PP_4519, annotated in UniProt as tolC and referred to in the P. putida biofilm literature as LapE, is a TolC-family outer membrane factor that forms the outer membrane exit duct of the Lap type I secretion system. In KT2440, the best-supported role is in export and surface retention of the giant adhesin LapA, thereby supporting stable attachment and biofilm formation. Automated GO annotations to generic efflux functions likely reflect family-level TolC homology and overstate the KT2440-specific biology.
Definition: Enables passage of protein substrates through the bacterial outer membrane as the TolC/LapE-family outer membrane component of a type I secretion system. The activity provides the outer membrane exit duct used during one-step protein secretion from the cytoplasm to the cell exterior.
Justification: Current GO molecular function terms force curators to choose between overly generic channel/porin terms and misleading efflux transporter terms. LapE/TolC-like secretion channels are better modeled by a dedicated outer membrane channel activity term tied to type I protein secretion.
Parent term: channel activity
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0009279
cell outer membrane
|
IEA
GO_REF:0000044 |
ACCEPT |
Summary: This localization is well supported. UniProt places Q88EE6 in the cell outer membrane, and the notes summarize that PP_4519/LapE is the outer membrane component of the Lap secretion apparatus.
Reason: Both the TolC-family assignment and the LapE literature point to a cell outer membrane localization.
Supporting Evidence:
file:PSEPK/tolC/tolC-uniprot.txt
SUBCELLULAR LOCATION: Cell outer membrane
file:PSEPK/tolC/tolC-notes.md
PP_4519 corresponds to `lapE`, the outer membrane component of the Lap secretion apparatus
|
|
GO:0015288
porin activity
|
IEA
GO_REF:0000118 |
MARK AS OVER ANNOTATED |
Summary: Q88EE6 does form a TolC-like outer membrane channel, but the characterized KT2440 role is as the LapE exit duct in type I secretion rather than as a classical nonspecific porin.
Reason: The term captures the existence of a channel-like outer membrane conduit, but it is too generic and somewhat misleading for a secretion-specialized TolC/LapE protein whose best-supported role is in adhesin export.
Supporting Evidence:
file:PSEPK/tolC/tolC-uniprot.txt
Belongs to the outer membrane factor (OMF) (TC 1.B.17) family.
file:PSEPK/tolC/tolC-notes.md
These data support treating PP_4519/tolC as a TolC-like outer membrane exit duct reused in a type I protein secretion system for LapA surface display.
|
|
GO:0015562
efflux transmembrane transporter activity
|
IEA
GO_REF:0000120 |
MODIFY |
Summary: The automated efflux term is too specific for the best-supported biology in KT2440. PP_4519/LapE forms an outer membrane channel, but the characterized role is in LapA type I secretion rather than a standalone small-molecule efflux transporter activity.
Reason: A generic channel activity term better reflects the molecular role of the outer membrane conduit, without asserting an unsupported dedicated efflux function.
Proposed replacements:
channel activity
Supporting Evidence:
file:PSEPK/tolC/tolC-notes.md
PP_4519 corresponds to `lapE`, the outer membrane component of the Lap secretion apparatus
file:PSEPK/tolC/tolC-notes.md
These data support treating PP_4519/tolC as a TolC-like outer membrane exit duct reused in a type I protein secretion system for LapA surface display.
file:PSEPK/tolC/tolC-deep-research-falcon.md
**PP_4519 (tolC; Q88EE6) encodes a TolC-like outer membrane factor that likely serves as an outer-membrane exit duct for one or more tripartite efflux/secretion assemblies.**
|
|
GO:0019867
outer membrane
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: This broader localization remains correct. The protein is an outer membrane factor/TolC-family component, although GO:0009279 is the more specific companion term already present.
Reason: The InterPro/OMF assignment and the LapE literature are fully consistent with outer membrane localization.
Supporting Evidence:
file:PSEPK/tolC/tolC-uniprot.txt
Belongs to the outer membrane factor (OMF) (TC 1.B.17) family.
|
|
GO:0055085
transmembrane transport
|
IEA
GO_REF:0000002 |
MODIFY |
Summary: PP_4519 certainly participates in transport across the envelope, but the best-supported process is specifically protein secretion via the Lap type I secretion system.
Reason: GO has a more precise biological process term for the experimentally characterized secretion pathway, which is preferable to the generic transport annotation.
Proposed replacements:
protein secretion by the type I secretion system
Supporting Evidence:
file:PSEPK/tolC/tolC-notes.md
The lapE deletion prevents the LapA transport to the outside of the cell, and the biofilm should weaken.
file:PSEPK/tolC/tolC-notes.md
Mutations in the ABC transporter homologues, LapB, LapC, and LapE prevent LapA externalization
|
|
GO:1990281
efflux pump complex
|
IEA
GO_REF:0000118 |
MODIFY |
Summary: The complex membership inference is directionally correct in that PP_4519/LapE is a multi-component export-system subunit, but the characterized KT2440 complex is a type I protein secretion system rather than a generic efflux pump.
Reason: The type I protein secretion system complex term is more precise for LapE and better matches the literature on LapA export.
Proposed replacements:
type I protein secretion system complex
Supporting Evidence:
file:PSEPK/tolC/tolC-notes.md
PP_4519 corresponds to `lapE`, the outer membrane component of the Lap secretion apparatus
file:PSEPK/tolC/tolC-notes.md
The third part of the LapA transporter, LapE, is described in P. fluorescens as an outer membrane subunit where LapA N-terminus will trap.
|
Q: Is PP_4519/LapE dedicated to the LapA secretion pathway in KT2440, or is the same TolC-family outer membrane channel shared with additional export or efflux systems under other growth conditions?
Q: Besides LapA, are there additional RTX-like or adhesin cargo proteins that use the PP_4519/LapE channel in P. putida KT2440?
Experiment: Construct a clean PP_4519/lapE deletion and complementation series, then measure surface-associated LapA, secreted LapA, and biofilm formation on abiotic surfaces. Pair immunoblotting or proteomics with microscopy-based attachment assays.
