NfxB (PA4600) is a TetR-family transcriptional repressor of Pseudomonas aeruginosa that controls the divergently transcribed mexC-mexD-oprJ operon, encoding the MexCD-OprJ RND multidrug efflux pump, and its own gene. The functional repressor is the 199-residue form translated from an upstream start codon. NfxB binds as a tetramer (a dimer of dimers, assembled through a C-terminal three-helix subdomain) to a region upstream of the mexCD-oprJ transcription start that contains two inverted repeats, and its N-terminal helix-turn-helix domain contacts the DNA. Loss-of-function nfxB mutations, which arise readily under fluoroquinolone selection and in cystic fibrosis isolates, derepress mexCD-oprJ and confer resistance to fluoroquinolones and other antimicrobials; many further phenotypes of nfxB mutants (reduced motility, virulence-factor production and fitness) are secondary consequences of pump overexpression.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000976 transcription cis-regulatory region binding | IBA GO_REF:0000033 | MODIFY | Summary: IBA from the PTN004738546 IBD (7 seeds). nfxB sits inside this TetR clade and is itself a seed, which is expected, not circular. nfxB binds an inverted-repeat operator in its target promoter. Reason: Correct and appropriate for the node, but binding to a defined inverted-repeat operator supports the child GO:0000987 cis-regulatory region sequence-specific DNA binding for this gene (the same refinement used for mexZ, which shares the node). The generic term stays right for the node. Propagation Review Root cause: NO FAILURE CORE Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN004738546 · PTN004738546 SUPPORTS TRANSFER TetR-family PAINT node (411 leaf IBAs, 33 taxa). Its IBDs rest on 19 seeds for GO:0006355, 7 for GO:0000976 and 7 for GO:0003700, including E. coli AcrR (P0ACS9, IDA operator binding and DNA-binding TF activity), RutR (P0ACU2), Vibrio VC_1746 (Q9KR96) and M. tuberculosis KstR, Mce3R and EthR. The generic node terms suit a TetR clade and stand without any single seed; node-level verdict recorded in the nfxB notes. UniProtKB:P32265 · nfxB (self; experimental seed) SUPPORTS TRANSFER Expected self-seed reflecting the target's own experimental evidence; not circular. Proposed replacements: cis-regulatory region sequence-specific DNA binding Supporting Evidence: PMID:23924707 NfxB binds upstream of the mexCD-oprJ transcription initiation site to a region containing two inverted repeats, both of which are required for binding PMID:7592337 NfxB specifically binds upstream of the nfxB coding region as demonstrated by gel retardation and DNase I footprinting |
| GO:0000976 transcription cis-regulatory region binding | IDA PMID:23924707 Functional characterization of the NfxB repressor of the mex... | MODIFY | Summary: PseudoCAP IDA and one of the 7 seeds of the PTN004738546 GO:0000976 IBD. The abstract reports EMSA binding of purified NfxB to a mexCD-oprJ upstream region containing two inverted repeats, both required for binding. Seed verdict SOUND. Reason: The seed is sound but under-specific: binding requires two defined inverted repeats, which supports GO:0000987 cis-regulatory region sequence-specific DNA binding. GO:0000987 is a child of GO:0000976, so refining the row would still support the IBD. Proposed replacements: cis-regulatory region sequence-specific DNA binding Supporting Evidence: PMID:23924707 NfxB binds upstream of the mexCD-oprJ transcription initiation site to a region containing two inverted repeats, both of which are required for binding |
| GO:0000976 transcription cis-regulatory region binding | IPI PMID:7592337 Purification and characterization of the Pseudomonas aerugin... | MODIFY | Summary: CollecTF row: gel retardation and DNase I footprinting of NfxB on the nfxB upstream region. Reason: Same claim as the IDA row; footprinting a defined site supports GO:0000987. The binding is shown with purified protein, so IDA would be a better code than IPI. Proposed replacements: cis-regulatory region sequence-specific DNA binding Supporting Evidence: PMID:7592337 NfxB specifically binds upstream of the nfxB coding region as demonstrated by gel retardation and DNase I footprinting |
