| Aspect | Summary | Key references with year and URL |
|---|---|---|
| Identity | **FlgM / PP_4395 / UniProt Q88EQ8** in *Pseudomonas putida* KT2440 matches the canonical **anti-σ28 factor / negative regulator of flagellin synthesis**. In KT2440 transcriptomic annotation, PP_4395 is explicitly labeled **“Negative regulator of flagellin synthesis FlgM”**; this agrees with the UniProt description and FlgM family/domain assignment. (pqac-00000003) | Navarro-Avilés & Van Dillewijn, 2010, https://doi.org/10.1111/j.1758-2229.2009.00084 |
| Molecular function | FlgM is a **protein anti-sigma factor** that binds the flagellar sigma factor **FliA (σ28)** and inhibits FliA-dependent transcription of late/class III-IV flagellar genes until assembly reaches the proper checkpoint. It is not an enzyme; its primary function is **regulatory sequestration of FliA**. (pqac-00000000, pqac-00000004, pqac-00000006, pqac-00000011) | Xiao et al., 2017, https://doi.org/10.1002/mbo3.402; Leal-Morales et al., 2022, https://doi.org/10.1111/1462-2920.15857 |
| Mechanism | Supported model for KT2440/Pseudomonas: FlgM acts in the **cytoplasm** to inhibit FliA; when the **hook-basal body / FT3SS checkpoint** is satisfied, **FlgM is exported/secretion-coupled**, releasing FliA and enabling late flagellar gene transcription. In KT2440 this is presented as the conserved activation logic of the final transcriptional tier. (pqac-00000004, pqac-00000006, pqac-00000008, pqac-00000011) | Xiao et al., 2017, https://doi.org/10.1002/mbo3.402; Leal-Morales et al., 2022, https://doi.org/10.1111/1462-2920.15857 |
| Localization | Best-supported localization is **cytoplasmic before export**, where FlgM binds FliA. After completion of the assembly checkpoint, FlgM is proposed to be **exported via the flagellar type III secretion system (FT3SS)**. Direct FlgM export was discussed for *Pseudomonas* and inferred as conserved for KT2440, but the gathered evidence did **not** include a KT2440-specific direct secretion assay for PP_4395. (pqac-00000000, pqac-00000004, pqac-00000006, pqac-00000011) | Xiao et al., 2017, https://doi.org/10.1002/mbo3.402; Leal-Morales et al., 2022, https://doi.org/10.1111/1462-2920.15857 |
| Pathway position | FlgM sits in the **late checkpoint of the flagellar transcriptional cascade** downstream of **FleQ/σ54-controlled early-class genes** and immediately upstream of **FliA-dependent late genes**. Figure-level synthesis for KT2440 supports a **three-tier hierarchy** in which FliA activation is the terminal step controlling filament/stator/final chemotaxis functions. (pqac-00000006, pqac-00000008, pqac-00000010) | Leal-Morales et al., 2022, https://doi.org/10.1111/1462-2920.15857 |
| Regulators / partners | Direct/near-direct partners and regulators supported in KT2440 evidence: **FliA (σ28)** is the cognate sigma factor inhibited by FlgM; **FlgN** and **PP4397/FlgZ** are co-transcribed with flgM in a **flgM-flgN-pp4397 tricistronic operon**; **σFliA** activates the promoter immediately upstream of flgM (**PflgM**); **σ54/RpoN** contributes via readthrough from upstream **flgA** transcription. KT2440 also encodes **HsbA/HsbR/HptB orthologues**, suggesting possible partner-switching regulation, but this was noted as a plausible mechanism rather than directly tested in the cited KT2440 study. (pqac-00000005, pqac-00000006, pqac-00000008, pqac-00000011) | Wirebrand et al., 2018, https://doi.org/10.1038/s41598-018-29785-w; Leal-Morales et al., 2022, https://doi.org/10.1111/1462-2920.15857 |