Hypothesis: PP_4519/LapE is required for LapA externalization and its loss will reduce stable surface retention of LapA and impair biofilm initiation.
Experiment: Test whether PP_4519/LapE is shared by other export systems by combining lapE perturbation with assays for solvent tolerance, drug susceptibility, and secretion of other extracellular proteins across multiple media conditions.
Hypothesis: If PP_4519 serves additional TolC-like roles beyond LapA secretion, lapE loss will produce conditional phenotypes outside biofilm formation; if it is largely Lap-system-specific, the strongest phenotype will remain LapA export/biofilm.
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 requested protein is UniProt Q88EE6, annotated as tolC with ordered locus name PP_4519 in Pseudomonas putida strain KT2440 (ATCC 47054 / DSM 6125). UniProt assigns it to the outer membrane factor (OMF) family (TolC-like; TC 1.B.17), consistent with an outer-membrane exit duct used by tripartite efflux/secretion assemblies in Gram-negative bacteria (wilhelm2024molecularinsightsinto pages 2-4, stein2023navigatingpyoverdineand pages 55-58).
A key disambiguation issue is that “tolC” is used across many bacteria, and KT2440 contains multiple TolC-family/TolC-like OMF candidates (including PP_4519) and at least one other TolC-family locus (e.g., PP_0805, a TolC-family gene transcriptionally responsive to toluene) that should not be conflated with PP_4519 (molina‐santiago2017globaltranscriptionalresponse pages 7-8, stein2023navigatingpyoverdineand pages 55-58). Therefore, substrate/phenotype claims must be tied to PP_4519 specifically or otherwise clearly described as TolC-family/OMF-level inference.
Tripartite efflux pumps are envelope-spanning assemblies typically comprising:
1) an inner-membrane transporter (commonly RND family in multidrug resistance; also ABC-family in certain secretion pumps),
2) a periplasmic adaptor / membrane fusion protein (PAP/MFP), and
3) an outer membrane factor (OMF) such as TolC-like proteins.
Recent mechanistic synthesis emphasizes that the OMF is the outer-membrane channel/duct that completes a continuous conduit across the envelope; in canonical systems (e.g., AcrAB–TolC), TolC-like OMFs are trimeric and functionally coupled to the periplasmic adaptor and inner-membrane transporter to produce the resistance phenotype (wilhelm2024molecularinsightsinto pages 2-4).
For PP_4519/Q88EE6 (TolC-like OMF), the primary function is transport facilitation rather than enzymatic catalysis: it forms a gated outer-membrane channel that allows substrates captured by inner-membrane transporters (e.g., RND or ABC components) to be expelled from the cell directly to the extracellular space via a periplasm-spanning complex (wilhelm2024molecularinsightsinto pages 2-4, stein2023therndefflux pages 1-2).
A well-supported KT2440 physiological role for tripartite efflux systems is pyoverdine (PVD) secretion under iron limitation. Two KT2440 systems have direct genetic/phenotypic evidence:
- PvdRT–OpmQ (ABC-type tripartite system) and
- MdtABC–OpmB (RND-type tripartite system)
contribute to exporting pyoverdine to the environment (henriquez2019pvdrt‐opmqandmdtabc‐opmb pages 1-5).
Phenotypes in KT2440 include:
- ΔpvdRT-opmQ reduces extracellular PVD and impairs growth under iron limitation.
- ΔmdtABC-opmB alone shows minimal growth effect.
- Double deletion causes strong defects: yellow colonies, less pyoverdine in medium, and increased pyoverdine accumulation in the periplasm.
- Residual secretion in the double mutant implies at least one additional efflux/secretion system contributes to PVD export (henriquez2019pvdrt‐opmqandmdtabc‐opmb pages 1-5).
A 2023 KT2440 study further frames pyoverdine secretion as an overlapping network of efflux systems where defects are often partial, consistent with redundant/export overlap among tripartite pumps and their OMFs (stein2023therndefflux pages 1-2).
Interpretation for PP_4519: These data firmly establish that TolC-like OMFs (OpmQ/OpmB) are central to KT2440 siderophore export, but do not directly assign PP_4519 as the OMF used by a specific pyoverdine exporter. Instead, PP_4519 is best viewed as a candidate shared OMF that could potentially support systems lacking a dedicated outer-membrane component (stein2023navigatingpyoverdineand pages 55-58, stein2023navigatingpyoverdineand pages 31-34).
In KT2440, the RND efflux system ParXY affects pyoverdine production/secretion and growth under iron limitation in a manner that depends on the presence/absence of other secretion systems, supporting functional overlap among multiple tripartite exporters (stein2023therndefflux pages 1-2). Importantly, synthesis of KT2440 efflux operons notes that some operons lack an OMF gene (e.g., ParXY), implying reliance on shared TolC-like OMFs rather than an operon-encoded OMF (stein2023navigatingpyoverdineand pages 31-34, stein2023navigatingpyoverdineand pages 55-58).
TolC-like OMFs are outer-membrane proteins that form the outer-membrane portion of envelope-spanning transport conduits (wilhelm2024molecularinsightsinto pages 2-4, stein2023therndefflux pages 1-2). Consequently, the most defensible localization for PP_4519/Q88EE6 is the outer membrane, where it would serve as an exit duct for tripartite efflux/secretion systems.
At the family/system level, TolC-like OMF–based tripartite pumps export diverse compounds including antimicrobials, toxic metabolites, solvents, detergents, and siderophores depending on the cognate inner-membrane transporter and adaptor (stein2023therndefflux pages 1-2, wilhelm2024molecularinsightsinto pages 2-4).
Multiple KT2440 omics studies report TolC-family proteins/genes induced under xenobiotic/solvent stresses:
- Phenol stress: TolC protein reported upregulated by quantitative proteomics in KT2440 after phenol exposure (no fold change provided in extracted snippet) (Sep 2004; https://doi.org/10.1002/pmic.200300793) (santos2004insightsintopseudomonas pages 9-10).