| GO:0001217 DNA-binding transcription repressor activity | IMP PMID:8878035 Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-t... | ACCEPT | Summary: NfxB represses mexC-lacZ and nfxB-lacZ fusions, and the cloned gene reverses an nfxB mutant phenotype. Reason: Repression plus direct operator binding (PMID:7592337, PMID:23924707) shows DNA-binding transcription repressor activity; core MF. Supporting Evidence: PMID:8878035 the cloned nfxB gene repressed synthesis of a mexC-lacZ fusion in Escherichia coli PMID:8878035 nfxB also repressed expression of a nfxB-lacZ fusion, indicating that NfxB negatively regulates its own expression PMID:7592337 NfxB specifically binds upstream of the nfxB coding region as demonstrated by gel retardation and DNase I footprinting |
| GO:0001217 DNA-binding transcription repressor activity | IPI PMID:7592337 Purification and characterization of the Pseudomonas aerugin... | ACCEPT | Summary: CollecTF row: NfxB binds the nfxB upstream region and represses an nfxB'-lacZ fusion. Reason: Consistent with the IMP row; core MF. Supporting Evidence: PMID:7592337 NfxB specifically binds upstream of the nfxB coding region as demonstrated by gel retardation and DNase I footprinting |
| GO:0003700 DNA-binding transcription factor activity | IBA GO_REF:0000033 | MODIFY | Summary: IBA from the PTN004738546 IBD (7 seeds). nfxB is a DNA-binding TetR-family repressor. Reason: Correct but general. The gene-level evidence (operator binding plus derepression in the mutant) supports GO:0001217 DNA-binding transcription repressor activity. The generic term remains right for the node. Propagation Review Root cause: NO FAILURE CORE Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN004738546 · PTN004738546 SUPPORTS TRANSFER TetR-family PAINT node (411 leaf IBAs, 33 taxa). Its IBDs rest on 19 seeds for GO:0006355, 7 for GO:0000976 and 7 for GO:0003700, including E. coli AcrR (P0ACS9, IDA operator binding and DNA-binding TF activity), RutR (P0ACU2), Vibrio VC_1746 (Q9KR96) and M. tuberculosis KstR, Mce3R and EthR. The generic node terms suit a TetR clade and stand without any single seed; node-level verdict recorded in the nfxB notes. UniProtKB:P32265 · nfxB (self; experimental seed) SUPPORTS TRANSFER Expected self-seed reflecting the target's own experimental evidence; not circular. Proposed replacements: DNA-binding transcription repressor activity Supporting Evidence: PMID:23924707 NfxB binds upstream of the mexCD-oprJ transcription initiation site to a region containing two inverted repeats, both of which are required for binding PMID:7592337 NfxB specifically binds upstream of the nfxB coding region as demonstrated by gel retardation and DNase I footprinting PMID:8878035 the cloned nfxB gene repressed synthesis of a mexC-lacZ fusion in Escherichia coli PMID:8878035 nfxB also repressed expression of a nfxB-lacZ fusion, indicating that NfxB negatively regulates its own expression |
| GO:0003700 DNA-binding transcription factor activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO (IPR050109 TetR-like regulator) mapping. True. Reason: Correct but general; the gene-level evidence supports GO:0001217 DNA-binding transcription repressor activity. Proposed replacements: DNA-binding transcription repressor activity Supporting Evidence: PMID:23924707 NfxB binds upstream of the mexCD-oprJ transcription initiation site to a region containing two inverted repeats, both of which are required for binding PMID:7592337 NfxB specifically binds upstream of the nfxB coding region as demonstrated by gel retardation and DNase I footprinting PMID:8878035 the cloned nfxB gene repressed synthesis of a mexC-lacZ fusion in Escherichia coli PMID:8878035 nfxB also repressed expression of a nfxB-lacZ fusion, indicating that NfxB negatively regulates its own expression |
| GO:0006355 regulation of DNA-templated transcription | IBA GO_REF:0000033 | MODIFY | Summary: IBA from the PTN004738546 IBD (19 seeds). nfxB is one of them, which is expected, not circular. The IBD stands on the other 18 seeds as well. Reason: Correct and appropriate for the node, but nfxB is a repressor; GO:0045892 negative regulation of DNA-templated transcription is supported by the gene's own experimental rows. Propagation Review Root cause: NO FAILURE CORE Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN004738546 · PTN004738546 SUPPORTS TRANSFER TetR-family PAINT node (411 leaf IBAs, 33 taxa). Its IBDs rest on 19 seeds for GO:0006355, 7 for GO:0000976 and 7 for GO:0003700, including E. coli AcrR (P0ACS9, IDA operator binding and DNA-binding TF activity), RutR (P0ACU2), Vibrio VC_1746 (Q9KR96) and M. tuberculosis KstR, Mce3R and EthR. The generic node terms suit a TetR clade and stand without any single seed; node-level verdict recorded in the nfxB notes. UniProtKB:P32265 · nfxB (self; experimental seed) SUPPORTS TRANSFER Expected