GacS is a membrane-associated hybrid sensor histidine kinase of the GacS/GacA global regulatory system. Its domain architecture indicates a two-pass inner-membrane sensor with HAMP, histidine kinase, receiver, and HPt domains, consistent with multistep phosphorelay signaling. In Pseudomonas putida KT2440, genetic studies link GacS to broad lifestyle control, including surface attachment and positive regulation of the K1 type VI secretion system, so its core role is signal sensing and phosphorelay rather than a pathway-specific metabolic function.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0000155
phosphorelay sensor kinase activity
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: This is the best existing molecular-function annotation for GacS. UniProt and domain architecture identify a membrane-bound hybrid sensor histidine kinase with histidine kinase, receiver, and HPt modules, exactly the architecture expected for a phosphorelay sensor kinase.
Reason: GO:0000155 captures the core biochemical role of GacS more precisely than the generic parent terms for histidine kinase or phosphorus-group transferase activity.
Supporting Evidence:
UniProt:Q88MC3
-!- CATALYTIC ACTIVITY: Reaction=ATP + protein L-histidine = ADP + protein N-phospho-L- histidine.; EC=2.7.13.3; Evidence={ECO:0000256|ARBA:ARBA00000085};
file:PSEPK/gacS/gacS-notes.md
The core molecular role is phosphorelay sensor kinase activity at the membrane.
|
|
GO:0004673
protein histidine kinase activity
|
IEA
GO_REF:0000003 |
MARK AS OVER ANNOTATED |
Summary: GacS does perform protein histidine kinase chemistry, but this annotation is less informative than GO:0000155 for a hybrid sensory phosphorelay protein. The sensor-kinase context is the biologically important distinction here.
Reason: This parent term is biochemically correct but redundant once GO:0000155 phosphorelay sensor kinase activity is present.
|
|
GO:0016772
transferase activity, transferring phosphorus-containing groups
|
IEA
GO_REF:0000002 |
MARK AS OVER ANNOTATED |
Summary: This is a very broad ancestral parent of histidine kinase activity. It does not add useful information beyond the much more specific phosphorelay sensor kinase annotation already present.
Reason: The term is correct only at a very high level and should not be treated as a core annotation for a well-characterized hybrid sensor kinase.
|
|
GO:0000160
phosphorelay signal transduction system
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: This is the correct process-level abstraction for GacS. In KT2440, GacS is part of the GacS/GacA phosphorelay that controls downstream programs such as surface attachment and K1 type VI secretion system expression.
Reason: GacS is a membrane hybrid sensor kinase whose core biological role is initiating phosphorelay signaling, with adhesion and secretion phenotypes representing downstream outputs of that regulatory cascade.
Supporting Evidence:
file:PSEPK/gacS/gacS-notes.md
Process terms should center on phosphorelay signal transduction; the documented adhesion and T6SS phenotypes are downstream outputs of that global signaling role rather than separate direct molecular activities.
file:PSEPK/gacS/gacS-notes.md
In KT2440, expression of the K1 type VI secretion system gene cluster is positively regulated by the GacS-GacA two-component system and repressed by RetS.
|
|
GO:0007165
signal transduction
|
IEA
GO_REF:0000002 |
MARK AS OVER ANNOTATED |
Summary: GacS certainly participates in signal transduction, but GO:0007165 is too broad for a protein whose signaling mode is already captured by GO:0000160 phosphorelay signal transduction system.
Reason: The more specific phosphorelay term should carry the biology here; the generic parent adds little value.
|
|
GO:0005886
plasma membrane
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: GacS is a transmembrane sensor located in the bacterial inner membrane. In bacterial GO usage, plasma membrane is the appropriate cellular-component term for this localization.
Reason: Membrane localization is essential to GacS function because it senses external/periplasmic cues and transmits them through a membrane-embedded signaling architecture.
Supporting Evidence:
UniProt:Q88MC3
-!- SUBCELLULAR LOCATION: Cell inner membrane {ECO:0000256|ARBA:ARBA00004429}; Multi-pass membrane protein {ECO:0000256|ARBA:ARBA00004429}.
|
|
GO:0016020
membrane
|
IEA
GO_REF:0000002 |
MARK AS OVER ANNOTATED |
Summary: This term is correct but unnecessarily general because the protein is already localized more specifically to the plasma membrane.
Reason: GO:0005886 plasma membrane is the more informative and appropriate cellular-component annotation for GacS.
|
Q: What signal or signals are detected directly by the periplasmic sensor region of GacS in KT2440?
Suggested experts: Patricia Bernal, María A. Llamas
Q: Which small RNAs and Rsm-family effectors mediate the adhesion versus K1-T6SS branches downstream of GacS/GacA in KT2440?
Suggested experts: Patricia Bernal, Estrella Duque, Juan-Luis Ramos
Experiment: Build phosphosite mutants in the predicted receiver Asp718 and HPt His863 modules, then test phosphotransfer to GacA in vitro and with in vivo transcriptional reporters for Gac-dependent outputs.
Hypothesis: GacS uses its hybrid receiver and HPt modules to relay phosphate to GacA in a defined multistep order.
Type: phosphotransfer biochemistry plus reporter genetics
Experiment: Compare RNA-seq and promoter-reporter responses in wild type, delta-gacS, and sensor-domain or relay-module mutants during surface growth and stationary phase to separate direct GacS outputs from secondary physiological effects.
Hypothesis: The adhesion and K1-T6SS outputs reflect distinct downstream branches of the same GacS/GacA signaling pathway.
Type: comparative transcriptomics and promoter-reporter analysis
The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.
You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.
We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.
We are interested in where in or outside the cell the gene product carries out its function.
We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.
Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.
