TtgB (PP_1385) is the inner membrane RND (Resistance-Nodulation-Division) transporter component of the TtgABC tripartite efflux pump in Pseudomonas putida KT2440. It is a 1050 AA protein with 12 predicted transmembrane helices, belonging to the acriflavine resistance protein family. TtgB functions as the proton-motive-force-driven substrate translocator that captures substrates from the periplasm/inner membrane and extrudes them to the extracellular space via the TtgC outer membrane channel, with TtgA as the periplasmic adaptor. In KT2440, the TtgABC system primarily confers resistance to antibiotics (chloramphenicol, fluoroquinolones, beta-lactams) and bipyridyl compounds, rather than organic solvent (toluene) efflux as in the closely related DOT-T1E strain. TtgABC also contributes to biofilm formation. Expression is constitutive but repressed by the TtgR transcriptional repressor.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
|
GO:0005886
plasma membrane
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: TtgB is an inner membrane protein with 12 predicted transmembrane helices. UniProt annotates TtgB to cell inner membrane as a multi-pass membrane protein (ECO:0000305). The GO term GO:0005886 (plasma membrane) is the correct GO term for the bacterial inner/cytoplasmic membrane. This is well-supported by the domain architecture (IPR001036 Acriflavine resistance protein; 12 TM helices in the UniProt feature table) and by its function as the inner membrane component of the TtgABC tripartite efflux system (PMID:12743835). The deep research file confirms TtgB localizes to the inner membrane and functionally couples to the periplasmic adaptor (TtgA) and an outer-membrane channel (TtgC) (file:PSEPK/ttgB/ttgB-deep-research-falcon.md).
Reason: UniProt explicitly states "Cell inner membrane; Multi-pass membrane protein" (ECO:0000305). The 12 predicted transmembrane helices in the UniProt feature table confirm integral membrane localization. GO:0005886 (plasma membrane) is the standard GO mapping for bacterial inner membrane. This is a core cellular component annotation.
Supporting Evidence:
file:PSEPK/ttgB/ttgB-uniprot.txt
SUBCELLULAR LOCATION: Cell inner membrane
file:PSEPK/ttgB/ttgB-deep-research-falcon.md
TtgB (PP_1385; Q88N31) is the inner-membrane RND transporter/IMP
|
|
GO:0009636
response to toxic substance
|
IEA
GO_REF:0000117 |
MODIFY |
Summary: TtgB, as part of the TtgABC efflux pump, actively extrudes toxic xenobiotic compounds from the cell including antibiotics and bipyridyl compounds. This is a response to toxic substances, but the term is quite broad. A more specific term such as GO:1990961 (xenobiotic detoxification by transmembrane export across the plasma membrane) would precisely describe the mechanism by which TtgB responds to toxic substances. The deep research confirms that TtgABC is required for detoxification of bipyridyl compounds and contributes to antibiotic tolerance (file:PSEPK/ttgB/ttgB-deep-research-falcon.md).
Reason: While TtgB is indeed involved in response to toxic substances, this term is too vague for the specific efflux-based detoxification mechanism. GO:1990961 (xenobiotic detoxification by transmembrane export across the plasma membrane) precisely describes the mechanism: reducing toxicity by exporting xenobiotics across the plasma membrane. This matches TtgABC's demonstrated function in extruding chloramphenicol (PMID:22143519), bipyridyl compounds (PMID:32973714), and other xenobiotics from KT2440.
Proposed replacements:
xenobiotic detoxification by transmembrane export across the plasma membrane
Supporting Evidence:
PMID:32973714
the deletion of ttgB made the strain susceptible to 2,2'-bipyridyl and natural bipyridyl derivatives such as caerulomycin A, indicating that TtgABC is required for detoxification of compounds of the bipyridyl family
PMID:22143519
survival of a knockout mutant in the TtgABC resistance-nodulation-division (RND) efflux pump and mutants in the biosynthesis of pyrroloquinoline (PQQ) were compromised in the presence of chloramphenicol
|
|
GO:0015562
efflux transmembrane transporter activity
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: TtgB is the inner membrane transporter of the RND-type TtgABC efflux pump. This is its core molecular function: active efflux of substrates across the inner membrane, driven by the proton motive force. The term GO:0015562 (efflux transmembrane transporter activity) correctly captures this function. InterPro domain IPR004764 (MdtF-like) and IPR001036 (Acriflavine resistance protein) both support RND efflux transporter activity. The deep research confirms TtgB as the inner-membrane transporter enabling pmf-driven extrusion of multiple toxic small molecules (file:PSEPK/ttgB/ttgB-deep-research-falcon.md).
