survival phenotypes of a gene that is induced under many stresses; the defining, reproducible role of YbjN is dosage-dependent negative regulation of the surface-appendage and multicellular programme, not a radiation-specific function. No DNA-repair or oxidative-defence activity has been shown for YbjN, and the paper's own framing is a broad stress role. Supporting Evidence: PMID:25049088 The genes in group 1 ( rdgC , ftsP , radA , rsxB , topB , recX , speA , yabI , yhgF , ybjN , tolA , and ompA ) exhibit relatively modest effects when deleted, with a decline in survival of just over an order of magnitude or less at 2,000 Gy. PMID:25049088 In three cases ( yabI , yhgF , and ybjN ), elimination of gene function has just a modest effect on IR survival and the genes fall into group 1. |
| GO:0030308 negative regulation of cell growth | IMP PMID:21980417 The orphan gene ybjN conveys pleiotropic effects on multicel... | MARK AS OVER ANNOTATED | Summary: CACAO IMP. The growth experiment (Fig. 2A) shows the ybjN deletion growing indistinguishably from wild type (rate constants 2.28 vs 2.26); only the multicopy overexpression strain is slowed (1.72). The claim rests entirely on a gene-dosage artefact, and the process measured is population growth rate, not cell growth in GO's sense. Reason: CACAO row, assessed against the paper. Two problems compound. First, the evidence code is IMP (ECO:0000315, mutant phenotype) but the mutant has no phenotype here: the authors state that BW and the ybjN mutant have similar growth curves, and the only effect comes from ~20-fold overexpression off a multicopy plasmid, which also induces the mqsR/ygiT and relE/relB toxin-antitoxin pairs 15.8-, 9.9-, 5.6- and 3.1-fold, so the authors themselves attribute the slowing to a toxin-antitoxin imbalance rather than to a physiological YbjN activity. Second, `GO:0030308` regulates `GO:0016049 cell growth`, the increase in cell size or mass, whereas the assay measures OD600 and exponential-phase rate constants, i.e. population proliferation; even taken at face value the measurement would fit `GO:0045967 negative regulation of growth rate` rather than this term. MARK_AS_OVER_ANNOTATED rather than REMOVE because YbjN does restrain growth-coupled processes when present at elevated levels and the over-expression observation is real; but no replacement term is proposed, since nothing in the loss-of-function data supports a growth-regulatory role for YbjN at native dosage. For the CACAO taxonomy this is a shape not previously catalogued: an overexpression-only phenotype annotated as the gene's process on a mutant-phenotype evidence code. Supporting Evidence: PMID:21980417 both BW and ybjN mutant showed a similar growth curves and growth rate constants for BW and the ybjN mutant were 2.26 and 2.28, respectively, compared to 1.72 for ybjN over-expressing strains grown in LB broth PMID:21980417 These results indicate that over-expression of ybjN slowed bacterial growth in E. coli. |
| GO:1900232 negative regulation of single-species biofilm formation on inanimate substrate | IMP PMID:21980417 The orphan gene ybjN conveys pleiotropic effects on multicel... | ACCEPT | Summary: CACAO IMP, and a good one. Crystal violet staining of 48 h biofilms on polyvinyl chloride microtitre plates (Fig. 6A, quantified in Fig. 6B) shows the ybjN deletion forming ~3-fold more biofilm than wild type and multicopy ybjN roughly halving it. Reason: CACAO row, assessed against the paper. The term matches the assay on every axis: a pure culture of Escherichia coli (single species), an abiotic PVC surface (inanimate substrate), and a deletion mutant that makes more biofilm, which is what negative regulation means. The direction is taken from the loss-of-function strain, not from overexpression, so the dosage-artefact objection that defeats the growth row does not apply here, and the overexpression result is a consistent reciprocal control. It is also mechanistically coherent with the rest of the paper, since the fimbrial, flagellar and colanic acid programmes that YbjN suppresses are the ones that drive attachment and biofilm maturation in E. coli K-12. Accepted as a core function. Supporting Evidence: PMID:21980417 The amount of biofilm formed by the ybjN mutant was much more than that of BW strain, whereas biofilm formation of BW strain with multi-copy plasmids containing ybjN was greatly impaired PMID:21980417 These results indicate that YbjN suppresses normal biofilm formation in E. coli. |