The literature reviewed here concerns the GacS sensor histidine kinase of the canonical GacS/GacA two-component system (TCS) upstream of the Gac/Rsm post-transcriptional regulatory cascade in pseudomonads. GacS is consistently described as an inner/plasma membrane-localized sensor kinase that phosphorylates the cytosolic response regulator GacA via a multi-step phosphorelay (latour2020theevanescentgacs pages 3-5, gallegos2024smallregulatoryrnas pages 4-6). This is consistent with the UniProt entry provided for Q88MC3 (gacS; PP_1650) in P. putida KT2440, including predicted membrane localization and the expected sensor-HK architecture and signaling domains (HAMP, His kinase catalytic modules, and receiver-like modules) (latour2020theevanescentgacs pages 3-5).
A bacterial TCS typically comprises a sensor histidine kinase (HK) and a response regulator (RR). In the Gac system, GacS is the sensor HK and GacA is the RR (latour2020theevanescentgacs pages 1-3, ferreiro2021distinctivefeaturesof pages 1-4). In pseudomonads, GacS is often described as a hybrid / non-orthodox HK, because its signaling can include additional phosphorelay steps (receiver and Hpt modules) before phosphate reaches the terminal RR (GacA) (ferreiro2021distinctivefeaturesof pages 6-9, latour2020theevanescentgacs pages 3-5).
In the Gac/Rsm cascade, phosphorylated GacA functions as a transcription factor that activates transcription of small regulatory RNAs (sRNAs) of the rsmX/rsmY/rsmZ families by binding a conserved upstream activation sequence (the Gac-box) (gallegos2024smallregulatoryrnas pages 4-6, ferreiro2021distinctivefeaturesof pages 1-4). The 2024 review summarizes the Gac-box consensus as TGTAAGN6CTTACA (gallegos2024smallregulatoryrnas pages 4-6).
These Rsm sRNAs then act as “sponge” RNAs: they sequester (titrate) RNA-binding proteins of the Rsm/CsrA family, relieving translational repression of many target mRNAs and enabling large lifestyle transitions (e.g., motile↔sessile and primary↔secondary metabolism) (gallegos2024smallregulatoryrnas pages 4-6, latour2020theevanescentgacs pages 1-3).
A detailed architecture is summarized in Latour (2020) (publication date: Nov 2020; URL https://doi.org/10.3390/microorganisms8111746). GacS is a homodimeric inner-membrane sensor kinase in which each monomer contains:
- an N-terminal transmembrane (TM) helix,
- a periplasmic detector domain,
- a second TM helix, and
- a large cytoplasmic region containing a HAMP domain and the histidine kinase catalytic core (HisKA + ATPase-like subdomain) (latour2020theevanescentgacs pages 3-5).
The periplasmic detector domain contains a putative ligand-binding pocket formed near a conserved flexible loop and a network of basic residues; residue-level functional mapping in P. aeruginosa implicated histidyl residues in this region in signal perception, and comparative analysis suggests divergence among Pseudomonas lineages (latour2020theevanescentgacs pages 3-5).
Cellular localization: the TM helices anchor GacS in the inner (plasma) membrane, positioning the detector domain in the periplasm and the regulatory/catalytic domains in the cytoplasm (latour2020theevanescentgacs pages 3-5, gallegos2024smallregulatoryrnas pages 4-6).
GacS is a histidine kinase (EC 2.7.13.3). Functionally, it converts an input signal into a phosphorylation state change by:
1) autophosphorylation on a conserved histidine within the HK domain (ATP-dependent), and
2) phosphotransfer through an internal phosphorelay to ultimately phosphorylate GacA on a conserved aspartate (latour2020theevanescentgacs pages 3-5, ferreiro2021distinctivefeaturesof pages 6-9).
Latour (2020) further notes that GacS autophosphorylation likely occurs in trans within the GacS homodimer, and that deletion of the HAMP domain can cause signal-independent activation of the downstream cascade (latour2020theevanescentgacs pages 3-5).
A key limitation in current knowledge is that the activating ligand(s) for GacS remain unknown. Latour (2020) synthesizes evidence suggesting the signal is likely a low-molecular-weight, relatively simple, moderately apolar metabolite and proposes that it may reflect nutritional stress/competition at high cell density (latour2020theevanescentgacs pages 1-3, latour2020theevanescentgacs pages 5-6). Ferreiro & Gallegos (2021) similarly emphasize that the activating environmental signal remains unidentified (ferreiro2021distinctivefeaturesof pages 6-9).
In pseudomonads, GacS output can be modulated by additional membrane kinases:
- LadS can stimulate GacS signaling by shuttling phosphate into the GacS phosphorelay (Hpt module) (latour2020theevanescentgacs pages 3-5, ferreiro2021distinctivefeaturesof pages 6-9).
- RetS inhibits GacS via several mechanisms (phosphate siphoning, dephosphorylation of internal modules, and physical blockage of the catalytic histidine region) (latour2020theevanescentgacs pages 3-5, ferreiro2021distinctivefeaturesof pages 6-9).
- PA1611 (in P. aeruginosa) can antagonize RetS inhibition (latour2020theevanescentgacs pages 3-5).
These components are best established in P. aeruginosa models and should be treated as mechanistic analogs unless validated directly in P. putida KT2440 (latour2020theevanescentgacs pages 3-5, ferreiro2021distinctivefeaturesof pages 6-9).
Huertas-Rosales et al. (2016) (publication date: Sep 2016; URL https://doi.org/10.1128/AEM.01724-16) established that P. putida KT2440 encodes three CsrA/Rsm-family RNA-binding proteins:
- RsmI (PP_1746),
- RsmE (PP_3832),
- RsmA (PP_4472),
with reported lengths and similarities (e.g., RsmA 62 aa; RsmE 65 aa; RsmI 59 aa) and pairwise identity relationships (huertasrosales2016selfregulationandinterplay pages 9-11).