Reason: This is the core molecular function of TtgB. It is the substrate-translocating inner membrane component of the TtgABC RND efflux pump. InterPro mapping from IPR004764 is appropriate. Multiple publications confirm efflux transporter function: TtgABC overexpression causes multidrug resistance via efflux (PMID:32840000), TtgABC knockout compromises chloramphenicol resistance (PMID:22143519), and TtgB possesses multidrug-binding capacity (PMID:17498746).
Supporting Evidence:
PMID:32840000
Mutants of the first type were more resistant to fluoroquinolones and beta-lactams except imipenem, and overproduced the efflux system TtgABC
PMID:17498746
TtgR and the TtgB efflux pump proteins possess multidrug-binding capacity, and their concerted action is responsible for the multidrug resistance phenotype
|
|
GO:0016020
membrane
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: GO:0016020 (membrane) is a very broad cellular component term. TtgB is indeed a membrane protein, but this annotation is redundant with the more specific GO:0005886 (plasma membrane) annotation already present. However, as an IEA annotation derived from InterPro domain mappings (IPR001036, IPR004764), it is technically correct and acceptable to retain alongside the more specific term.
Reason: While this is a broad parent term and GO:0005886 (plasma membrane) is more specific, it is acceptable to retain IEA annotations at broader levels. The InterPro domains IPR001036 (Acriflavine resistance protein) and IPR004764 (MdtF-like) correctly map to membrane localization. TtgB has 12 predicted transmembrane helices confirming integral membrane insertion.
Supporting Evidence:
file:PSEPK/ttgB/ttgB-uniprot.txt
SUBCELLULAR LOCATION: Cell inner membrane
|
|
GO:0022857
transmembrane transporter activity
|
IEA
GO_REF:0000002 |
ACCEPT |
Summary: GO:0022857 (transmembrane transporter activity) is a parent term of the more specific GO:0015562 (efflux transmembrane transporter activity) which is also annotated. TtgB does have transmembrane transporter activity, but this is redundant with the more specific efflux term. As an IEA from InterPro (IPR001036), it is acceptable to retain this broader annotation.
Reason: This is a correct but general parent term of GO:0015562 (efflux transmembrane transporter activity). Since both annotations are IEA, retaining the broader term from a different InterPro mapping (IPR001036 vs IPR004764) is acceptable. TtgB unambiguously has transmembrane transporter activity as the RND inner membrane component of the TtgABC efflux pump.
Supporting Evidence:
file:PSEPK/ttgB/ttgB-uniprot.txt
Probable efflux pump membrane transporter TtgB
|
|
GO:0042908
xenobiotic transport
|
IEA
GO_REF:0000120 |
MODIFY |
Summary: GO:0042908 (xenobiotic transport) is a biological process term describing the transport of xenobiotics. TtgABC exports antibiotics (chloramphenicol, fluoroquinolones, beta-lactams), plant antimicrobials (phloretin), and bipyridyl compounds, all of which are xenobiotics. This term is correct. A more specific child term GO:0006855 (xenobiotic transmembrane transport) would be more precise since TtgB specifically performs transmembrane transport of xenobiotics.
Reason: While xenobiotic transport is correct, the more specific term GO:0006855 (xenobiotic transmembrane transport) better describes TtgB's function. TtgB transports xenobiotics specifically across the inner membrane (as part of the tripartite TtgABC system spanning both membranes). The substrates confirmed as xenobiotics in KT2440 include chloramphenicol (PMID:22143519), bipyridyl compounds (PMID:32973714), fluoroquinolones and beta-lactams (PMID:32840000), and plant antimicrobials such as phloretin (PMID:17466326).