| GO:1902201 negative regulation of bacterial-type flagellum-dependent cell motility | IMP PMID:21980417 The orphan gene ybjN conveys pleiotropic effects on multicel... | ACCEPT | Summary: EcoCyc IMP. Soft-agar (0.25%) migration assays (Fig. 4A, 4B) give migration diameters of 0.84 mm for wild type, 1.88 mm for the ybjN deletion and 0.41 mm for the overexpression strain, so loss of ybjN increases swimming motility. Reason: Directly supported by the loss-of-function assay, with a reciprocal overexpression control and a mechanistic correlate in the 12.5- and 11.3-fold induction of fliA and flgD in the mutant. The term's granularity is right: swimming on soft agar in E. coli K-12 is bacterial-type flagellum-dependent motility, and YbjN is a regulator of it rather than a motor or chemotaxis component, which `acts_upstream_of_or_within` correctly conveys. Independently corroborated outside K-12, where loss of the adhesin FdeC in avian pathogenic E. coli raises motility via YbjN down-regulation. Accepted as a core function. Supporting Evidence: PMID:21980417 The ybjN mutant exhibited enhanced motility compared with that of the BW strain 12 h following inoculation PMID:21980417 These results suggest that the YbjN is a negative regulator of bacterial motility in E. coli. PMID:38822378 a protein known to supress motility |
| GO:1902209 negative regulation of bacterial-type flagellum assembly | IMP PMID:21980417 The orphan gene ybjN conveys pleiotropic effects on multicel... | ACCEPT | Summary: CACAO IMP. The ybjN deletion up-regulates the flagellar regulon: fliA (sigma-28) and flgD 12.5- and 11.3-fold by qRT-PCR (Fig. 4C), and in the microarray (Table 1) the structural and assembly genes flgB (basal-body rod), flgD and flgE (hook), fliL and fliM rise 7- to 16-fold, all falling again on overexpression. Reason: CACAO row, assessed against the paper. This row is distinct from the motility row and is not a parent-level restatement of it: the genes measured are flagellar structural and assembly components downstream of the FliA sigma factor, so the claim is about building the organelle, not only about swimming. The direction comes from the deletion strain, and the term's sign and granularity both match. The one caveat is that flagella were not themselves imaged, so the assembly step is inferred from transcript levels of assembly genes plus the motility phenotype rather than from counting flagella as the paper does for fimbriae; that is a normal basis for an IMP on a regulator and does not change the action. Accepted as a core function. Supporting Evidence: PMID:21980417 Expression of fliA and flgD in the ybjN mutant was up-regulated by 12.5 and 11.3 fold, as compared to that of the BW PMID:21980417 These findings indicate that YbjN negatively regulates flagellar gene expression PMID:21980417 Several FliA controlled genes including flgB, flgD, flgE, fliL and fliM were up-regulated in the ybjN mutant, but down-regulated in the ybjN over-expression strains |
| GO:1901914 negative regulation of capsule organization | IMP PMID:21980417 The orphan gene ybjN conveys pleiotropic effects on multicel... | NEW | Summary: Proposed new annotation. On LB plus 700 mM NaCl the ybjN deletion is strongly mucoid, characteristic of colanic acid over-production, while multicopy ybjN makes colonies non-mucoid and additionally suppresses the mucoid phenotype of ackA and ackA/rcsC mutants (Fig. 5A, 5B). The authors conclude that YbjN negatively regulates capsule formation. Reason: This is the one well-supported phenotype from the ybjN paper that no GOA row captures, and it is of exactly the same kind and strength as the biofilm and flagellar rows that are accepted above: a loss-of-function mucoidy phenotype with a reciprocal multicopy control, plus a genetic epistasis test in which extra ybjN suppresses the constitutive mucoidy of ackA and ackA/rcsC. On the participation test, YbjN is being annotated as a regulator and not as a biosynthetic enzyme, which is what `GO:1901914 negative regulation of capsule organization` asserts; the colanic acid synthases themselves carry the biosynthetic terms. The sign is independently reproduced in the ortholog: deleting ybjN/amyR in Erwinia amylovora raises amylovoran exopolysaccharide about eight-fold. The more specific `GO:0062084 regulation of capsule polysaccharide biosynthetic process` has only a positive child, so `GO:1901914` is the most specific term of the right sign available. Kept deliberately at capsule organization rather than asserting a mechanism, since whether YbjN acts through RcsB phosphorylation is speculation in the paper. Supporting Evidence: PMID:21980417 These observations indicate that YbjN negatively regulates capsule formation. PMID:23028751 a mutation of ybjN gene in E. amylovora, an ortholog of E. coli ybjN gene, results in about eight-fold more amylovoran than that of the wild type strain, suggesting that this gene may act as a negative regulator of amylovoran production |
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Download this section (compressed HTML)Q: Does YbjN act as a chaperone or holdase on specific partner proteins, as the class I type III secretion chaperone-like fold of its Erwinia ortholog AmyR and the multicopy suppression of unstable CoaA alleles both suggest, and if so what are its clients in Escherichia coli?
