| Aspect | Summary | Key quantitative data | Key references (year/URL) |
|---|---|---|---|
| Identity / domains | **Verified target:** FleQ encoded by **fleQ / PP_4373**, UniProt **Q88ET0**, in **Pseudomonas putida KT2440**. Literature for KT2440 matches the UniProt description: FleQ is an **atypical NtrC/NifA-family bacterial enhancer-binding protein (bEBP)** with an **N-terminal REC-like domain** lacking the canonical phospho-Asp, a **central AAA+/ATPase domain** that interfaces with **σ54/RpoN**, and a **C-terminal HTH DNA-binding domain**. FleQ is also described as a **c-di-GMP-binding** multimeric regulator, consistent with AAA+_ATPase, FleQ, and CheY-like/receiver-like domain annotations. (pqac-00000004, pqac-00000012, pqac-00000011) | Domain organization is tripartite; noncanonical REC-like domain lacks conserved phosphoacceptor Asp; oligomerization states reported for FleQ family include **dimers/trimers/tetramers/hexamers**, with spontaneous hexamerization described in recent mechanistic review. (pqac-00000011, pqac-00000012) | Blanco-Romero et al., 2018, https://doi.org/10.1038/s41598-018-31371-z; Oladosu et al., 2024, https://doi.org/10.1128/jb.00365-23 |
| Primary function | FleQ is the **top-level/master regulator of flagellar biogenesis** in P. putida KT2440 and also a major switch controlling the **motile-to-sessile transition** by oppositely regulating **flagellar genes** and **biofilm matrix/adhesion genes**. It activates many **σ54-dependent class II flagellar promoters** and directly controls biofilm determinants such as **lapA** and the **bcs cellulose operon**. (pqac-00000001, pqac-00000006, pqac-00000014) | In heterologous promoter assays, FleQ increased expression from **PflgB, PflhA, and PflgA by ~4- to 7-fold**. Deletion of fleQ caused a **~2-fold drop in lapA mRNA** and a **~32-fold increase in bcsD mRNA**. (pqac-00000006) | Jiménez-Fernández et al., 2016, https://doi.org/10.1371/journal.pone.0163142; Leal-Morales et al., 2022, https://doi.org/10.1111/1462-2920.15857 |
| Regulated pathways / direct targets | Direct and/or strongly supported FleQ targets in KT2440 include **flagellar export and structural genes** (**flhA**, **fliLMNOPQ**, **fliEFG**, **flhF**, **fleN**, **fleSR**), chemotaxis-associated genes, **lapA** and its secretion-linked functions, and **bcs/cellulose** loci. Genome-wide ChIP-seq further indicates a broad direct regulon extending to **adhesion**, **exopolysaccharide production**, and **iron-homeostasis** genes. FleQ acts as a **bifunctional regulator**: typically activating motility/adhesion genes and repressing some EPS genes. (pqac-00000004, pqac-00000005, pqac-00000007, pqac-00000010) | ChIP-seq in KT2440 identified **279** initial peaks, narrowed to **103** peaks at **≥5-fold enrichment**; **69.31%** of peaks were intergenic and **98%** upstream of ORFs, yielding **~160 likely target genes**. Across P. fluorescens F113 and KT2440, **41** promoter regions overlapped, and **56.1%** of shared targets related to motility, iron homeostasis, or cell wall functions. (pqac-00000007, pqac-00000010) | Blanco-Romero et al., 2018, https://doi.org/10.1038/s41598-018-31371-z |
| Mechanism & modulators | FleQ is a **σ54-dependent transcriptional activator** for class II flagellar genes but also regulates some **σ70-like promoter** outputs for biofilm genes. **c-di-GMP** binds FleQ’s AAA+ region/Walker A-associated ATPase module and **inhibits ATPase activity**, shifting FleQ from flagellar activation toward biofilm-associated regulation. **FleN** acts as an **antiactivator/modulator** that antagonizes FleQ at flagellar promoters and collaborates with FleQ (plus ATP/c-di-GMP) at biofilm promoters such as **lapA** and **bcs**. In promoter models, low c-di-GMP favors DNA distortion/repression at matrix loci, while high c-di-GMP relieves distortion and promotes activation of selected sessility genes. (pqac-00000009, pqac-00000012, pqac-00000013, pqac-00000016) | At **PlapA**, wild type showed **~4-fold** higher expression than fleQ mutant under low c-di-GMP and **~10-fold** higher under high c-di-GMP; at **PbcsD**, low c-di-GMP caused only **~1.4-fold** higher expression without FleQ, but at high c-di-GMP PbcsD became highly expressed and largely FleQ-unresponsive. Mutation of a critical FleQ motif in PlapA caused **>3-fold** decreased expression; full fleQ deletion caused **~6-fold** reduced PlapA reporter activity in that assay. (pqac-00000006, pqac-00000013) | Navarrete et al., 2019, https://doi.org/10.1371/journal.pone.0214166; Jiménez-Fernández et al., 2016, https://doi.org/10.1371/journal.pone.0163142; Oladosu et al., 2024, https://doi.org/10.1128/jb.00365-23 |
