FleQ is an NtrC-family, sigma-54-associated AAA+ transcriptional regulator that acts as the master controller of flagellar biogenesis in Pseudomonas putida KT2440. Under low c-di-GMP it activates the flagellar transcriptional cascade through sigma-N-dependent promoters, whereas c-di-GMP-responsive promoter control redirects FleQ activity toward adhesin and exopolysaccharide loci such as lapA, lapE, and bcs to coordinate the transition from motility to surface attachment and biofilm growth. ChIP-seq and targeted promoter studies support a broader regulon that includes additional adhesion, exopolysaccharide, iron-homeostasis, and type VI secretion genes, but the clearest core functions remain flagellum regulation and c-di-GMP-responsive control of biofilm-associated transcription.
Definition: Catalysis of transcriptional activation by a bacterial enhancer-binding protein that binds specific DNA sequences and uses ATP-dependent remodeling to activate RNA polymerase containing sigma-54 at target promoters.
Justification: FleQ and related enhancer-binding proteins are currently forced into generic DNA-binding transcription activator activity and ATP binding terms, which do not capture the sigma-54-specific promoter class or ATP-dependent remodeling mechanism.
Parent term: DNA-binding transcription activator activity
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: Accurate and core. FleQ is an AAA+ enhancer-binding transcriptional regulator with a central ATPase/sigma-54 interaction domain, and ATP binding is mechanistically required for its transcriptional control of flagellar promoters. Reason: Although generic, ATP binding is part of the core mechanism of a sigma-54 bacterial enhancer-binding protein rather than an incidental property. Supporting Evidence: file:PSEPK/fleQ/fleQ-uniprot.txt UniProt and InterPro annotate FleQ, AAA+ ATPase, and sigma-54 interaction domains in Q88ET0 PMID:30177764 central AAA+/ATPase Ο54 (RpoN)-interaction domain file:PSEPK/fleQ/fleQ-deep-research-falcon.md **FleN is required for full c-di-GMP-dependent activation** at certain matrix promoters and that **ATP hydrolysis is required for flagellar gene expression**, but not necessarily for matrix gene regulation. |
| GO:0006355 regulation of DNA-templated transcription | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Correct but overly broad for FleQ. The gene product is a global transcriptional regulator with separable flagellar-activation and biofilm-control branches, so this parent term is best retained as non-core while more informative child terms are added below. Reason: FleQ clearly regulates transcription, but this term is too unspecific to represent the biologically informative functions of the protein. Supporting Evidence: PMID:27636892 coordinate regulation of multiple functions related to motility and surface colonization in Pseudomonas putida PMID:30177764 Putative genes regulated by FleQ included, as expected, flagellar and motility-related genes file:PSEPK/fleQ/fleQ-deep-research-openai.md master activator of flagellar biogenesis in pseudomonads file:PSEPK/fleQ/fleQ-deep-research-falcon.md FleQ acts as a **bifunctional regulator**: typically activating motility/adhesion genes and repressing some EPS genes. |
| GO:0043565 sequence-specific DNA binding | IEA GO_REF:0000002 | ACCEPT | Summary: Accurate and core. FleQ directly binds specific promoters in the flagellar region and at biofilm-associated loci including lapA, bcs, lapE, and cyaA, establishing sequence-specific promoter recognition as a central molecular function. Reason: Direct DNA-binding evidence exists in KT2440 for multiple named targets, and this function underlies both the motility and biofilm branches of FleQ regulation. Supporting Evidence: PMID:27120564 In vitro experiments show that FleQ binds to lapA and bcs promoter DNA PMID:33975969 Both in vitro and in vivo assays show that FleQ binds directly to promoters of the five genes file:PSEPK/fleQ/fleQ-deep-research-falcon.md promoter fragments predicted to contain FleQ boxes shifted upon incubation with purified FleQ, while control fragments lacking predicted motifs did not. |
| GO:0001216 DNA-binding transcription activator activity | IDA PMID:27636892 Complex Interplay between FleQ, Cyclic Diguanylate and Multi... | NEW | Summary: FleQ is a bona fide DNA-binding transcription activator for the flagellar cascade. Under low c-di-GMP it activates sigma-N-dependent promoters in the flagellar regulon, and multiple KT2440 studies describe FleQ as the master activator of flagellar gene expression. Reason: The seeded IEA set captures DNA binding and generic transcription regulation but misses FleQ's specific activator role. Supporting Evidence: PMID:27636892 Low cyclic diguanylate levels favor FleQ interaction with ΟN-dependent promoters to activate the flagellar cascade PMID:30177764 It has been described that FleQ is able to activate the expression of genes involved in flagellar export file:PSEPK/fleQ/fleQ-deep-research-falcon.md FleQ directly activated a subset of Ο54-class promoters, including **PflgB, PflhA, and PflgA** |