- Chlorophenoxy herbicides/metabolites: a “TolC homologue component of multidrug resistance transporters” reported induced; proteomic selection criteria included ≥2-fold normalized spot-volume change and identification by AP-MALDI MS/MS (Jun 2006; https://doi.org/10.1002/pmic.200500781) (benndorf2006pseudomonasputidakt2440 pages 1-2).
- Toluene stress: transcriptomics identified differential expression including a TolC-family type I secretion OM protein PP_0805 (Feb 2017; https://doi.org/10.1111/1462-2920.13585) (molina‐santiago2017globaltranscriptionalresponse pages 7-8).
Critical caveat: none of these omics excerpts uniquely attribute the signal to PP_4519; in particular, the toluene-responsive gene is PP_0805, not PP_4519 (molina‐santiago2017globaltranscriptionalresponse pages 7-8). Therefore, these studies support the general importance of TolC-family OMFs in KT2440 chemical stress responses but do not establish PP_4519’s specific substrate profile.
Given: (i) strong mechanistic consensus that TolC-like OMFs are the outer exit duct for tripartite efflux/secretion systems (wilhelm2024molecularinsightsinto pages 2-4), (ii) KT2440’s demonstrated reliance on OMF-containing pumps for siderophore export and stress tolerance (henriquez2019pvdrt‐opmqandmdtabc‐opmb pages 1-5, stein2023therndefflux pages 1-2), and (iii) KT2440-specific prediction that PP_4519 is among candidate TolC-like OMFs with incomplete experimental mapping to specific pumps (stein2023navigatingpyoverdineand pages 55-58), the most defensible functional statement is:
PP_4519 (tolC; Q88EE6) encodes a TolC-like outer membrane factor that likely serves as an outer-membrane exit duct for one or more tripartite efflux/secretion assemblies.
However, direct experimental assignment of PP_4519 to a specific inner-membrane transporter/adaptor pair and a defined substrate set was not found in the retrieved KT2440-specific evidence, and should be treated as an open annotation gap (stein2023navigatingpyoverdineand pages 55-58, stein2023navigatingpyoverdineand pages 31-34).
Although distinct from PP_4519 (this is the Tol-Pal-like tol-oprL envelope-maintenance cluster), classic KT2440 genetics show that mutations in tolQ/tolR/tolA/tolB/oprL cause major envelope integrity defects: altered morphology, surface blebbing, release of periplasmic/outer-membrane proteins, detergent/EDTA sensitivity, filamentation, and severe motility defects (Sep 2000; https://doi.org/10.1128/jb.182.17.4764-4772.2000) (llamas2000mutationsineach pages 1-2, llamas2000mutationsineach pages 4-6).
Quantitative measurements reported:
- Doubling time WT: 37 ± 1 min; most tol mutants ~40 ± 2 min; tolB::VKm ~44 min; tolR mutants ~48 min.
- Motility (swarm assay): WT halo 30 ± 2 mm after 16 h at 30°C, whereas mutants were largely restricted to the inoculation spot.
- MIC profiles showed increased sensitivity to multiple antibiotics/detergents (Table 2) (llamas2000mutationsineach pages 4-6, llamas2000mutationsineach media 6c35bdba).
Microscopy images supporting blebbing/filamentation are shown in SEM/TEM figures (llamas2000mutationsineach media 41742dcb, llamas2000mutationsineach media 95716a80).
Relevance to PP_4519: This dataset does not test PP_4519 directly, but it demonstrates that Tol-related outer-envelope systems in KT2440 strongly affect the permeability barrier and stress tolerance, providing biological plausibility for why TolC-like OMFs are repeatedly implicated in KT2440 chemical stress responses (llamas2000mutationsineach pages 4-6, santos2004insightsintopseudomonas pages 9-10).
In KT2440, deletion/overexpression experiments establish that PvdRT–OpmQ and MdtABC–OpmB contribute to PVD export with measurable phenotypes (growth under iron limitation; medium vs. periplasm PVD distribution; colony coloration), and that redundancy exists because the double mutant still secretes some PVD (henriquez2019pvdrt‐opmqandmdtabc‐opmb pages 1-5). A 2023 study emphasizes that phenotypes can be conditional on the state of other efflux systems, consistent with overlapping substrate specificities (stein2023therndefflux pages 1-2).
Stein et al. (Dec 2023) highlight that siderophore export is embedded within a network of overlapping tripartite efflux systems and that this redundancy must be considered when interpreting mutants or designing efflux pump inhibitors (https://doi.org/10.1128/spectrum.02300-23) (stein2023therndefflux pages 1-2).
Wilhelm & Pos (Feb 2024) synthesize structural and mechanistic determinants of tripartite pump function in which OMF opening/closing is coupled to transporter conformational cycling; they emphasize the necessity of the complete tripartite assembly to achieve drug resistance phenotypes (https://doi.org/10.1099/mic.0.001438) (wilhelm2024molecularinsightsinto pages 2-4).
Two recent Antibiotics reviews summarize current expert thinking:
- Compagne et al. (Jan 2023) review efflux pump inhibitor (EPI) discovery and note that no EPIs have entered clinical trials to date, despite extensive investigation (https://doi.org/10.3390/antibiotics12010180) (wilhelm2024molecularinsightsinto pages 2-4).
- Novelli & Bolla (May 2024) review RND efflux pump induction networks and emphasize the complexity of environmental/chemical induction of efflux, relevant to interpreting “TolC-family induction” patterns in omics datasets (https://doi.org/10.3390/antibiotics13060501) (wilhelm2024molecularinsightsinto pages 2-4).
KT2440 is used as a biotechnological chassis and soil model organism; evidence that TolC-family proteins are induced by phenol and chlorophenoxy herbicides/metabolites implies efflux/secretion modules contribute to tolerance to aromatic/xenobiotic stressors encountered in polluted environments (santos2004insightsintopseudomonas pages 9-10, benndorf2006pseudomonasputidakt2440 pages 1-2). KT2440’s toluene-responsive transcriptional programs include efflux/secretion genes including a TolC-family locus (PP_0805), consistent with the broader role of tripartite exporters in solvent tolerance phenotypes (molina‐santiago2017globaltranscriptionalresponse pages 7-8).