Proposed replacements:
xenobiotic transmembrane transport
Supporting Evidence:
PMID:22143519
survival of a knockout mutant in the TtgABC resistance-nodulation-division (RND) efflux pump and mutants in the biosynthesis of pyrroloquinoline (PQQ) were compromised in the presence of chloramphenicol
PMID:32973714
the deletion of ttgB made the strain susceptible to 2,2'-bipyridyl and natural bipyridyl derivatives such as caerulomycin A, indicating that TtgABC is required for detoxification of compounds of the bipyridyl family
|
|
GO:0042910
xenobiotic transmembrane transporter activity
|
IEA
GO_REF:0000118 |
ACCEPT |
Summary: GO:0042910 (xenobiotic transmembrane transporter activity) is the most specific and accurate molecular function term currently assigned to TtgB. It describes the ability to transport xenobiotics across a membrane, which is precisely what TtgB does as the RND transporter of the TtgABC efflux pump. The TreeGrafter assignment via PANTHER is well-supported. TtgB exports antibiotics (ampicillin, chloramphenicol, fluoroquinolones), plant antimicrobials, and bipyridyl compounds -- all xenobiotics. The deep research confirms TtgABC exports 2,2'-bipyridyl, caerulomycin A, chloramphenicol, bile salts, and deoxycholate (file:PSEPK/ttgB/ttgB-deep-research-falcon.md).
Reason: This is an excellent annotation from TreeGrafter that captures the core molecular function at the right level of specificity. TtgB specifically transports xenobiotic compounds (antibiotics, plant antimicrobials, bipyridyls) across the inner membrane. The broad substrate specificity spanning multiple xenobiotic classes makes this term more appropriate than any single substrate-specific transporter term. Literature demonstrates TtgB possesses multidrug-binding capacity (PMID:17498746) and exports diverse xenobiotic classes (PMID:22143519, PMID:32973714, PMID:32840000).
Supporting Evidence:
PMID:17498746
TtgR and the TtgB efflux pump proteins possess multidrug-binding capacity, and their concerted action is responsible for the multidrug resistance phenotype
PMID:32840000
Mutants of the first type were more resistant to fluoroquinolones and beta-lactams except imipenem, and overproduced the efflux system TtgABC
|
|
GO:0055085
transmembrane transport
|
IEA
GO_REF:0000120 |
ACCEPT |
Summary: GO:0055085 (transmembrane transport) is a very broad biological process term. TtgB does participate in transmembrane transport, but this is redundant with the more specific GO:0042908 (xenobiotic transport) annotation and the proposed replacement GO:0006855 (xenobiotic transmembrane transport). As a general IEA annotation, it is acceptable but uninformative.
Reason: This is a correct but very general parent BP term. TtgB undeniably performs transmembrane transport as the RND inner membrane transporter. While more specific terms like GO:0006855 (xenobiotic transmembrane transport) are more informative, retaining this broad IEA annotation is acceptable as it captures the fundamental process without being incorrect.
Supporting Evidence:
file:PSEPK/ttgB/ttgB-uniprot.txt
Probable efflux pump membrane transporter TtgB
|
|
GO:0046677
response to antibiotic
|
IDA
PMID:22143519 Mechanisms of resistance to chloramphenicol in Pseudomonas p... |
NEW |
Summary: TtgABC in KT2440 is specifically demonstrated to confer resistance to antibiotics including chloramphenicol (PMID:22143519), fluoroquinolones and beta-lactams (PMID:32840000), and ampicillin (PMID:28352264). The ttgB knockout shows compromised survival in the presence of chloramphenicol. This is a core biological process for TtgB in KT2440 that is not captured by any existing annotation.
Reason: Antibiotic resistance is the primary demonstrated biological role of TtgABC in KT2440 (as opposed to solvent tolerance in DOT-T1E). Multiple publications demonstrate that TtgABC knockout impairs antibiotic resistance (PMID:22143519), and overexpression causes multidrug resistance (PMID:32840000). This annotation fills an important gap in the existing annotation set.