self-seed reflecting the target's own experimental evidence; not circular. Proposed replacements: negative regulation of DNA-templated transcription Supporting Evidence: PMID:8878035 the cloned nfxB gene repressed synthesis of a mexC-lacZ fusion in Escherichia coli PMID:8878035 nfxB also repressed expression of a nfxB-lacZ fusion, indicating that NfxB negatively regulates its own expression |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO (IPR050109) mapping. True. Reason: Correct but general; nfxB is shown to be a repressor, so GO:0045892 applies. Proposed replacements: negative regulation of DNA-templated transcription Supporting Evidence: PMID:8878035 the cloned nfxB gene repressed synthesis of a mexC-lacZ fusion in Escherichia coli PMID:8878035 nfxB also repressed expression of a nfxB-lacZ fusion, indicating that NfxB negatively regulates its own expression |
| GO:0032410 negative regulation of transporter activity | IMP PMID:20373734 Mutation of nfxB causes global changes in the physiology and... | REMOVE | Summary: PseudoCAP IMP from a study of the global physiology of an nfxB mutant. Reason: The abstract attributes the nfxB mutant phenotypes to overexpression of the MexCD-OprJ pump; NfxB lowers the amount of pump by repressing its operon, not its activity. Supporting Evidence: PMID:20373734 suggesting that inappropriate overexpression of a functional MexCD-OprJ efflux pump causes pleiotropic changes |
| GO:0032410 negative regulation of transporter activity | IMP PMID:8878035 Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-t... | REMOVE | Summary: PseudoCAP IMP. The paper shows NfxB represses transcription of the mexC-mexD-oprJ operon. Reason: GO:0032410 is a process that reduces the activity of a transporter. NfxB represses transcription of mexCD-oprJ and so lowers the amount of the pump; the abstract shows the effect through lacZ fusions. The transcriptional role is captured by GO:0045892. Supporting Evidence: PMID:8878035 the cloned nfxB gene repressed synthesis of a mexC-lacZ fusion in Escherichia coli |
| GO:0032993 protein-DNA complex | IPI PMID:7592337 Purification and characterization of the Pseudomonas aerugin... | KEEP AS NON CORE | Summary: CollecTF row for the NfxB-operator complex from gel retardation and footprinting. Reason: NfxB forms a protein-DNA complex on its operator; a CollecTF convention, true but peripheral. Supporting Evidence: PMID:7592337 NfxB specifically binds upstream of the nfxB coding region as demonstrated by gel retardation and DNase I footprinting |
| GO:0045892 negative regulation of DNA-templated transcription | IMP PMID:8878035 Overexpression of the mexC-mexD-oprJ efflux operon in nfxB-t... | ACCEPT | Summary: PseudoCAP and UniProt IMP and one of the 19 seeds of the PTN004738546 GO:0006355 IBD. The abstract states that the cloned nfxB gene represses mexC-lacZ and nfxB-lacZ fusions and reverses OprJ overproduction in the nfxB mutant K385. Seed verdict SOUND. Reason: Direct repressor of mexCD-oprJ and of its own gene; core BP. Supporting Evidence: PMID:8878035 OprJ overproduction and multidrug resistance in K385 was reversed by the cloned nfxB gene, suggesting that nfxB encodes a repressor of mexC-mexD-oprJ expression PMID:8878035 the cloned nfxB gene repressed synthesis of a mexC-lacZ fusion in Escherichia coli PMID:8878035 nfxB also repressed expression of a nfxB-lacZ fusion, indicating that NfxB negatively regulates its own expression |
| GO:0046677 response to antibiotic | IMP PMID:1330820 Cloning and nucleotide sequence of the Pseudomonas aeruginos... | MARK AS OVER ANNOTATED | Summary: UniProt IMP: an nfxB point mutant alters quinolone permeability and resistance. Reason: A resistance phenotype of a repressor mutant reflects derepression of the MexCD-OprJ pump, not a response of NfxB to antibiotic. Induction of mexCD-oprJ by membrane-damaging agents is not known to act through NfxB sensing a drug. Supporting Evidence: PMID:1330820 The gene nfxB is one of the genes which affect the cell membrane permeability of quinolones in Pseudomonas aeruginosa PAO |
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Download this section (compressed HTML)Q: What effector, if any, binds the NfxB C-terminal domain and relieves repression of mexCD-oprJ?
Q: Does NfxB regulate genes other than mexCD-oprJ and nfxB directly?
Experiment: Ligand screen (thermal shift, EMSA with candidate effectors) on purified NfxB tetramer and ChIP-seq in PAO1.
Type: ligand screening and ChIP-seq
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