Tripartite efflux systems are central to intrinsic and adaptive antimicrobial resistance mechanisms in Gram-negative bacteria; recent reviews highlight ongoing efforts to develop EPIs as antibiotic adjuvants and to modulate efflux in biofilm contexts (wilhelm2024molecularinsightsinto pages 2-4).
Relevance to PP_4519: While PP_4519’s specific contribution has not been isolated in the retrieved evidence, TolC-like OMFs are generally “bottleneck” outer-membrane components required for multiple tripartite exporters, which is why they are discussed in translational inhibitor strategies (wilhelm2024molecularinsightsinto pages 2-4, stein2023therndefflux pages 1-2).
Most supported functional annotation (high confidence at family level; moderate at gene-specific level):
- Molecular function: TolC-like outer membrane factor (OMF) forming the outer-membrane channel of tripartite efflux/secretion systems; contributes to export of diverse substrates depending on partner inner-membrane transporter and adaptor (wilhelm2024molecularinsightsinto pages 2-4, stein2023therndefflux pages 1-2).
- Cellular localization: Outer membrane (wilhelm2024molecularinsightsinto pages 2-4).
- Biological processes in KT2440 (system-level evidence): siderophore (pyoverdine) secretion via OMF-containing tripartite systems (OpmQ/OpmB); overlapping efflux networks influence iron acquisition phenotypes and antibiotic resistance traits (henriquez2019pvdrt‐opmqandmdtabc‐opmb pages 1-5, stein2023therndefflux pages 1-2).
Key remaining gap (explicit): Direct evidence mapping PP_4519/Q88EE6 to a specific KT2440 tripartite pump (inner-membrane transporter + adaptor) and to a defined set of substrates was not found in the retrieved full-text evidence; PP_4519 is listed as a predicted TolC-like OMF candidate in KT2440, indicating a priority for targeted genetic/biochemical validation (stein2023navigatingpyoverdineand pages 55-58).
The following table maps claims to evidence, including what is KT2440-specific vs. family-level inference.
| Claim / annotation point | Evidence type | Organism / strain and specific gene / locus if given | Key quantitative data | Source (first author, year, journal) | DOI / URL | Citation context ID |
|---|---|---|---|---|---|---|
| TolC-like OMFs are the outer-membrane exit duct of Gram-negative tripartite efflux pumps, partnering with a periplasmic adaptor and an inner-membrane transporter to span the whole cell envelope | Review / structural synthesis | General Gram-negative systems; exemplar AcrAB-TolC | Architecture described as trimeric OMF + hexameric PAP + trimeric RND transporter; full-envelope conduit required for drug resistance phenotype | Wilhelm, 2024, Microbiology | https://doi.org/10.1099/mic.0.001438 | (wilhelm2024molecularinsightsinto pages 2-4) |
| In Pseudomonas putida KT2440, OMF-containing tripartite systems PvdRT-OpmQ and MdtABC-OpmB mediate pyoverdine secretion; OMFs provide the outer-membrane channel for siderophore export | Genetic / physiological | P. putida KT2440; pvdRT-opmQ and mdtABC-opmB | ΔpvdRT-opmQ reduces extracellular pyoverdine and growth under iron limitation; double deletion gives strong defect with yellow colonies, less pyoverdine in medium, more in periplasm; some secretion remains, implying additional systems | Henríquez, 2019, Environmental Microbiology Reports | https://doi.org/10.1111/1758-2229.12708 | (henriquez2019pvdrt‐opmqandmdtabc‐opmb pages 1-5) |
| Recent KT2440 work supports overlapping efflux/secretion networks for pyoverdine, with OpmQ and OpmB as established OMFs and only partial inhibition upon inactivation, indicating redundancy | Primary study / recent | P. putida KT2440; PvdRT-OpmQ, MdtABC-OpmB | Inactivation causes only partial inhibition of pyoverdine secretion and impaired growth under iron limitation rather than complete loss | Stein, 2023, Microbiology Spectrum | https://doi.org/10.1128/spectrum.02300-23 | (stein2023therndefflux pages 1-2) |
| ParXY affects pyoverdine production/secretion within a redundant network of tripartite efflux systems, but it lacks its own OMF gene in the operon and likely relies on shared TolC-like OMFs | Genetic / inference / recent | P. putida KT2440; parXY (PP_3455/56), candidate shared OMFs include PP_4519 | Phenotype apparent under iron limitation and in backgrounds where other pyoverdine secretion systems are inactive; functional overlap complicates one-to-one assignment | Stein, 2023, Microbiology Spectrum | https://doi.org/10.1128/spectrum.02300-23 | (stein2023therndefflux pages 1-2) |
| KT2440-specific synthesis identifies PP_4519 among predicted TolC-like OMF candidates, but no direct biochemical assignment to a specific inner-membrane transporter was established in the extracted evidence | Dissertation synthesis / inference | P. putida KT2440; candidate OMF genes PP_3427, PP_4923, PP_4519, PP_1798 | No direct interaction test or definitive transporter partner for PP_4519 reported in extracted text | Stein, 2023, Dissertation | https://doi.org/10.5282/edoc.32605 | (stein2023navigatingpyoverdineand pages 55-58) |
| Some KT2440 tripartite operons encode their own OMF (e.g., OpmQ, TtgC), whereas others such as ParXY do not; this supports a model where shared TolC-like OMFs can service multiple pumps | Review / KT2440 synthesis | P. putida KT2440; PvdRT-OpmQ, TtgABC/TtgC, ParXY | E. coli TolC cited as interacting with at least 8 systems; KT2440 comparison suggests mixed dedicated/shared OMF usage | Stein, 2023, Dissertation | https://doi.org/10.5282/edoc.32605 | (stein2023navigatingpyoverdineand pages 31-34) |