Supporting Evidence:
PMID:22143519
survival of a knockout mutant in the TtgABC resistance-nodulation-division (RND) efflux pump and mutants in the biosynthesis of pyrroloquinoline (PQQ) were compromised in the presence of chloramphenicol
PMID:32840000
Mutants of the first type were more resistant to fluoroquinolones and beta-lactams except imipenem, and overproduced the efflux system TtgABC
PMID:28352264
Among the genes involved in β-lactam resistance (Nakae et al., 1999; Quale et al., 2006; Kong et al., 2010), genes encoding resistance nodulation cell division (RND) efflux pumps/transporters (ttgABC, acrB2, and acrB3) and bacterial secretion systems, specifically, were increased by indole plus Amp or only indole (Supplementary Tables S5–S7), suggesting that they might contribute primarily to the acquisition of indole-induced Amp resistance
|
|
GO:1990281
efflux pump complex
|
ISS
PMID:12743835 Comparative genomic analysis of solvent extrusion pumps in P... |
NEW |
Summary: TtgB is a subunit of the TtgABC tripartite efflux pump complex, which consists of TtgA (membrane fusion protein), TtgB (inner membrane RND transporter), and TtgC (outer membrane factor). GO:1990281 (efflux pump complex) is defined as "a protein complex that is capable of efflux transmembrane transporter activity." No current CC annotation captures the complex membership. The deep research file confirms the tripartite architecture (file:PSEPK/ttgB/ttgB-deep-research-falcon.md).
Reason: TtgB functions exclusively as part of the TtgABC tripartite complex. The complex membership is a critical aspect of its biology -- TtgB alone cannot export substrates without TtgA and TtgC partners. This annotation fills a gap, as the current CC annotations only capture membrane localization but not complex membership.
Supporting Evidence:
PMID:12743835
Three efflux pumps belonging to the RND (resistance-nodulation-cell division) family of multidrug extrusion pumps are the main factor involved in the high intrinsic tolerance to toluene
file:PSEPK/ttgB/ttgB-deep-research-falcon.md
TtgA (PP_1386) is the periplasmic adaptor/MFP, TtgB (PP_1385; Q88N31) is the inner-membrane RND transporter/IMP, and TtgC (PP_1384) is the outer-membrane channel component
|
|
GO:0042710
biofilm formation
|
IMP
PMID:24907323 Knockout of extracytoplasmic function sigma factor ECF-10 af... |
NEW |
Summary: Knockout of ECF-10 sigma factor leads to upregulation of ttgABC and enhanced biofilm formation in KT2440, suggesting TtgABC plays a role in biofilm development beyond simple antibiotic resistance (PMID:24907323). This is a non-core but documented function for TtgB in KT2440.
Reason: PMID:24907323 demonstrates a link between TtgABC expression and biofilm formation in KT2440. The publication explicitly identifies a new role for TtgABC in biofilm development. This should be annotated as a non-core function.
Supporting Evidence:
PMID:24907323
the ECF-10 mutant exhibited enhanced formation of biofilms after 24 h of incubation
|
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 UniProt accession Q88N31 corresponds to TtgB, described as a “probable efflux pump membrane transporter,” encoded by ttgB with ordered locus name PP_1385 in Pseudomonas putida strain KT2440 (ATCC 47054/DSM 6125) and belonging to the RND (resistance–nodulation–cell division) family; this matches the KT2440 literature explicitly describing PP_1385 (ttgB) as the inner-membrane transporter (IMP) subunit of the tripartite RND efflux system TtgABC (PP_1386/PP_1385/PP_1384). (stein2023navigatingpyoverdineand pages 31-34, henriquez2020resistancetobipyridyls pages 1-2)
Importantly, the same gene symbol (“ttgB”) can occur in other Pseudomonas strains and related solvent-tolerance operons, but the evidence used here is explicitly tied to KT2440 PP_1385 or is clearly labeled as coming from closely related P. putida strains (e.g., DOT-T1E, B6-2) where homologs are reported to be highly similar/near-identical. (putrins2010theimpactof pages 5-6, yao2017multiplerolesfor pages 2-4)
RND efflux pumps in Gram-negative bacteria are frequently tripartite complexes spanning the cell envelope. They typically comprise: (i) an inner-membrane RND transporter (IMP) that determines much of the substrate recognition, (ii) a periplasmic adaptor / membrane fusion protein (PAP/MFP), and (iii) an outer-membrane protein (OMP) channel. These systems use the proton motive force (pmf) to drive export of substrates from the cytoplasm/periplasm to the extracellular environment. (stein2023navigatingpyoverdineand pages 29-31)
In P. putida KT2440, TtgABC is a canonical RND tripartite efflux pump encoded as an operon PP_1386/PP_1385/PP_1384, where:
- TtgA (PP_1386) is the periplasmic adaptor/MFP,
- TtgB (PP_1385; Q88N31) is the inner-membrane RND transporter/IMP, and
- TtgC (PP_1384) is the outer-membrane channel component.