| GO:1902210 positive regulation of bacterial-type flagellum assembly | IMP PMID:30177764 Genome-wide analysis of the FleQ direct regulon in Pseudomon... | NEW | Summary: FleQ positively regulates flagellum assembly by driving transcription of flagellar biogenesis genes. fleQ mutants are non-motile and aflagellated, and ChIP-seq/promoter analyses place FleQ directly upstream of multiple flagellar loci required for assembly. Reason: This term captures the specific biological outcome of FleQ's best-established regulatory role better than generic motility or transcription terms. Supporting Evidence: PMID:30177764 mutations in the fleQ gene result in non-motile, aflagellated bacteria PMID:28968917 FleQ positively regulates flagella biosynthesis but negatively regulates the type VI secretion system in P. putida file:PSEPK/fleQ/fleQ-deep-research-falcon.md In KT2440-focused work, FleQ activates multiple flagellar promoters and sits at the top of a multi-tier regulatory cascade. |
| GO:1900190 regulation of single-species biofilm formation | IMP PMID:27636892 Complex Interplay between FleQ, Cyclic Diguanylate and Multi... | NEW | Summary: FleQ directly regulates the transition into the biofilm state by controlling transcription of adhesin and matrix-associated loci such as lapA, lapE, and bcs in a c-di-GMP-responsive manner. The literature shows clear FleQ dependence for biofilm formation, but because FleQ can activate some matrix genes and repress others, the broader regulation term is safer than forcing a purely positive label. Reason: Biofilm control is a major experimentally supported branch of FleQ function, but the directionality is promoter- and c-di-GMP-state dependent. Supporting Evidence: PMID:27636892 Disruption of fleQ caused strong defects in flagellar motility, biofilm formation and surface attachment PMID:27120564 FleQ acts as an activator of lapA, but a repressor of bcs PMID:33975969 These results demonstrated that FleQ was essential for c-di-GMP-mediated biofilm formation and LapA secretion file:PSEPK/fleQ/fleQ-deep-research-falcon.md Deletion of fleQ caused a **~2-fold drop in lapA mRNA** and a **~32-fold increase in bcsD mRNA**. |
| GO:0035438 cyclic-di-GMP binding | IDA PMID:27503246 FleQ of Pseudomonas putida KT2440 is a multimeric cyclic dig... | NEW | Summary: FleQ of P. putida KT2440 directly binds the second messenger c-di-GMP, as demonstrated by isothermal titration calorimetry. c-di-GMP binding to the AAA+ ATPase module is the molecular switch that redirects FleQ between flagellar activation and biofilm/matrix gene control, making this a core molecular function distinct from generic ATP binding. Reason: The seeded IEA set captures ATP binding but misses FleQ's experimentally demonstrated direct c-di-GMP binding, which is the key ligand-sensing function underlying its lifestyle-switch behavior. Supporting Evidence: PMID:27503246 we show in this work that FleQ of P. putida interacts with c-di-GMP and directly binds the promoter regions of flagellar and EPS genes file:PSEPK/fleQ/fleQ-deep-research-falcon.md FleQ is also described as a **c-di-GMP-binding** multimeric regulator, consistent with AAA+_ATPase, FleQ, and CheY-like/receiver-like domain annotations. file:PSEPK/fleQ/fleQ-deep-research-falcon.md **c-di-GMP** binds FleQβs AAA+ region/Walker A-associated ATPase module and **inhibits ATPase activity**, shifting FleQ from flagellar activation toward biofilm-associated regulation. |
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Download this section (compressed HTML)Q: Which FleQ-regulated iron-homeostasis genes are direct physiological targets in KT2440 under rhizosphere-relevant iron limitation, and are these roles core or context-specific?
Q: What promoter or cofactor features determine whether c-di-GMP-bound FleQ activates versus represses a given surface-associated operon in P. putida?
Q: How much of the reported type VI secretion phenotype reflects direct FleQ control versus secondary remodeling of the motility-biofilm regulatory network?
Experiment: ChIP-seq across low- and high-c-di-GMP states with matched RNA-seq in KT2440 to separate constitutive FleQ binding from c-di-GMP-dependent transcriptional output
Hypothesis: FleQ occupancy will remain at many promoters while c-di-GMP changes the transcriptional outcome and cofactor usage rather than simple DNA binding
Type: Differential ChIP-seq plus transcriptomics
Experiment: Promoter mutagenesis and in vitro transcription assays for fleS-fliF, flhA, lapA, bcs, and lapE promoters using purified FleQ with ATP, c-di-GMP, and FleN
Hypothesis: Distinct promoter architectures encode the activator-versus-repressor logic of FleQ and explain the opposite responses of lapA and bcs
Type: Promoter mutagenesis, EMSA, and in vitro transcription
Experiment: Separation-of-function FleQ mutants targeting ATPase versus c-di-GMP-binding surfaces tested for motility, attachment, biofilm, and root colonization phenotypes
Hypothesis: ATPase-deficient alleles will preferentially collapse the flagellar branch, whereas c-di-GMP-response mutants will uncouple motility from biofilm regulation
Type: Allelic replacement with phenotypic profiling
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