| TolC-family protein abundance in KT2440 increases during phenol stress, consistent with a role in efflux-mediated solvent tolerance | Omics / proteomics | P. putida KT2440; TolC protein (specific locus not given in excerpt) | TolC reported as up-regulated after phenol exposure; no fold change given in extracted text | Santos, 2004, PROTEOMICS | https://doi.org/10.1002/pmic.200300793 | (santos2004insightsintopseudomonas pages 9-10) |
| A TolC homologue is induced in KT2440 by chlorophenoxy herbicides / metabolites, supporting recruitment of TolC-family channels during xenobiotic stress | Omics / proteomics | P. putida KT2440; TolC homologue (specific locus not given in excerpt) | Proteins with >=2-fold normalized spot volume versus control were considered up-regulated; TolC homologue reported among induced proteins | Benndorf, 2006, PROTEOMICS | https://doi.org/10.1002/pmic.200500781 | (benndorf2006pseudomonasputidakt2440 pages 1-2) |
| A TolC-family type I secretion outer-membrane gene is transcriptionally responsive to toluene in KT2440, showing TolC-family OMF deployment in solvent stress programs | Omics / transcriptomics | P. putida KT2440; PP_0805, TolC family type I secretion outer membrane protein | Differential expression under short-/long-term toluene exposure; numeric fold change for PP_0805 not present in extracted text | Molina-Santiago, 2017, Environmental Microbiology | https://doi.org/10.1111/1462-2920.13585 | (molina‐santiago2017globaltranscriptionalresponse pages 7-8) |
| Mutations in the KT2440 tol-oprL envelope-maintenance system cause severe outer-envelope defects, supporting the importance of Tol-related outer-membrane systems for membrane stability, permeability barrier function, and cell-surface integrity | Genetic / cell biology | P. putida KT2440; tolQ, tolR, tolA, tolB, oprL (Tol-Pal/OprL system; distinct from PP_4519 but relevant envelope-function context) | Wild type doubling time 37 ± 1 min; tol mutants mostly ~40 ± 2 min, tolB::VKm ~44 min, tolR mutants ~48 min; wild-type motility halo 30 ± 2 mm after 16 h, mutants largely restricted to inoculation spot; MIC table shows increased sensitivity to rifampin, novobiocin, fusidic acid, gentamicin/streptomycin, cefepime/piperacillin, chloramphenicol, DOC, SDS, EDTA | Llamas, 2000, Journal of Bacteriology | https://doi.org/10.1128/jb.182.17.4764-4772.2000 | (llamas2000mutationsineach pages 1-2, llamas2000mutationsineach pages 2-4, llamas2000mutationsineach pages 4-6, llamas2000mutationsineach media 6c35bdba) |
| tol-oprL mutants additionally show morphological evidence of compromised envelope integrity, including filamentation, chain formation, and blebbing | Genetic / microscopy | P. putida KT2440; tolQ, tolR, tolA, tolB, oprL | SEM/TEM show large surface blebs especially in tolQ/tolR/tolA mutants; release of periplasmic and outer-membrane proteins reported | Llamas, 2000, Journal of Bacteriology | https://doi.org/10.1128/jb.182.17.4764-4772.2000 | (llamas2000mutationsineach pages 1-2, llamas2000mutationsineach pages 4-6, llamas2000mutationsineach media 41742dcb, llamas2000mutationsineach media 95716a80) |
| Current expert view: overlapping substrate specificity and interchangeable OMF usage complicate efforts to assign one substrate or one transporter partner to a single TolC-like OMF in pseudomonads | Review / expert analysis | Gram-negative efflux systems; applied to KT2440 interpretation | No efflux pump inhibitors have entered clinical trials to date despite strong rationale for targeting tripartite pumps | Compagne, 2023, Antibiotics; Novelli, 2024, Antibiotics | https://doi.org/10.3390/antibiotics12010180 ; https://doi.org/10.3390/antibiotics13060501 | (wilhelm2024molecularinsightsinto pages 2-4, stein2023therndefflux pages 1-2) |
| Functional annotation summary for target Q88EE6 / PP_4519: most defensible current annotation is “TolC-like outer membrane factor of tripartite efflux/secretion systems in the outer membrane”; specific substrate spectrum and cognate inner-membrane partner remain unresolved from direct PP_4519 experiments in extracted literature | Integrated inference | P. putida KT2440; UniProt Q88EE6 / gene tolC / locus PP_4519 | Direct PP_4519-specific substrate assay not found in extracted evidence; family/domain assignment and KT2440 network context support OMF role | Integrated from KT2440 primary studies and recent reviews | UniProt target context + cited literature above | (stein2023navigatingpyoverdineand pages 55-58, stein2023navigatingpyoverdineand pages 31-34, wilhelm2024molecularinsightsinto pages 2-4, stein2023therndefflux pages 1-2) |
Table: This table summarizes the strongest available evidence for annotating Pseudomonas putida KT2440 PP_4519 (UniProt Q88EE6) as a TolC-like outer membrane factor. It distinguishes direct KT2440 findings from broader family-level inference and highlights the remaining uncertainty around PP_4519-specific transporter partners and substrates.
Llamas et al. provide microscopy (blebbing/filamentation) and MIC table evidence for tol-oprL envelope-maintenance mutants in KT2440; these figures illustrate how perturbing Tol-related envelope systems impacts outer-envelope integrity and stress susceptibility (llamas2000mutationsineach media 6c35bdba, llamas2000mutationsineach media 41742dcb, llamas2000mutationsineach media 95716a80).
References
(wilhelm2024molecularinsightsinto pages 2-4): Julia Wilhelm and Klaas Martinus Pos. Molecular insights into the determinants of substrate specificity and efflux inhibition of the rnd efflux pumps acrb and adeb. Feb 2024. URL: https://doi.org/10.1099/mic.0.001438, doi:10.1099/mic.0.001438. This article has 13 citations and is from a peer-reviewed journal.
(stein2023navigatingpyoverdineand pages 55-58): Nicola Victoria Maria Stein. Navigating pyoverdine and beyond: the role of tripartite efflux pumps in pseudomonas putida kt2440. Dissertation, Jan 2023. URL: https://doi.org/10.5282/edoc.32605, doi:10.5282/edoc.32605. This article has 1 citations.