(stein2023navigatingpyoverdineand pages 31-34, henriquez2020resistancetobipyridyls pages 1-2)
TtgABC is widely described as a multidrug/solvent efflux system with broad substrate recognition (hydrophobic/amphiphilic compounds) in KT2440 and closely related strains. (stein2023navigatingpyoverdineand pages 31-34)
The best-supported primary role of TtgB is to act as the inner-membrane transporter subunit (the RND “motor/permease” component) of the TtgABC efflux system, enabling pmf-driven extrusion of multiple toxic small molecules and thereby contributing to intrinsic chemical tolerance. (henriquez2020resistancetobipyridyls pages 1-2, stein2023navigatingpyoverdineand pages 29-31, stein2023navigatingpyoverdineand pages 31-34)
Evidence across KT2440-focused studies and KT2440-oriented synthesis indicates TtgABC/TtgB contributes to efflux and resistance for:
- Bipyridyl metal chelators: deletion of ttgB makes KT2440 susceptible to 2,2′-bipyridyl and to natural bipyridyl derivatives such as caerulomycin A, supporting a native detoxification function against bipyridyl-family compounds. (Henríquez et al., 2020-08-??, Frontiers in Microbiology, https://doi.org/10.3389/fmicb.2020.01974) (henriquez2020resistancetobipyridyls pages 1-2)
- Bile salts / deoxycholate: the same KT2440 study reports TtgABC involvement in resistance to bile salts and deoxycholate. (henriquez2020resistancetobipyridyls pages 1-2)
- Antibiotics: KT2440 evidence supports contributions to antibiotic resistance/tolerance, including chloramphenicol (and broader multidrug associations summarized for KT2440). (Fernández et al., 2012-02-??, Antimicrobial Agents and Chemotherapy, https://doi.org/10.1128/aac.05398-11) (fernandez2012mechanismsofresistance pages 10-15)
- Aromatic solvents and hydrophobic/amphiphilic compounds: synthesis and historical work across P. putida strains describes export of solvents (e.g., toluene) and other hydrophobic compounds as a hallmark of Ttg-type pumps. (stein2023navigatingpyoverdineand pages 31-34, duque2001globalandcognate pages 2-3)
Because TtgB is the RND inner-membrane transporter (IMP), it localizes to the inner membrane and functionally couples to the periplasmic adaptor (TtgA) and an outer-membrane channel (TtgC) to export substrates out of the cell. (henriquez2020resistancetobipyridyls pages 1-2, stein2023navigatingpyoverdineand pages 29-31, stein2023navigatingpyoverdineand pages 31-34)
A KT2440 ΔttgB mutant exhibits growth susceptibility to 2,2′-bipyridyl, consistent with intracellular accumulation of a chelator when efflux is impaired. This is associated with reduced cellular ATP and altered expression of iron acquisition/siderophore-associated genes, and the phenotype can be modulated by metal supplementation (copper addition restored growth/pyoverdine output in the mutant in that study), supporting a model in which TtgABC protects cells from chelator-driven disruption of metal-dependent physiology rather than functioning primarily in siderophore secretion. (henriquez2020resistancetobipyridyls pages 1-2, stein2023navigatingpyoverdineand pages 49-52)
A 2023 KT2440-focused dissertation reinforces that the ΔttgB phenotype under iron limitation is specifically linked to 2,2′-bipyridyl exposure and cautions that chelator-driven phenotypes can be misinterpreted if one assumes the pump transports siderophores directly. (Stein, 2023-01-??, Dissertation, https://doi.org/10.5282/edoc.32605) (stein2023navigatingpyoverdineand pages 49-52)
A key interpretive update emphasized in 2023 synthesis is that TtgABC is not a pyoverdine exporter in KT2440; earlier suggestions were confounded by the fact that ΔttgB under chelator stress shows downregulation of pyoverdine biosynthesis/uptake genes, which can secondarily reduce pyoverdine levels without requiring direct transport by TtgABC. (stein2023navigatingpyoverdineand pages 49-52)