Mechanistically, the same study situates these Rsm proteins within the GacS/GacA-controlled cascade: the Rsm family proteins and their cognate small RNAs in Pseudomonas are described as part of the GacS/GacA pathway, where GacS phosphorylates GacA and GacA activates rsmX/Y/Z-family sRNAs (huertasrosales2016selfregulationandinterplay pages 3-5).
Huertas-Rosales et al. (2021) (publication date: Feb 2021; URL https://doi.org/10.3389/fmolb.2021.624061) performed in vivo affinity purification of Rsm-associated RNAs coupled to sequencing. Key quantitative outcomes:
- 437 unique RNA molecules were identified as in vivo Rsm-associated targets,
- 75 targets were common to all three Rsm proteins, and
- under the tested conditions, at least 12% of transcripts in KT2440 were bound in vivo by Rsm proteins (huertasrosales2021genomewideanalysisof pages 1-2, huertasrosales2021genomewideanalysisof pages 5-7).
These targets span metabolism, transport/secretion, stress responses, and multiple regulatory nodes, including c-di-GMP turnover and secretion system components (e.g., Type VI secretion system targets among common sets) (huertasrosales2021genomewideanalysisof pages 5-7).
Huertas-Rosales et al. (2016) demonstrated that Rsm proteins modulate KT2440 lifestyles: a triple rsm mutant displayed reduced swimming and swarming and increased biofilm formation, while overexpression of RsmE or RsmI reduced attachment. The authors also observed surface-dependent biofilm properties (glass vs plastic) indicating altered extracellular matrix composition/timing (huertasrosales2016selfregulationandinterplay pages 1-3).
Huertas-Rosales et al. (2021) further connected Rsm regulation to biofilm via c-di-GMP circuitry and adhesins (e.g., lapA), and provided a schematic pathway model integrating regulators (RpoS, FleQ), c-di-GMP enzymes, and matrix components (huertasrosales2021genomewideanalysisof pages 9-11). The requested corroborating figure is available (huertasrosales2021genomewideanalysisof media 3d0e1063).
A plant-associated Pseudomonas review compiles KT2440 observations that perturbations in gacS/gacA are associated with increased expression of lapA, lapF, rpoS and cfcR and increased biofilm formation, and that overexpression of rsmA/E/I decreases swimming/swarming (ferreiro2021distinctivefeaturesof pages 28-29). This provides KT2440-relevant directionality for the GacS→Rsm→biofilm/motility axis, though the review table does not provide fold changes (ferreiro2021distinctivefeaturesof pages 28-29).
Gallegos et al. (2024) (publication date: Sep 2024; URL https://doi.org/10.1111/mmi.15313) provides an up-to-date review and large comparative analysis of Rsm sRNAs in Pseudomonas. Key points relevant to functional annotation:
- GacS is described as a membrane histidine kinase sensing a periplasmic stimulus and activating GacA (gallegos2024smallregulatoryrnas pages 4-6).
- The review compiles genus-wide statistics from 245 complete chromosomes: rsmY and rsmZ appear broadly conserved (245 alleles each in their dataset), while rsmX is more variable (259 alleles; often multi-copy) and allele sizes average ~121 nt (rsmY), ~132 nt (rsmZ), ~113 nt (rsmX) (gallegos2024smallregulatoryrnas pages 8-11).
- Typical Rsm sRNAs carry multiple protein-binding motifs (often 4–7 binding sites) supporting their sponge role (gallegos2024smallregulatoryrnas pages 4-6).
These updates strengthen confidence that the GacS/GacA→Rsm sRNA→Rsm protein architecture is a conserved “core logic” while allowing KT2440-specific elaborations in the composition of Rsm proteins and sRNAs.
Thompson et al. (2023) (publication date: Feb 14, 2023; URL https://doi.org/10.1371/journal.pbio.3001988) showed that a plasmid-encoded translational regulator (RsmQ) can cause large-scale proteome remodeling in Pseudomonas fluorescens by interfering with the host’s translational regulatory network connected to Gac/Rsm. Under their conditions, deletion of rsmQ altered abundance of 581 proteins (≥2-fold increased) and 203 proteins (≥2-fold decreased) (thompson2023plasmidsmanipulatebacterial pages 8-10). While not in KT2440, this is a recent, high-profile demonstration that the Gac/Rsm post-transcriptional layer is an interaction hub that can be manipulated by mobile elements—relevant to strain engineering and stability considerations.
The Gac/Rsm cascade is repeatedly linked to production of extracellular/secondary metabolites and behaviors critical for plant-associated fitness and biocontrol, including antibiotics, lipopeptide surfactants, and enzymes, as well as motility/biofilm traits (ferreiro2021distinctivefeaturesof pages 11-14, ferreiro2021distinctivefeaturesof pages 14-16). Ferreiro & Gallegos (2021) specifically notes Gac/Rsm control of putisolvins in P. putida PCL1445 (lipopeptide surfactants) and a role in biofilm formation and other plant-associated traits in P. putida (ferreiro2021distinctivefeaturesof pages 11-14).
In an applied biocontrol organism (P. chlororaphis O6), the Gac/Rsm system coordinates beneficial metabolite expression during root colonization; a quantitative colonization statistic reported is recovery at approximately 10^7–10^8 CFU per gram of root after seed inoculation (anderson2017thegacrsmsignaling pages 1-3). This illustrates that Gac/Rsm-controlled traits can be deployed in real agricultural contexts.
Ferreiro & Gallegos (2021) emphasizes that spontaneous gacS/gacA variants can arise and may alter biocontrol performance and survival; therefore, monitoring for Gac/Rsm functionality (e.g., rapid surfactant/siderophore phenotyping) is relevant in practical strain development and deployment (ferreiro2021distinctivefeaturesof pages 14-16).