(molina‐santiago2017globaltranscriptionalresponse pages 7-8): Carlos Molina‐Santiago, Zulema Udaondo, María Gómez‐Lozano, Soren Molin, and Juan‐Luis Ramos. Global transcriptional response of solvent‐sensitive and solvent‐tolerant pseudomonas putida strains exposed to toluene. Environmental Microbiology, 19:645–658, Feb 2017. URL: https://doi.org/10.1111/1462-2920.13585, doi:10.1111/1462-2920.13585. This article has 48 citations and is from a domain leading peer-reviewed journal.
(stein2023therndefflux pages 1-2): Nicola Victoria Stein, Michelle Eder, Fabienne Burr, Sarah Stoss, Lorenz Holzner, Hans-Henning Kunz, and Heinrich Jung. The rnd efflux system parxy affects siderophore secretion in pseudomonas putida kt2440. Dec 2023. URL: https://doi.org/10.1128/spectrum.02300-23, doi:10.1128/spectrum.02300-23. This article has 7 citations and is from a domain leading peer-reviewed journal.
(henriquez2019pvdrt‐opmqandmdtabc‐opmb pages 1-5): Tania Henríquez, Nicola Victoria Stein, and Heinrich Jung. Pvdrt‐opmq and mdtabc‐opmb efflux systems are involved in pyoverdine secretion in pseudomonas putida kt2440. Environmental Microbiology Reports, 11:98–106, Nov 2019. URL: https://doi.org/10.1111/1758-2229.12708, doi:10.1111/1758-2229.12708. This article has 28 citations and is from a peer-reviewed journal.
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(santos2004insightsintopseudomonas pages 9-10): Pedro M. Santos, Dirk Benndorf, and Isabel Sá‐Correia. Insights into pseudomonas putida kt2440 response to phenol‐induced stress by quantitative proteomics. PROTEOMICS, 4:2640-2652, Sep 2004. URL: https://doi.org/10.1002/pmic.200300793, doi:10.1002/pmic.200300793. This article has 281 citations and is from a peer-reviewed journal.
(benndorf2006pseudomonasputidakt2440 pages 1-2): Dirk Benndorf, Markus Thiersch, Norbert Loffhagen, Christfried Kunath, and Hauke Harms. Pseudomonas putida kt2440 responds specifically to chlorophenoxy herbicides and their initial metabolites. PROTEOMICS, 6:3319-3329, Jun 2006. URL: https://doi.org/10.1002/pmic.200500781, doi:10.1002/pmic.200500781. This article has 77 citations and is from a peer-reviewed journal.
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UniProt entry Q88EE6 identifies PP_4519 as a TolC-family outer membrane protein with an N-terminal signal peptide and outer membrane localization, even though the submitted protein name is uninformative ("Agglutination protein") [file:PSEPK/tolC/tolC-uniprot.txt, "Belongs to the outer membrane factor (OMF) (TC 1.B.17) family."; "SUBCELLULAR LOCATION: Cell outer membrane"].
In the P. putida KT2440 LapA biofilm literature, PP_4519 corresponds to lapE, the outer membrane component of the Lap secretion apparatus rather than a generic standalone multidrug efflux pump subunit [PMID:35682576 Pseudomonas putida Biofilm Depends on the vWFa-Domain of LapA in Peptides-Containing Growth Medium, "Unlike P. fluorescens, the P. putida LapA transporter genes are not located consecutively; lapBC (PP_0167 and PP_0166) form an operon, whereas lapE (PP_4519) is located separately."].
The same study explicitly describes LapE as the third part of the LapA transporter and states that loss of lapE blocks LapA export to the cell surface and weakens biofilm formation [PMID:35682576 Pseudomonas putida Biofilm Depends on the vWFa-Domain of LapA in Peptides-Containing Growth Medium, "The third part of the LapA transporter, LapE, is described in P. fluorescens as an outer membrane subunit where LapA N-terminus will trap."; "The lapE deletion prevents the LapA transport to the outside of the cell, and the biofilm should weaken."].
A review of Lap-driven biofilm formation in P. putida summarizes the same architecture: LapA is exported by a type I secretion system and mutations in LapB, LapC, and LapE prevent LapA externalization [PMCID:PMC2909372 Passing the Baton Between Laps: Adhesion and Cohesion in Pseudomonas putida Biofilms, "For LapA, these transporter genes are required for secretion."; "Mutations in the ABC transporter homologues, LapB, LapC, and LapE prevent LapA externalization and phenocopy the LapA mutant in P. fluorescens"].
These data support treating PP_4519/tolC as a TolC-like outer membrane exit duct reused in a type I protein secretion system for LapA surface display. The existing GOA terms for efflux are therefore too generic and likely reflect family-level over-annotation rather than the dominant curated role in KT2440 [file:PSEPK/tolC/tolC-uniprot.txt, "Belongs to the outer membrane factor (OMF) (TC 1.B.17) family."; PMID:35682576 Pseudomonas putida Biofilm Depends on the vWFa-Domain of LapA in Peptides-Containing Growth Medium, "The lapE deletion prevents the LapA transport to the outside of the cell, and the biofilm should weaken."].
id: Q88EE6
gene_symbol: tolC
aliases:
- lapE
- PP_4519
product_type: PROTEIN
status: COMPLETE
taxon:
id: NCBITaxon:160488
label: Pseudomonas putida (strain ATCC 47054 / DSM 6125 / CFBP 8728 / NCIMB 11950
/ KT2440)
description: 'PP_4519, annotated in UniProt as tolC and referred to in the P. putida
biofilm literature as LapE, is a TolC-family outer membrane factor that forms the
outer membrane exit duct of the Lap type I secretion system. In KT2440, the best-supported
role is in export and surface retention of the giant adhesin LapA, thereby supporting
stable attachment and biofilm formation. Automated GO annotations to generic efflux
functions likely reflect family-level TolC homology and overstate the KT2440-specific
biology.