| Localization | FleQ is a **cytoplasmic DNA-binding transcriptional regulator** acting at **promoter regions on the chromosome**. Its biological effects are exerted in the **cytoplasm/nucleoid** through transcriptional control of surface organelles (flagella) and extracellular matrix determinants (LapA secretion/cellulose synthesis), rather than by being a membrane or extracellular protein. This localization is inferred from its domain architecture and DNA-binding/ChIP/EMSA evidence. (pqac-00000004, pqac-00000003, pqac-00000015) | ChIP-seq peak enrichment was overwhelmingly promoter-associated: **98%** of retained KT2440 peaks mapped upstream of ORFs; EMSA confirmed direct binding to promoter fragments containing predicted FleQ boxes. (pqac-00000003, pqac-00000010) | Blanco-Romero et al., 2018, https://doi.org/10.1038/s41598-018-31371-z; Jiménez-Fernández et al., 2016, https://doi.org/10.1371/journal.pone.0163142 |
| Key quantitative data | Experimental evidence for direct DNA interaction includes EMSA with purified native FleQ on **PlapA** and **PbcsD** fragments containing predicted FleQ motifs. FleQ directly bound motif-containing fragments but not control fragments lacking motifs. Reporter and qRT-PCR data support direct activation of **lapA** and repression of **bcsD**, with c-di-GMP changing the output. (pqac-00000003, pqac-00000006) | EMSA used **0, 0.45, and 4.5 μM FleQ**; **4.5 μM** fully shifted the **550 bp** and **297 bp** PlapA fragments and the **263 bp** PbcsD fragment, while a **152 bp** PlapA fragment and **200 bp** PbcsD fragment lacking predicted motifs were not shifted. ChIP-seq used **20,558,997** reads for KT2440 with **70.8%** alignment; peak calling threshold **FDR q=0.01** and **≥5-fold** enrichment. (pqac-00000003, pqac-00000010, pqac-00000015) | Jiménez-Fernández et al., 2016, https://doi.org/10.1371/journal.pone.0163142; Blanco-Romero et al., 2018, https://doi.org/10.1038/s41598-018-31371-z |
| Recent developments / applications | Recent work has mainly refined the **mechanistic model** rather than redefining the core annotation. A **2024 review** synthesizes structural and biochemical evidence that c-di-GMP binding to FleQ’s AAA+ ATPase domain **obstructs the ATP-binding pocket**, destabilizes hexamers, and explains switching between flagellar and biofilm outputs in Pseudomonas; these concepts are considered applicable to **P. putida** orthologs. A **2023 P. putida study** showed FleQ also **represses K1-T6SS expression**, indicating FleQ integrates motility/biofilm decisions with interbacterial competition traits. This is relevant for **biocontrol and chassis engineering**, because KT2440 is used in environmental/biotechnological contexts where motility, adhesion, biofilm formation, and antimicrobial competition affect root colonization and process performance. (pqac-00000011, pqac-00000008, pqac-00000012) | 2023 study reports K1-T6SS transcription is indirectly repressed by **RpoN** and **FleQ**; 2024 review emphasizes FleQ as a central switch between planktonic and sessile modes. No newer 2023–2024 study in the retrieved set overturns the established KT2440 functional annotation. (pqac-00000008, pqac-00000011) | Bernal et al., 2023, https://doi.org/10.1099/mic.0.001295; Oladosu et al., 2024, https://doi.org/10.1128/jb.00365-23 |


*Table: This table summarizes the experimentally supported functional annotation of FleQ (UniProt Q88ET0; PP_4373) in Pseudomonas putida KT2440, including identity, regulatory roles, mechanism, localization, and key quantitative data. It highlights direct evidence from ChIP-seq, reporter assays, qRT-PCR, and DNA-binding experiments, with recent contextual updates from 2023–2024.*