1) Central role, but unknown primary signal. Across authoritative reviews, the upstream “sensor” role of GacS is clear, but the identity of the activating ligand remains a major unresolved problem, limiting precise mapping of environmental inputs to regulatory outputs in KT2440 (latour2020theevanescentgacs pages 1-3, ferreiro2021distinctivefeaturesof pages 6-9).
2) GacS is best annotated as a global regulatory hub rather than a pathway-specific enzyme. The phosphorelay is biochemically well defined (HK autophosphorylation and transfer to GacA), but the biological meaning is expressed through Rsm sRNAs/proteins controlling broad regulons and lifestyle transitions (latour2020theevanescentgacs pages 3-5, gallegos2024smallregulatoryrnas pages 4-6).
3) In KT2440, downstream Rsm post-transcriptional control appears extensive and mechanistically grounded. The KT2440 Rsm binding datasets (hundreds of direct RNA interactions; ≥12% of transcripts bound) support that many pleiotropic phenotypes of gacS/gacA perturbation plausibly arise through Rsm-dependent translational/post-transcriptional reprogramming and c-di-GMP/biofilm control nodes (huertasrosales2021genomewideanalysisof pages 1-2, huertasrosales2021genomewideanalysisof pages 9-11).
| Component/claim | Evidence summary | Organism/strain | Quantitative detail | Source (with DOI URL and year) |
|---|---|---|---|---|
| GacS identity, domains, localization | GacS is the membrane/inner-membrane sensor histidine kinase upstream of the Gac/Rsm cascade; described as a homodimer with N-terminal TM helix, periplasmic detector domain, second TM helix, cytoplasmic HAMP domain, HK (HisKA + ATPase) core, internal receiver (REC/RR) and Hpt modules characteristic of a hybrid/non-orthodox HK (latour2020theevanescentgacs pages 3-5) | Pseudomonas spp.; applicable to P. putida KT2440 | 2 TM helices; homodimeric sensor | Latour 2020, DOI: https://doi.org/10.3390/microorganisms8111746 |
| GacS→GacA phosphorelay mechanism | Ligand-triggered conformational change is transmitted through HAMP to the HK domain, causing autophosphorylation on conserved His, followed by phosphorelay through REC and Hpt domains to a conserved Asp on GacA; autophosphorylation likely occurs in trans within the dimer (latour2020theevanescentgacs pages 3-5, ferreiro2021distinctivefeaturesof pages 6-9) | Pseudomonas spp. | Multi-step His→Asp phosphorelay; terminal transfer to GacA Asp | Latour 2020, DOI: https://doi.org/10.3390/microorganisms8111746; Ferreiro & Gallegos 2021, DOI: https://doi.org/10.1111/1462-2920.15558 |
| GacS signal and modulation | The direct activating ligand of GacS remains unknown; reviews describe it as likely a low-molecular-weight, moderately apolar metabolite. GacS output is modulated by LadS (stimulatory phosphotransfer into GacS Hpt) and RetS (inhibitory phosphate siphoning/heterointerference), with PA1611 counteracting RetS in some pseudomonads (latour2020theevanescentgacs pages 1-3, latour2020theevanescentgacs pages 3-5, ferreiro2021distinctivefeaturesof pages 6-9) | Pseudomonas spp. | Signal unresolved as of 2020–2024 | Latour 2020, DOI: https://doi.org/10.3390/microorganisms8111746; Ferreiro & Gallegos 2021, DOI: https://doi.org/10.1111/1462-2920.15558 |
| GacA target promoter motif (Gac-box) | Activated GacA binds a conserved upstream activation sequence/Gac-box to induce transcription of Rsm sRNAs (gallegos2024smallregulatoryrnas pages 4-6, ferreiro2021distinctivefeaturesof pages 1-4) | Pseudomonas spp. | Consensus TGTAAGN6CTTACA | Gallegos et al. 2024, DOI: https://doi.org/10.1111/mmi.15313; Ferreiro & Gallegos 2021, DOI: https://doi.org/10.1111/1462-2920.15558 |
| rsmX/rsmY/rsmZ sRNAs function | Rsm sRNAs are induced by GacS/GacA and act as “sponge” RNAs that sequester Rsm/Csr proteins, relieving translational repression of target mRNAs and enabling lifestyle/metabolic transitions (gallegos2024smallregulatoryrnas pages 4-6, latour2020theevanescentgacs pages 1-3, huertasrosales2016selfregulationandinterplay pages 3-5) | Pseudomonas spp.; P. putida KT2440 pathway context | Typical size range ~100–130 nt; usually 4–7 Rsm-binding motifs in Pseudomonas sRNAs | Gallegos et al. 2024, DOI: https://doi.org/10.1111/mmi.15313; Latour 2020, DOI: https://doi.org/10.3390/microorganisms8111746 |
| Distribution of Rsm sRNAs in the genus | Comparative 2024 analysis found broad conservation of rsmY and rsmZ and patchier distribution of rsmX across Pseudomonas genomes, supporting conserved core Gac/Rsm logic with lineage-specific expansions (gallegos2024smallregulatoryrnas pages 8-11) | 245 complete Pseudomonas chromosomes | 245 rsmZ alleles, 245 rsmY alleles, 259 rsmX alleles; average sizes: rsmZ 132 nt, rsmY 121 nt, rsmX 113 nt | Gallegos et al. 2024, DOI: https://doi.org/10.1111/mmi.15313 |
| P. putida KT2440 Rsm proteins | KT2440 encodes three CsrA/Rsm-family proteins: RsmI (PP_1746), RsmE (PP_3832), and RsmA (PP_4472); they are small RNA-binding proteins involved in post-transcriptional control downstream of GacS/GacA (huertasrosales2016selfregulationandinterplay pages 9-11, huertasrosales2016selfregulationandinterplay pages 3-5) | P. putida KT2440 | Identities to E. coli CsrA: 46% (RsmI), 70% (RsmE), 75% (RsmA); lengths 59, 65, and 62 aa | Huertas-Rosales et al. 2016, DOI: https://doi.org/10.1128/AEM.01724-16 |