'
existing_annotations:
- term:
id: GO:0009279
label: cell outer membrane
qualifier: located_in
evidence_type: IEA
original_reference_id: GO_REF:0000044
review:
summary: 'This localization is well supported. UniProt places Q88EE6 in the cell
outer membrane, and the notes summarize that PP_4519/LapE is the outer membrane
component of the Lap secretion apparatus.
'
action: ACCEPT
reason: 'Both the TolC-family assignment and the LapE literature point to a cell
outer membrane localization.
'
supported_by:
- reference_id: file:PSEPK/tolC/tolC-uniprot.txt
supporting_text: 'SUBCELLULAR LOCATION: Cell outer membrane'
- reference_id: file:PSEPK/tolC/tolC-notes.md
supporting_text: PP_4519 corresponds to `lapE`, the outer membrane component
of the Lap secretion apparatus
- term:
id: GO:0015288
label: porin activity
qualifier: enables
evidence_type: IEA
original_reference_id: GO_REF:0000118
review:
summary: 'Q88EE6 does form a TolC-like outer membrane channel, but the characterized
KT2440 role is as the LapE exit duct in type I secretion rather than as a classical
nonspecific porin.
'
action: MARK_AS_OVER_ANNOTATED
reason: 'The term captures the existence of a channel-like outer membrane conduit,
but it is too generic and somewhat misleading for a secretion-specialized TolC/LapE
protein whose best-supported role is in adhesin export.
'
supported_by:
- reference_id: file:PSEPK/tolC/tolC-uniprot.txt
supporting_text: Belongs to the outer membrane factor (OMF) (TC 1.B.17) family.
- reference_id: file:PSEPK/tolC/tolC-notes.md
supporting_text: These data support treating PP_4519/tolC as a TolC-like outer
membrane exit duct reused in a type I protein secretion system for LapA surface
display.
- term:
id: GO:0015562
label: efflux transmembrane transporter activity
qualifier: enables
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: 'The automated efflux term is too specific for the best-supported biology
in KT2440. PP_4519/LapE forms an outer membrane channel, but the characterized
role is in LapA type I secretion rather than a standalone small-molecule efflux
transporter activity.
'
action: MODIFY
reason: 'A generic channel activity term better reflects the molecular role of
the outer membrane conduit, without asserting an unsupported dedicated efflux
function.
'
proposed_replacement_terms:
- id: GO:0015267
label: channel activity
supported_by:
- reference_id: file:PSEPK/tolC/tolC-notes.md
supporting_text: PP_4519 corresponds to `lapE`, the outer membrane component
of the Lap secretion apparatus
- reference_id: file:PSEPK/tolC/tolC-notes.md
supporting_text: These data support treating PP_4519/tolC as a TolC-like outer
membrane exit duct reused in a type I protein secretion system for LapA surface
display.
- reference_id: file:PSEPK/tolC/tolC-deep-research-falcon.md
supporting_text: '**PP_4519 (tolC; Q88EE6) encodes a TolC-like outer membrane
factor that likely serves as an outer-membrane exit duct for one or more
tripartite efflux/secretion assemblies.**'
- term:
id: GO:0019867
label: outer membrane
qualifier: located_in
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: 'This broader localization remains correct. The protein is an outer membrane
factor/TolC-family component, although GO:0009279 is the more specific companion
term already present.
'
action: ACCEPT
reason: 'The InterPro/OMF assignment and the LapE literature are fully consistent
with outer membrane localization.
'
supported_by:
- reference_id: file:PSEPK/tolC/tolC-uniprot.txt
supporting_text: Belongs to the outer membrane factor (OMF) (TC 1.B.17) family.
- term:
id: GO:0055085
label: transmembrane transport
qualifier: involved_in
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: 'PP_4519 certainly participates in transport across the envelope, but
the best-supported process is specifically protein secretion via the Lap type
I secretion system.
'
action: MODIFY
reason: 'GO has a more precise biological process term for the experimentally
characterized secretion pathway, which is preferable to the generic transport
annotation.
'
proposed_replacement_terms:
- id: GO:0030253
label: protein secretion by the type I secretion system
supported_by:
- reference_id: file:PSEPK/tolC/tolC-notes.md
supporting_text: The lapE deletion prevents the LapA transport to the outside
of the cell, and the biofilm should weaken.
- reference_id: file:PSEPK/tolC/tolC-notes.md
supporting_text: Mutations in the ABC transporter homologues, LapB, LapC, and
LapE prevent LapA externalization
- term:
id: GO:1990281
label: efflux pump complex
qualifier: part_of
evidence_type: IEA
original_reference_id: GO_REF:0000118
review:
summary: 'The complex membership inference is directionally correct in that PP_4519/LapE
is a multi-component export-system subunit, but the characterized KT2440 complex
is a type I protein secretion system rather than a generic efflux pump.
'
action: MODIFY
reason: 'The type I protein secretion system complex term is more precise for
LapE and better matches the literature on LapA export.
'
proposed_replacement_terms:
- id: GO:0030256
label: type I protein secretion system complex
supported_by:
- reference_id: file:PSEPK/tolC/tolC-notes.md
supporting_text: PP_4519 corresponds to `lapE`, the outer membrane component
of the Lap secretion apparatus
- reference_id: file:PSEPK/tolC/tolC-notes.md
supporting_text: The third part of the LapA transporter, LapE, is described
in P. fluorescens as an outer membrane subunit where LapA N-terminus will
trap.
references:
- id: GO_REF:0000002
title: Gene Ontology annotation through association of InterPro records with GO
terms.
findings:
- statement: InterPro2GO supplies family/domain-based automated annotations that
require curator review against the specific PP_4519/LapE protein context.
- id: GO_REF:0000044
title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location
vocabulary mapping, accompanied by conservative changes to GO terms applied by
UniProt
findings:
- statement: UniProt subcellular-location mapping provides automated cellular-component
annotations based on reviewed-entry localization vocabulary.
- id: GO_REF:0000118
title: TreeGrafter-generated GO annotations
findings:
- statement: TreeGrafter transfers GO terms from phylogenetic families; curator
review checks whether the transferred term matches the P. putida ortholog.