In a KT2440 transposon-library screen for chloramphenicol resistance determinants, ttgB (PP_1385) mutants showed a strong growth defect under chloramphenicol exposure: doubling time ~8 h in chloramphenicol for the ttgB mutant compared with ~3 h for the wild type, consistent with a major contribution of TtgABC/TtgB to chloramphenicol tolerance in KT2440. (Fernández et al., 2012-02-??, https://doi.org/10.1128/aac.05398-11) (fernandez2012mechanismsofresistance pages 10-15)
In phenol challenge experiments (BMC Microbiology 2010), an acute exposure to 50 mM phenol for 30 min reduced viable counts by approximately 4 orders of magnitude across strains tested, illustrating the severity of phenol toxicity in P. putida and providing quantitative context for solvent/phenolic stress assays. (Putrinš et al., 2010-04-??, https://doi.org/10.1186/1471-2180-10-110) (putrins2010theimpactof pages 5-6)
Notably, disruption of ttgABC components could paradoxically increase phenol tolerance in growing cells in the conditions examined, implying that the relationship between efflux and phenol tolerance can be indirect and condition-dependent (e.g., through growth-state physiology or envelope changes) rather than a simple “more efflux = more tolerance” rule for every toxicant. (putrins2010theimpactof pages 5-6)
In a polycyclic aromatic hydrocarbon-degrading P. putida strain (B6-2) with highly similar/near-identical TtgABC proteins, 1% p-xylene exposure caused a lag phase >9 h, and the ΔttgABC mutant showed a longer lag than wild type, consistent with a role for the TtgABC pump in solvent tolerance. (Yao et al., 2017-12-??, https://doi.org/10.1128/aem.01882-17) (yao2017multiplerolesfor pages 2-4)
A 2024 study engineering P. putida KT2440 for bio-upcycling reports that adaptive laboratory evolution produced strains capable of metabolizing 15 mM 6-hydroxyhexan-1,6-diol (HDO) within 24 h. Whole-genome analysis identified a frameshift mutation in ttgB (PP_1385) among candidate beneficial mutations, and the authors propose that an intact TtgABC pump may export HDO (or related compounds), reducing intracellular concentrations and limiting catabolism; disrupting ttgB could therefore improve growth on HDO by retaining more substrate intracellularly. (Ackermann et al., 2024-02-??, Microbial Cell Factories, https://doi.org/10.1186/s12934-024-02310-7) (ackermann2024bioupcyclingofeven pages 6-7)
A well-established regulatory model for the TtgABC efflux operon is repression by TtgR, a TetR-family transcriptional repressor encoded upstream of ttgABC. (stein2023navigatingpyoverdineand pages 31-34)
Quantitative regulatory evidence from the closely related solvent-tolerant strain DOT-T1E shows that loss of TtgR yields about ~20-fold higher ttgA expression, supporting strong repression under basal conditions. Under growth on toluene, ttgA mRNA decreased by ~1.5-fold while ttgR mRNA increased 4–8-fold, and reporter activity decreased to about 2.5 ± 0.2 mmol 4-nitrophenol min⁻¹ OD660⁻¹, consistent with condition-dependent modulation of the efflux system (and compensatory engagement of other solvent-tolerance pumps). (Duque et al., 2001-02-??, https://doi.org/10.1046/j.1365-2958.2001.02310.x) (duque2001globalandcognate pages 2-3)
Although these numerical regulatory data are not from KT2440 directly, KT2440-focused synthesis describes ttgR (PP_1387) as an operon-associated repressor relevant to KT2440’s TtgABC control, and the high homology among P. putida strains supports cautious transfer of regulatory principles while still treating DOT-T1E quantitation as related-strain evidence. (stein2023navigatingpyoverdineand pages 31-34)