| KT2440 Rsm-regulon size | In vivo affinity purification + RNA-seq identified a large direct Rsm target set in KT2440, including regulatory, metabolic, transport, stress, c-di-GMP and secretion-related RNAs; rsmY itself and rsmE were among common targets (huertasrosales2021genomewideanalysisof pages 1-2, huertasrosales2021genomewideanalysisof pages 5-7) | P. putida KT2440 | 437 unique RNA targets total; 75 common to all three Rsm proteins; at least 12% of transcripts bound in vivo under tested conditions | Huertas-Rosales et al. 2021, DOI: https://doi.org/10.3389/fmolb.2021.624061 |
| KT2440 downstream pathway links to biofilm/c-di-GMP | Rsm proteins directly or indirectly control c-di-GMP signaling and biofilm determinants through targets including cfcA/cfcR-related circuitry, lapA, cellulose/EPS loci, and other regulators; schematic pathway integration was presented in Figure 4 (huertasrosales2021genomewideanalysisof pages 9-11, huertasrosales2021genomewideanalysisof media 3d0e1063) | P. putida KT2440 | cfcR RsmA FE values reported below cutoff at 1.82 and 1.88 in vivo; multiple c-di-GMP regulators identified among targets | Huertas-Rosales et al. 2021, DOI: https://doi.org/10.3389/fmolb.2021.624061 |
| KT2440 Rsm mutant phenotypes | The triple rsm mutant shows reduced swimming and swarming and increased biofilm formation; biofilms on glass are more labile/easily detached, while overexpression of RsmE or RsmI reduces attachment, indicating dosage-sensitive control of sessile behavior (huertasrosales2016selfregulationandinterplay pages 1-3) | P. putida KT2440 | 3 Rsm proteins analyzed via single/double/triple mutants and overexpression | Huertas-Rosales et al. 2016, DOI: https://doi.org/10.1128/AEM.01724-16 |
| KT2440 gacS/gacA-linked phenotypes | Reviews compiling KT2440 studies report that gacS/gacA mutants increase expression of lapA, lapF, rpoS and cfcR and enhance biofilm formation and use of L-lysine, arginine, and histidine; overexpression of rsmA/E/I decreases swimming and swarming (ferreiro2021distinctivefeaturesof pages 28-29) | P. putida KT2440 | Directional effects reported; no fold-change values given in reviewed table | Ferreiro & Gallegos 2021, DOI: https://doi.org/10.1111/1462-2920.15558 |
| Broader pathway output | Gac/Rsm controls major lifestyle switches including motile↔sessile behavior, primary↔secondary metabolism, stress tolerance, secretion, and biofilm traits; in plant-associated pseudomonads it can affect ~10% of genes in some strains (ferreiro2021distinctivefeaturesof pages 14-16, latour2020theevanescentgacs pages 1-3) | Plant-associated Pseudomonas spp. | “About 10% of genes” in some cases; high-density activation associated with metabolic switch | Ferreiro & Gallegos 2021, DOI: https://doi.org/10.1111/1462-2920.15558; Latour 2020, DOI: https://doi.org/10.3390/microorganisms8111746 |
| Recent development: 2024 Rsm sRNA synthesis | A 2024 authoritative review updated the phylogeny, structure, nomenclature and mechanistic understanding of Rsm-clan sRNAs, reinforcing the central place of GacS/GacA-controlled sRNAs in Pseudomonas regulation (gallegos2024smallregulatoryrnas pages 4-6, gallegos2024smallregulatoryrnas pages 8-11) | Pseudomonas genus | Dataset of 245 complete chromosomes; 70 candidate novel rsm-family loci | Gallegos et al. 2024, DOI: https://doi.org/10.1111/mmi.15313 |
| Recent development: 2023 translational crosstalk | A 2023 study showed plasmid-encoded RsmQ rewires the host Gac/Rsm-linked translational network, including interaction with RsmY/RsmZ and altered abundance of RetS/Hfq-linked outputs, illustrating new layers of post-transcriptional crosstalk relevant to Gac/Rsm biology (thompson2023plasmidsmanipulatebacterial pages 8-10) | P. fluorescens SBW25 | 581 proteins increased and 203 decreased by ≥2-fold in ΔrsmQ comparison; ~50% of affected genes had AnGGA motifs | Thompson et al. 2023, DOI: https://doi.org/10.1371/journal.pbio.3001988 |
Table: This table condenses the verified identity and mechanism of GacS, the core downstream Gac/Rsm regulatory elements, and the most relevant Pseudomonas putida KT2440-specific findings and quantitative data. It also highlights recent 2023–2024 developments that update understanding of the broader Gac/Rsm network.
Despite strong pathway-level evidence, two KT2440-specific gaps remain prominent: (i) direct biochemical identification of the GacS ligand(s) in KT2440 and (ii) quantitative mapping from defined stimuli to GacA phosphorylation and rsm sRNA transcription kinetics in KT2440. These are highlighted as open problems in authoritative reviews (latour2020theevanescentgacs pages 1-3, ferreiro2021distinctivefeaturesof pages 6-9).
References
(latour2020theevanescentgacs pages 3-5): Xavier Latour. The evanescent gacs signal. Microorganisms, 8:1746, Nov 2020. URL: https://doi.org/10.3390/microorganisms8111746, doi:10.3390/microorganisms8111746. This article has 46 citations.