| Downstream genes / outputs | Through inhibition of FliA, FlgM negatively controls **late flagellar outputs**, including **flagellin/filament genes** and motility functions. In KT2440 and related Pseudomonas evidence, FliA-dependent targets include **fliC**, **flaG**, **fliD**, **fliS**, multiple **chemotaxis genes**, and motility-linked functions. FliA also contributes to **bifA** transcription, linking the flagellar cascade to **c-di-GMP lowering** and enhanced swimming. (pqac-00000003, pqac-00000004, pqac-00000006, pqac-00000008) | Navarro-Avilés & Van Dillewijn, 2010, https://doi.org/10.1111/j.1758-2229.2009.00084; Xiao et al., 2017, https://doi.org/10.1002/mbo3.402 |
| Experimental evidence / data points | Quantitative and direct evidence in KT2440 includes: **PP_4395/flgM downregulated ~1.8-fold** in a **fliA mutant** transcriptome, with a putative upstream FliA motif; **bifA transcription decreased ~2-fold** in a **fliA deletion** mutant; mutation of the **σ28 promoter** reduced promoter activity in wild type but not in the fliA mutant; **in vitro single-round transcription** confirmed **σFliA-RNAP** can drive **PflgM**; **RT-PCR** showed **flgM-flgN-pp4397** co-transcription; reporter assays in wild type versus **FliA-null** and **RpoN-null** backgrounds showed reduced, but not abolished, flgM operon expression, consistent with dual control. (pqac-00000003, pqac-00000004, pqac-00000005) | Navarro-Avilés & Van Dillewijn, 2010, https://doi.org/10.1111/j.1758-2229.2009.00084; Xiao et al., 2017, https://doi.org/10.1002/mbo3.402; Wirebrand et al., 2018, https://doi.org/10.1038/s41598-018-29785-w |
| Applications / implementation notes | No 2023-2024 KT2440 paper in the gathered evidence used **FlgM itself** as a standalone biotechnology part, but the **FlgM–FliA checkpoint** is functionally relevant to **motility engineering**, **biofilm–planktonic switching**, and **c-di-GMP control** in *Pseudomonas*. In KT2440, FliA-dependent control of **bifA** connects late flagellar regulation to intracellular signaling affecting swimming; more broadly, the cascade is useful for interpreting phenotypes in chassis engineering where motility or surface colonization matters. Because direct PP_4395 perturbation data in KT2440 were limited, implementation conclusions should be considered **mechanism-informed rather than directly engineered demonstrations**. (pqac-00000004, pqac-00000005, pqac-00000008, pqac-00000010) | Xiao et al., 2017, https://doi.org/10.1002/mbo3.402; Wirebrand et al., 2018, https://doi.org/10.1038/s41598-018-29785-w; Leal-Morales et al., 2022, https://doi.org/10.1111/1462-2920.15857 |
| Evidence strength / caveats | Confidence is **high** for identity, anti-σ28 function, operon context, FliA-dependent transcription, and placement in the KT2440 flagellar cascade. Confidence is **moderate** for the exact export behavior of KT2440 FlgM because the gathered evidence mainly presents a **conserved model/inference** rather than a direct KT2440 secretion assay. Claims about partner-switching via **HsbA/HsbR/HptB** are **hypothesis-supported/background-supported** in KT2440, not directly demonstrated for PP_4395 in the cited papers. (pqac-00000005, pqac-00000006, pqac-00000008, pqac-00000011) | Wirebrand et al., 2018, https://doi.org/10.1038/s41598-018-29785-w; Leal-Morales et al., 2022, https://doi.org/10.1111/1462-2920.15857 |


*Table: This table summarizes the functional annotation of *Pseudomonas putida* KT2440 FlgM (PP_4395; UniProt Q88EQ8), focusing on experimentally supported identity, mechanism, localization, regulation, and pathway context. It emphasizes what is directly shown in the gathered evidence and flags where conclusions are based on conserved models rather than direct KT2440 assays.*