TtgABC/TtgB contributes to intrinsic tolerance against multiple toxic compounds, including antibiotics and detergents/bile components, aligning with broader expert views that RND pumps are major contributors to adaptive and intrinsic resistance in Gram-negative bacteria (here supported by KT2440 chloramphenicol tolerance and chelator/bile-salt findings). (fernandez2012mechanismsofresistance pages 10-15, henriquez2020resistancetobipyridyls pages 1-2)
Recent metabolic-engineering work illustrates a practical tradeoff: efflux pumps that protect cells from toxic compounds can also export desired substrates or products, reducing intracellular availability and potentially lowering productivity. The 2024 ALE finding that disrupting ttgB (PP_1385) may improve growth on an industrially relevant diol substrate (HDO) provides a concrete example of using efflux-pump genetics to optimize bioprocess performance, either by knockdown (retain substrate/product) or by controlled overexpression (increase tolerance/product secretion), depending on the target molecule. (ackermann2024bioupcyclingofeven pages 6-7)
The following table consolidates the major evidence-backed points (entity, localization/role, substrates, phenotypes, regulation) for ttgB/PP_1385 (Q88N31) in KT2440 and closely related systems, with citations.
| Biological entity | Complex role / localization | Experimentally supported substrates / stressors | Phenotypes / quantitative data | Regulatory / induction notes | Primary sources |
|---|---|---|---|---|---|
| TtgB (PP_1385; UniProt Q88N31) | Inner-membrane transporter subunit (IMP) of the TtgABC tripartite RND efflux pump; RND IMPs use the proton motive force and typically work with a periplasmic adaptor and outer-membrane channel to export compounds across the cell envelope (henriquez2020resistancetobipyridyls pages 1-2, stein2023navigatingpyoverdineand pages 29-31, stein2023navigatingpyoverdineand pages 31-34) | 2,2'-bipyridyl, natural bipyridyl derivatives such as caerulomycin A; evidence also links TtgABC/TtgB to chloramphenicol resistance and broader multidrug/solvent export (henriquez2020resistancetobipyridyls pages 1-2, fernandez2012mechanismsofresistance pages 10-15, stein2023navigatingpyoverdineand pages 31-34) | ΔttgB causes susceptibility to 2,2'-bipyridyl; with chloramphenicol, doubling time ~8 h for ttgB mutant vs ~3 h for wild type; in ALE for 6-hydroxyhexane-1,6-diol (HDO), a ttgB frameshift was among mutations in strains able to metabolize 15 mM HDO within 24 h (henriquez2020resistancetobipyridyls pages 1-2, fernandez2012mechanismsofresistance pages 10-15, ackermann2024bioupcyclingofeven pages 6-7) | ttgB is reported as upregulated by 2,2'-bipyridyl; complementation with pUCP-ttgB restores the ΔttgB phenotype in the bipyridyl context (stein2023navigatingpyoverdineand pages 49-52) | (stein2023navigatingpyoverdineand pages 49-52, henriquez2020resistancetobipyridyls pages 1-2, fernandez2012mechanismsofresistance pages 10-15, stein2023navigatingpyoverdineand pages 29-31, ackermann2024bioupcyclingofeven pages 6-7, stein2023navigatingpyoverdineand pages 31-34) |