(gallegos2024smallregulatoryrnas pages 4-6): María Trinidad Gallegos, Matías Garavaglia, and Claudio Valverde. Small regulatory rnas of the rsm clan in pseudomonas. Molecular Microbiology, 122:563-582, Sep 2024. URL: https://doi.org/10.1111/mmi.15313, doi:10.1111/mmi.15313. This article has 5 citations and is from a domain leading peer-reviewed journal.
(latour2020theevanescentgacs pages 1-3): Xavier Latour. The evanescent gacs signal. Microorganisms, 8:1746, Nov 2020. URL: https://doi.org/10.3390/microorganisms8111746, doi:10.3390/microorganisms8111746. This article has 46 citations.
(ferreiro2021distinctivefeaturesof pages 1-4): María‐Dolores Ferreiro and María‐Trinidad Gallegos. Distinctive features of the
(ferreiro2021distinctivefeaturesof pages 6-9): María‐Dolores Ferreiro and María‐Trinidad Gallegos. Distinctive features of the
(latour2020theevanescentgacs pages 5-6): Xavier Latour. The evanescent gacs signal. Microorganisms, 8:1746, Nov 2020. URL: https://doi.org/10.3390/microorganisms8111746, doi:10.3390/microorganisms8111746. This article has 46 citations.
(huertasrosales2016selfregulationandinterplay pages 9-11): Óscar Huertas-Rosales, María Isabel Ramos-González, and Manuel Espinosa-Urgel. Self-regulation and interplay of rsm family proteins modulate the lifestyle of pseudomonas putida. Applied and Environmental Microbiology, 82:5673-5686, Sep 2016. URL: https://doi.org/10.1128/aem.01724-16, doi:10.1128/aem.01724-16. This article has 31 citations and is from a peer-reviewed journal.
(huertasrosales2016selfregulationandinterplay pages 3-5): Óscar Huertas-Rosales, María Isabel Ramos-González, and Manuel Espinosa-Urgel. Self-regulation and interplay of rsm family proteins modulate the lifestyle of pseudomonas putida. Applied and Environmental Microbiology, 82:5673-5686, Sep 2016. URL: https://doi.org/10.1128/aem.01724-16, doi:10.1128/aem.01724-16. This article has 31 citations and is from a peer-reviewed journal.
(huertasrosales2021genomewideanalysisof pages 1-2): Óscar Huertas-Rosales, Manuel Romero, Kok-Gan Chan, Kar-Wai Hong, Miguel Cámara, Stephan Heeb, Laura Barrientos-Moreno, María Antonia Molina-Henares, María L. Travieso, María Isabel Ramos-González, and Manuel Espinosa-Urgel. Genome-wide analysis of targets for post-transcriptional regulation by rsm proteins in pseudomonas putida. Frontiers in Molecular Biosciences, Feb 2021. URL: https://doi.org/10.3389/fmolb.2021.624061, doi:10.3389/fmolb.2021.624061. This article has 14 citations.
(huertasrosales2021genomewideanalysisof pages 5-7): Óscar Huertas-Rosales, Manuel Romero, Kok-Gan Chan, Kar-Wai Hong, Miguel Cámara, Stephan Heeb, Laura Barrientos-Moreno, María Antonia Molina-Henares, María L. Travieso, María Isabel Ramos-González, and Manuel Espinosa-Urgel. Genome-wide analysis of targets for post-transcriptional regulation by rsm proteins in pseudomonas putida. Frontiers in Molecular Biosciences, Feb 2021. URL: https://doi.org/10.3389/fmolb.2021.624061, doi:10.3389/fmolb.2021.624061. This article has 14 citations.
(huertasrosales2016selfregulationandinterplay pages 1-3): Óscar Huertas-Rosales, María Isabel Ramos-González, and Manuel Espinosa-Urgel. Self-regulation and interplay of rsm family proteins modulate the lifestyle of pseudomonas putida. Applied and Environmental Microbiology, 82:5673-5686, Sep 2016. URL: https://doi.org/10.1128/aem.01724-16, doi:10.1128/aem.01724-16. This article has 31 citations and is from a peer-reviewed journal.
(huertasrosales2021genomewideanalysisof pages 9-11): Óscar Huertas-Rosales, Manuel Romero, Kok-Gan Chan, Kar-Wai Hong, Miguel Cámara, Stephan Heeb, Laura Barrientos-Moreno, María Antonia Molina-Henares, María L. Travieso, María Isabel Ramos-González, and Manuel Espinosa-Urgel. Genome-wide analysis of targets for post-transcriptional regulation by rsm proteins in pseudomonas putida. Frontiers in Molecular Biosciences, Feb 2021. URL: https://doi.org/10.3389/fmolb.2021.624061, doi:10.3389/fmolb.2021.624061. This article has 14 citations.
(huertasrosales2021genomewideanalysisof media 3d0e1063): Óscar Huertas-Rosales, Manuel Romero, Kok-Gan Chan, Kar-Wai Hong, Miguel Cámara, Stephan Heeb, Laura Barrientos-Moreno, María Antonia Molina-Henares, María L. Travieso, María Isabel Ramos-González, and Manuel Espinosa-Urgel. Genome-wide analysis of targets for post-transcriptional regulation by rsm proteins in pseudomonas putida. Frontiers in Molecular Biosciences, Feb 2021. URL: https://doi.org/10.3389/fmolb.2021.624061, doi:10.3389/fmolb.2021.624061. This article has 14 citations.
(ferreiro2021distinctivefeaturesof pages 28-29): María‐Dolores Ferreiro and María‐Trinidad Gallegos. Distinctive features of the
(gallegos2024smallregulatoryrnas pages 8-11): María Trinidad Gallegos, Matías Garavaglia, and Claudio Valverde. Small regulatory rnas of the rsm clan in pseudomonas. Molecular Microbiology, 122:563-582, Sep 2024. URL: https://doi.org/10.1111/mmi.15313, doi:10.1111/mmi.15313. This article has 5 citations and is from a domain leading peer-reviewed journal.
