| Aspect | Key findings | Best supporting citations | Primary source |
|---|---|---|---|
| Enzyme reaction / substrates | • RelA is the primary **(p)ppGpp synthetase** in γ-proteobacteria, including the P. putida context  • Catalyzes transfer of the **βγ-pyrophosphate from ATP to the 3′-OH of GDP or GTP**, yielding **ppGpp or pppGpp + AMP**  • Fits UniProt Q88MB8 annotation: GTP pyrophosphokinase / ATP:GTP 3′-pyrophosphotransferase / ppGpp synthase I | (pqac-00000023, pqac-00000024, pqac-00000029) | Urwin et al. 2024, https://doi.org/10.1099/mic.0.001483; Ray 2023; Becker et al. 2025, https://doi.org/10.1159/000546200 |
| Protein family & domains | • Q88MB8 matches the **RelA/SpoT homolog (RSH)** family  • Long RSH architecture: N-terminal **HD/pseudo-HD** and **synthetase (SYNTH/SD)** regions plus C-terminal **helical/AH-RIS, ZFD, TGS, ACT** regulatory modules  • In RelA, the HD is typically **degenerate/inactive**, so the protein functions mainly as a synthetase  • Domain logic is consistent with UniProt domain/family assignments | (pqac-00000022, pqac-00000024, pqac-00000028, pqac-00000029) | Urwin et al. 2024, https://doi.org/10.1099/mic.0.001483; Becker et al. 2025, https://doi.org/10.1159/000546200 |
| Activation / regulation | • Activated during nutrient stress, especially **amino-acid starvation**, when **deacylated tRNA** accumulates in the ribosomal A site  • RelA undergoes ribosome-coupled conformational activation; (p)ppGpp can also support **allosteric positive feedback** in long RSH proteins  • In Pseudomonas-related systems, **DksA** collaborates with (p)ppGpp to remodel transcription  • In P. putida biofilm dispersal conditions tested, **SpoT-derived (p)ppGpp** rather than RelA was sufficient/required for the starvation response | (pqac-00000022, pqac-00000024, pqac-00000028, pqac-00000032, pqac-00000034) | Urwin et al. 2024, https://doi.org/10.1099/mic.0.001483; Díaz-Salazar et al. 2017, https://doi.org/10.1038/s41598-017-18518-0 |
| Cellular localization | • RelA is understood as a **cytosolic, ribosome-associated** enzyme rather than a membrane or extracellular protein  • Activation depends on interaction with **stalled ribosomes** and uncharged tRNA  • Direct KT2440 localization imaging was not retrieved, but Pseudomonas evidence supports ribosome association as the operative localization for function | (pqac-00000022, pqac-00000024, pqac-00000027) | Urwin et al. 2024, https://doi.org/10.1099/mic.0.001483; Pletzer et al. 2020, https://doi.org/10.1128/msystems.00495-20 |
| Pathway roles | • (p)ppGpp controls the **stringent response**, altering transcription, translation, DNA-replication-linked physiology, and metabolic allocation  • In P. putida, (p)ppGpp rapidly remodels **central carbon metabolism** and strongly **downregulates de novo purine biosynthesis**  • In biofilms, stringent-response signaling promotes dispersal through **bifA upregulation**, c-di-GMP reduction, and reduced **LapA** synthesis/secretion  • In nitrogen-limited P. putida, stringent-response deficiency alters **PHA-linked transcriptional programs** | (pqac-00000022, pqac-00000030, pqac-00000033, pqac-00000031) | Urwin et al. 2024, https://doi.org/10.1099/mic.0.001483; Vogeleer & Létisse 2022, https://doi.org/10.3389/fmicb.2022.872749; Díaz-Salazar et al. 2017, https://doi.org/10.1038/s41598-017-18518-0; Dabrowska et al. 2020, https://doi.org/10.3390/ijms22010152 |
| P. putida experimental evidence | • SHX treatment in KT2440 caused rapid **ppGpp and pppGpp accumulation within minutes** and growth arrest while cells remained metabolically active  • **ΔrelA** and **ppGpp0** strains failed to show the WT purine-pathway decrease after SHX, implicating RelA/(p)ppGpp in purine control  • **ppGpp0** and **ΔdksA ppGpp0** strains were strongly defective in starvation-induced biofilm dispersal; **ΔrelA** resembled WT in that assay  • relA/spoT deficiency altered pha operon regulation during nitrogen-responsive mcl-PHA physiology | (pqac-00000035, pqac-00000030, pqac-00000032, pqac-00000031) | Vogeleer & Létisse 2022, https://doi.org/10.3389/fmicb.2022.872749; Díaz-Salazar et al. 2017, https://doi.org/10.1038/s41598-017-18518-0; Dabrowska et al. 2020, https://doi.org/10.3390/ijms22010152 |
| Quantitative data points | • After stringent-response induction, WT purine intermediates fell to about **GAR 11%**, **FGAR 21%**, **AICAR 27%**, **AS 7%**, **IMP 50%** of baseline  • ppGpp0-related biofilm biomass peaked at **2–3× WT** and dispersal remained defective over ~20–26 h  • In ppGpp0, **PlapA / PlapBC / PlapE** reporter outputs increased about **4× / 2× / 3×** versus WT  • At **PbifA**, **1 µM DksA** alone gave up to **~2-fold** stimulation; **1 µM DksA + 200–600 µM ppGpp** gave about **~3-fold** combined stimulation; figure-based summary indicates up to **~9-fold** maximal in vitro activation across tested conditions | (pqac-00000030, pqac-00000013, pqac-00000012, pqac-00000019) | Vogeleer & Létisse 2022, https://doi.org/10.3389/fmicb.2022.872749; Díaz-Salazar et al. 2017, https://doi.org/10.1038/s41598-017-18518-0 |
| Applications / implementations | • For biotechnology, stringent-response signaling is relevant to **industrial stress adaptation** in P. putida, a major chassis organism  • Repeated glucose starvation in large-scale-like conditions induced a stringent-response-like program; only **0.4% of glucose uptake** was estimated to build 3-HA energy buffers and cellular maintenance increased by about **17%** under the tested regime  • relA/spoT-linked control of **biofilm dispersal** and **PHA-associated metabolism** makes the pathway relevant to bioprocess robustness, surface colonization, and carbon-storage engineering | (pqac-00000006, pqac-00000031, pqac-00000033) | Ankenbauer et al. 2020, https://doi.org/10.1111/1751-7915.13571; Dabrowska et al. 2020, https://doi.org/10.3390/ijms22010152; Díaz-Salazar et al. 2017, https://doi.org/10.1038/s41598-017-18518-0 |


*Table: This table summarizes the best-supported functional annotation points for Pseudomonas putida KT2440 RelA (UniProt Q88MB8 / PP_1656), integrating mechanism, regulation, organism-specific experiments, and key quantitative findings useful for annotation.*