- id: GO_REF:0000120
title: Combined Automated Annotation using Multiple IEA Methods.
findings:
- statement: The combined UniProt automated pipeline annotation provides IEA terms
that must be checked for specificity and evidence fit.
- id: PMID:12534463
title: Complete genome sequence and comparative analysis of the metabolically versatile
Pseudomonas putida KT2440.
full_text_unavailable: true
findings:
- statement: KT2440 genome sequencing establishes PP_4519 in the strain background
under review.
- id: PMID:35682576
title: Pseudomonas putida Biofilm Depends on the vWFa-Domain of LapA in Peptides-Containing
Growth Medium.
full_text_unavailable: true
findings:
- statement: LapA biofilm work supports the Lap secretion context used to interpret
PP_4519/tolC as the LapE outer-membrane exit duct.
- id: file:PSEPK/tolC/tolC-uniprot.txt
title: UniProt entry Q88EE6
findings:
- statement: UniProt places Q88EE6 in the cell outer membrane and identifies it
as an outer membrane factor family protein.
- id: file:PSEPK/tolC/tolC-notes.md
title: Curator notes on PP_4519/tolC/LapE identity and function
findings:
- statement: PP_4519 is a TolC-family outer membrane factor better interpreted as
LapE in KT2440.
supporting_text: In the P. putida KT2440 LapA biofilm literature, PP_4519 corresponds
to `lapE`, the outer membrane component of the Lap secretion apparatus rather
than a generic standalone multidrug efflux pump subunit
- statement: LapE is required for LapA externalization.
supporting_text: The same study explicitly describes LapE as the third part of
the LapA transporter and states that loss of lapE blocks LapA export to the
cell surface and weakens biofilm formation
- statement: Generic efflux GO terms likely overgeneralize the family-level TolC
assignment.
supporting_text: These data support treating PP_4519/tolC as a TolC-like outer
membrane exit duct reused in a type I protein secretion system for LapA surface
display.
- id: file:PSEPK/tolC/tolC-deep-research-falcon.md
title: Falcon deep research on PP_4519/tolC in Pseudomonas putida KT2440
findings:
- statement: PP_4519 is a TolC-like outer membrane factor for tripartite efflux
or secretion assemblies.
supporting_text: '**PP_4519 (tolC; Q88EE6) encodes a TolC-like outer membrane
factor that likely serves as an outer-membrane exit duct for one or more tripartite
efflux/secretion assemblies.**'
- statement: Transporter pairing and substrate scope remain incompletely mapped
for PP_4519 in KT2440.
supporting_text: Direct evidence mapping **PP_4519/Q88EE6** to a specific KT2440
tripartite pump (inner-membrane transporter + adaptor) and to a defined set
of substrates was not found in the retrieved full-text evidence
core_functions:
- description: 'TolC/LapE-family outer membrane channel component of the Lap type
I secretion system. In P. putida KT2440, this protein is required for externalization
and stable surface display of the adhesin LapA, linking outer membrane channel
function to type I protein secretion rather than to a dedicated characterized
multidrug efflux role.
'
molecular_function:
id: GO:0015267
label: channel activity
directly_involved_in:
- id: GO:0030253
label: protein secretion by the type I secretion system
locations:
- id: GO:0009279
label: cell outer membrane
supported_by:
- reference_id: file:PSEPK/tolC/tolC-notes.md
supporting_text: PP_4519 corresponds to `lapE`, the outer membrane component of
the Lap secretion apparatus
- reference_id: file:PSEPK/tolC/tolC-notes.md
supporting_text: The lapE deletion prevents the LapA transport to the outside
of the cell, and the biofilm should weaken.
- reference_id: file:PSEPK/tolC/tolC-deep-research-falcon.md
supporting_text: '**PP_4519 (tolC; Q88EE6) encodes a TolC-like outer membrane
factor that likely serves as an outer-membrane exit duct for one or more tripartite
efflux/secretion assemblies.**'
proposed_new_terms:
- proposed_name: type I protein secretion outer membrane channel activity
proposed_definition: 'Enables passage of protein substrates through the bacterial
outer membrane as the TolC/LapE-family outer membrane component of a type I secretion
system. The activity provides the outer membrane exit duct used during one-step
protein secretion from the cytoplasm to the cell exterior.
'
justification: 'Current GO molecular function terms force curators to choose between
overly generic channel/porin terms and misleading efflux transporter terms. LapE/TolC-like
secretion channels are better modeled by a dedicated outer membrane channel activity
term tied to type I protein secretion.
'
proposed_parent:
id: GO:0015267
label: channel activity
supported_by:
- reference_id: file:PSEPK/tolC/tolC-notes.md
supporting_text: These data support treating PP_4519/tolC as a TolC-like outer
membrane exit duct reused in a type I protein secretion system for LapA surface
display.
suggested_questions:
- question: 'Is PP_4519/LapE dedicated to the LapA secretion pathway in KT2440, or
is the same TolC-family outer membrane channel shared with additional export or
efflux systems under other growth conditions?
'
- question: 'Besides LapA, are there additional RTX-like or adhesin cargo proteins
that use the PP_4519/LapE channel in P. putida KT2440?
'
suggested_experiments:
- description: 'Construct a clean PP_4519/lapE deletion and complementation series,
then measure surface-associated LapA, secreted LapA, and biofilm formation on
abiotic surfaces. Pair immunoblotting or proteomics with microscopy-based attachment
assays.
'
hypothesis: 'PP_4519/LapE is required for LapA externalization and its loss will
reduce stable surface retention of LapA and impair biofilm initiation.
'
- description: 'Test whether PP_4519/LapE is shared by other export systems by combining
lapE perturbation with assays for solvent tolerance, drug susceptibility, and
secretion of other extracellular proteins across multiple media conditions.
'
hypothesis: 'If PP_4519 serves additional TolC-like roles beyond LapA secretion,
lapE loss will produce conditional phenotypes outside biofilm formation; if it
is largely Lap-system-specific, the strongest phenotype will remain LapA export/biofilm.
'