(thompson2023plasmidsmanipulatebacterial pages 8-10): Catriona M. A. Thompson, James P. J. Hall, Govind Chandra, Carlo Martins, Gerhard Saalbach, Supakan Panturat, Susannah M. Bird, Samuel Ford, Richard H. Little, Ainelen Piazza, Ellie Harrison, Robert W. Jackson, Michael A. Brockhurst, and Jacob G. Malone. Plasmids manipulate bacterial behaviour through translational regulatory crosstalk. Feb 2023. URL: https://doi.org/10.1371/journal.pbio.3001988, doi:10.1371/journal.pbio.3001988. This article has 34 citations and is from a highest quality peer-reviewed journal.
(ferreiro2021distinctivefeaturesof pages 11-14): María‐Dolores Ferreiro and María‐Trinidad Gallegos. Distinctive features of the
(ferreiro2021distinctivefeaturesof pages 14-16): María‐Dolores Ferreiro and María‐Trinidad Gallegos. Distinctive features of the
(anderson2017thegacrsmsignaling pages 1-3): Anne J. Anderson, Beom Ryong Kang, and Young Cheol Kim. The gac/rsm signaling pathway of a biocontrol bacterium, pseudomonas chlororaphis o6. Radiation Protection Dosimetry, 23:212-227, Sep 2017. URL: https://doi.org/10.5423/rpd.2017.23.3.212, doi:10.5423/rpd.2017.23.3.212. This article has 24 citations and is from a peer-reviewed journal.
id: Q88MC3
gene_symbol: gacS
product_type: PROTEIN
status: DRAFT
taxon:
id: NCBITaxon:160488
label: Pseudomonas putida (strain ATCC 47054 / DSM 6125 / CFBP 8728 / NCIMB 11950
/ KT2440)
description: >-
GacS is a membrane-associated hybrid sensor histidine kinase of the GacS/GacA
global regulatory system. Its domain architecture indicates a two-pass inner-membrane
sensor with HAMP, histidine kinase, receiver, and HPt domains, consistent with
multistep phosphorelay signaling. In Pseudomonas putida KT2440, genetic studies
link GacS to broad lifestyle control, including surface attachment and positive
regulation of the K1 type VI secretion system, so its core role is signal sensing
and phosphorelay rather than a pathway-specific metabolic function.
existing_annotations:
- term:
id: GO:0000155
label: phosphorelay sensor kinase activity
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
This is the best existing molecular-function annotation for GacS. UniProt
and domain architecture identify a membrane-bound hybrid sensor histidine
kinase with histidine kinase, receiver, and HPt modules, exactly the architecture
expected for a phosphorelay sensor kinase.
action: ACCEPT
reason: >-
GO:0000155 captures the core biochemical role of GacS more precisely than
the generic parent terms for histidine kinase or phosphorus-group transferase
activity.
supported_by:
- reference_id: UniProt:Q88MC3
supporting_text: "-!- CATALYTIC ACTIVITY: Reaction=ATP + protein L-histidine = ADP + protein N-phospho-L- histidine.; EC=2.7.13.3; Evidence={ECO:0000256|ARBA:ARBA00000085};"
- reference_id: file:PSEPK/gacS/gacS-notes.md
supporting_text: "The core molecular role is phosphorelay sensor kinase activity at the membrane."
- term:
id: GO:0004673
label: protein histidine kinase activity
evidence_type: IEA
original_reference_id: GO_REF:0000003
review:
summary: >-
GacS does perform protein histidine kinase chemistry, but this annotation is
less informative than GO:0000155 for a hybrid sensory phosphorelay protein.
The sensor-kinase context is the biologically important distinction here.
action: MARK_AS_OVER_ANNOTATED
reason: >-
This parent term is biochemically correct but redundant once GO:0000155 phosphorelay
sensor kinase activity is present.
- term:
id: GO:0016772
label: transferase activity, transferring phosphorus-containing groups
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
This is a very broad ancestral parent of histidine kinase activity. It does
not add useful information beyond the much more specific phosphorelay sensor
kinase annotation already present.
action: MARK_AS_OVER_ANNOTATED
reason: >-
The term is correct only at a very high level and should not be treated as
a core annotation for a well-characterized hybrid sensor kinase.
- term:
id: GO:0000160
label: phosphorelay signal transduction system
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
This is the correct process-level abstraction for GacS. In KT2440, GacS is
part of the GacS/GacA phosphorelay that controls downstream programs such as
surface attachment and K1 type VI secretion system expression.
action: ACCEPT
reason: >-
GacS is a membrane hybrid sensor kinase whose core biological role is initiating
phosphorelay signaling, with adhesion and secretion phenotypes representing
downstream outputs of that regulatory cascade.
supported_by:
- reference_id: file:PSEPK/gacS/gacS-notes.md
supporting_text: "Process terms should center on phosphorelay signal transduction; the documented adhesion and T6SS phenotypes are downstream outputs of that global signaling role rather than separate direct molecular activities."
- reference_id: file:PSEPK/gacS/gacS-notes.md
supporting_text: "In KT2440, expression of the K1 type VI secretion system gene cluster is positively regulated by the GacS-GacA two-component system and repressed by RetS."
- term:
id: GO:0007165
label: signal transduction
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
GacS certainly participates in signal transduction, but GO:0007165 is too broad
for a protein whose signaling mode is already captured by GO:0000160 phosphorelay
signal transduction system.
action: MARK_AS_OVER_ANNOTATED
reason: >-
The more specific phosphorelay term should carry the biology here; the generic
parent adds little value.