Suggested experts: Stefano Benini, Joseph D Bartho, Bing Zhou
Q: Is the suppression of fimbrial, flagellar and colanic acid gene expression by YbjN exerted through the Rcs phosphorelay - for example by affecting RcsB phosphorylation, as proposed from the suppression of ackA/rcsC mucoidy - or through an independent route?
Suggested experts: Youfu Zhao, Dongping Wang
Q: How much of the YbjN overexpression phenotype (slowed growth, acid susceptibility, induction of mqsR/ygiT and relE/relB) reflects a genuine physiological activity at native dosage, and how much is titration of a partner by excess YbjN?
Suggested experts: Youfu Zhao
Q: By what mechanism does loss of ybjN reduce survival of high-dose ionizing radiation, given that no DNA-repair or oxidative-defence activity has been assigned to YbjN and the effect is modest?
Suggested experts: Michael M Cox, Robert T Byrne
Q: Is the two-hybrid interaction between YbjN and the YpjF toxin (UniProtKB:Q46953) physiologically relevant, and does it explain the toxin-antitoxin induction seen on ybjN overexpression?
Suggested experts: Peter Uetz, Seesandra V Rajagopala
Experiment: Identify YbjN's in vivo partners without relying on two-hybrid: express chromosomally tagged YbjN at native levels in Escherichia coli K-12 and perform affinity purification followed by mass spectrometry, in parallel with in vivo crosslinking (for example DSSO or photo-crosslinking with an incorporated unnatural amino acid) to capture transient clients, under both unstressed and stress conditions where ybjN is induced. Validate candidates by reciprocal pull-down and by testing whether purified YbjN suppresses thermal aggregation of the candidate in vitro.
Hypothesis: YbjN acts as a chaperone-like protein that binds and stabilizes one or a few specific partner proteins, and its regulatory effects on fimbriae, flagella and capsule are indirect consequences of that binding.
Type: affinity purification mass spectrometry and in vivo crosslinking
Experiment: Combine a ybjN deletion and a multicopy ybjN plasmid with rcsB, rcsC, rcsD and rcsF deletions and measure mucoidy, fimD/fliA/wca transcript levels and biofilm formation in each background, to establish whether the YbjN effect requires the Rcs system. In the same strains, measure RcsB phosphorylation directly by Phos-tag electrophoresis, and test whether YbjN binds RcsB or RcsD by purified-protein assays. Independently, run ChIP-seq on tagged YbjN to test for the direct DNA binding that a "sensory transduction regulator" name implies but that has never been demonstrated.
Hypothesis: YbjN suppresses the fimbrial, flagellar and colanic acid regulons by acting on the Rcs phosphorelay rather than by binding DNA.
Type: epistasis analysis with phosphorylation and ChIP-seq readouts
Experiment: Replace the multicopy plasmid with a chromosomally integrated, titratable promoter (for example an arabinose-inducible single copy) and measure growth rate, toxin-antitoxin transcript levels and fimbriation as a continuous function of YbjN level, including levels within the physiological range observed between log and stationary phase and between 28 and 42 degrees. If the growth effect appears only above the natural maximum while fimbrial and flagellar suppression tracks the physiological range, the growth phenotype is a dosage artefact.
Hypothesis: The slowed growth seen on multicopy ybjN is a titration artefact of excess protein, not a physiological growth-regulatory function of YbjN.
Type: titratable single-copy expression series
Experiment: Solve the structure of E. coli YbjN itself by crystallography or cryo-EM and compare it with AmyR (PDB 5FRK), then test the functional importance of the divergent surface residues and the dimer interface by mutating them in the chromosomal copy and scoring fimbriation, motility, mucoidy and biofilm formation, together with cross-complementation between the E. coli and E. amylovora orthologs.
Hypothesis: Escherichia coli YbjN has the same fold and dimerization behaviour as Erwinia amylovora AmyR, and the surface residues that differ between them determine partner specificity.
Type: structure determination with structure-guided mutagenesis
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