- term:
id: GO:0005886
label: plasma membrane
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
GacS is a transmembrane sensor located in the bacterial inner membrane. In bacterial
GO usage, plasma membrane is the appropriate cellular-component term for this
localization.
action: ACCEPT
reason: >-
Membrane localization is essential to GacS function because it senses external/periplasmic
cues and transmits them through a membrane-embedded signaling architecture.
supported_by:
- reference_id: UniProt:Q88MC3
supporting_text: "-!- SUBCELLULAR LOCATION: Cell inner membrane {ECO:0000256|ARBA:ARBA00004429}; Multi-pass membrane protein {ECO:0000256|ARBA:ARBA00004429}."
- term:
id: GO:0016020
label: membrane
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
This term is correct but unnecessarily general because the protein is already
localized more specifically to the plasma membrane.
action: MARK_AS_OVER_ANNOTATED
reason: >-
GO:0005886 plasma membrane is the more informative and appropriate cellular-component
annotation for GacS.
references:
- id: GO_REF:0000002
title: Gene Ontology annotation through association of InterPro records with GO terms
findings: []
- id: GO_REF:0000003
title: Gene Ontology annotation based on Enzyme Commission mapping
findings: []
- id: GO_REF:0000120
title: Combined Automated Annotation using Multiple IEA Methods
findings: []
- id: UniProt:Q88MC3
title: UniProtKB Q88MC3 histidine kinase entry
findings:
- statement: GacS is a membrane-associated histidine kinase
supporting_text: "-!- SUBCELLULAR LOCATION: Cell inner membrane {ECO:0000256|ARBA:ARBA00004429}; Multi-pass membrane protein {ECO:0000256|ARBA:ARBA00004429}."
- statement: GacS is annotated with histidine kinase catalytic activity
supporting_text: "-!- CATALYTIC ACTIVITY: Reaction=ATP + protein L-histidine = ADP + protein N-phospho-L- histidine.; EC=2.7.13.3; Evidence={ECO:0000256|ARBA:ARBA00000085};"
- id: PMID:22458445
title: Identification of reciprocal adhesion genes in pathogenic and non-pathogenic Pseudomonas.
findings:
- statement: gacS mutants are defective in attachment-related phenotypes in KT2440
- id: PMID:36748579
title: Transcriptional organization and regulation of the Pseudomonas putida K1 type VI secretion system gene cluster.
findings:
- statement: GacS-GacA positively regulates the KT2440 K1 type VI secretion system
- id: PMID:31931111
title: Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440.
findings:
- statement: Loss of gacS can improve engineered growth and muconate production in KT2440
- id: PMID:39701409
title: Adaptive laboratory evolution and genetic engineering improved terephthalate utilization in Pseudomonas putida KT2440.
findings:
- statement: Converged loss-of-function mutations in gacS and gacA improve engineered TPA utilization
- id: file:PSEPK/gacS/gacS-notes.md
title: Curated notes on gacS in Pseudomonas putida KT2440
findings:
- statement: The core molecular role is membrane phosphorelay sensor kinase activity
supporting_text: "The core molecular role is phosphorelay sensor kinase activity at the membrane."
- statement: Adhesion and K1-T6SS regulation are downstream outputs of global GacS signaling
supporting_text: "Process terms should center on phosphorelay signal transduction; the documented adhesion and T6SS phenotypes are downstream outputs of that global signaling role rather than separate direct molecular activities."
core_functions:
- molecular_function:
id: GO:0000155
label: phosphorelay sensor kinase activity
directly_involved_in:
- id: GO:0000160
label: phosphorelay signal transduction system
locations:
- id: GO:0005886
label: plasma membrane
description: >-
GacS is a membrane-spanning hybrid sensor histidine kinase that initiates the
GacS/GacA phosphorelay in Pseudomonas putida KT2440. Its core role is signal
sensing and phosphotransfer at the membrane, while organism-specific downstream
outputs documented in this strain include control of surface attachment and
activation of the K1 type VI secretion system.
supported_by:
- reference_id: UniProt:Q88MC3
supporting_text: "-!- CATALYTIC ACTIVITY: Reaction=ATP + protein L-histidine = ADP + protein N-phospho-L- histidine.; EC=2.7.13.3; Evidence={ECO:0000256|ARBA:ARBA00000085};"
- reference_id: file:PSEPK/gacS/gacS-notes.md
supporting_text: "In KT2440, expression of the K1 type VI secretion system gene cluster is positively regulated by the GacS-GacA two-component system and repressed by RetS."
suggested_questions:
- question: What signal or signals are detected directly by the periplasmic sensor region of GacS in KT2440?
experts:
- Patricia Bernal
- María A. Llamas
- question: Which small RNAs and Rsm-family effectors mediate the adhesion versus K1-T6SS branches downstream of GacS/GacA in KT2440?
experts:
- Patricia Bernal
- Estrella Duque
- Juan-Luis Ramos
suggested_experiments:
- hypothesis: GacS uses its hybrid receiver and HPt modules to relay phosphate to GacA in a defined multistep order.
description: >-
Build phosphosite mutants in the predicted receiver Asp718 and HPt His863 modules,
then test phosphotransfer to GacA in vitro and with in vivo transcriptional reporters
for Gac-dependent outputs.
experiment_type: phosphotransfer biochemistry plus reporter genetics
- hypothesis: The adhesion and K1-T6SS outputs reflect distinct downstream branches of the same GacS/GacA signaling pathway.
description: >-
Compare RNA-seq and promoter-reporter responses in wild type, delta-gacS, and
sensor-domain or relay-module mutants during surface growth and stationary phase
to separate direct GacS outputs from secondary physiological effects.
experiment_type: comparative transcriptomics and promoter-reporter analysis