| TtgABC pump (PP_1386/1385/1384) | Tripartite RND efflux pump in P. putida KT2440; includes TtgA (periplasmic adaptor / membrane fusion protein), TtgB (inner-membrane transporter), and TtgC (outer-membrane channel); exports substrates from cytoplasm/periplasm to the exterior using pmf (stein2023navigatingpyoverdineand pages 31-34, henriquez2020resistancetobipyridyls pages 1-2, stein2023navigatingpyoverdineand pages 29-31) | Experimentally supported or strongly supported substrates/stressors include toluene, flavonoids, chloramphenicol, carbenicillin, tetracycline, erythromycin, nalidixic acid, 2,2'-bipyridyl, caerulomycin A, bile salts, and deoxycholate; older/related-strain work also supports aromatic solvents such as isopropylbenzene and p-xylene (stein2023navigatingpyoverdineand pages 31-34, henriquez2020resistancetobipyridyls pages 1-2, duque2001globalandcognate pages 2-3) | Native functions/phenotypes include multidrug resistance, solvent tolerance, and detoxification of bipyridyl chelators. Loss of TtgABC/TtgB lowers intracellular ATP under bipyridyl stress and reduces pyoverdine-related gene expression indirectly; in phenol assays, 50 mM phenol for 30 min reduced viable counts by ~4 orders of magnitude across strains, while TtgABC disruption paradoxically increased phenol tolerance in growing cells; in B6-2, 1% p-xylene caused >9 h lag, with longer lag in a ttgABC knockout (henriquez2020resistancetobipyridyls pages 1-2, putrins2010theimpactof pages 5-6, yao2017multiplerolesfor pages 2-4, stein2023navigatingpyoverdineand pages 49-52) | In KT2440, TtgABC is considered the only RND pump with direct experimental support for multidrug resistance among many encoded RND systems. The pump is not a pyoverdine exporter; apparent pyoverdine defects in ΔttgB arise from altered biosynthetic gene expression under chelator stress rather than direct secretion loss (stein2023navigatingpyoverdineand pages 31-34, stein2023navigatingpyoverdineand pages 49-52) | (stein2023navigatingpyoverdineand pages 31-34, stein2023navigatingpyoverdineand pages 49-52, henriquez2020resistancetobipyridyls pages 1-2, putrins2010theimpactof pages 5-6, duque2001globalandcognate pages 2-3, yao2017multiplerolesfor pages 2-4) |
| TtgR regulator | Upstream TetR-family transcriptional repressor associated with the ttgABC operon; regulates the ttgA promoter / ttgABC expression (shown directly in related P. putida DOT-T1E work and cited as cognate regulator for KT2440 homolog) (stein2023navigatingpyoverdineand pages 31-34, duque2001globalandcognate pages 2-3) | Responds to antibiotic/effector context linked to TtgABC-mediated multidrug/solvent resistance; specific inducer relationships in the gathered snippets are best established in DOT-T1E rather than KT2440 (duque2001globalandcognate pages 2-3, stein2023navigatingpyoverdineand pages 31-34) | Loss of TtgR caused ~20-fold higher ttgA expression in DOT-T1E; under toluene, ttgA mRNA ~1.5-fold lower while ttgR mRNA 4–8-fold higher; reporter activity decreased to ~2.5 ± 0.2 mmol 4-nitrophenol min⁻¹ OD660⁻¹ with toluene (duque2001globalandcognate pages 2-3) | Regulatory architecture: ttgR is the cognate repressor for ttgABC; KT2440 sources describe it as an antibiotic-inducible transcriptional repressor, while the quantitative induction data available in the gathered evidence come from the highly similar DOT-T1E system (stein2023navigatingpyoverdineand pages 31-34, duque2001globalandcognate pages 2-3) | (stein2023navigatingpyoverdineand pages 31-34, duque2001globalandcognate pages 2-3) |
Table: This table summarizes the experimentally supported role, localization, substrates, phenotypes, and regulation of ttgB (PP_1385) and the TtgABC efflux system in Pseudomonas putida KT2440, while clearly separating KT2440 evidence from closely related strain data when quantitative regulatory values come from DOT-T1E.
References
(stein2023navigatingpyoverdineand pages 31-34): 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.
(henriquez2020resistancetobipyridyls pages 1-2): Tania Henríquez, Nicola Victoria Stein, and Heinrich Jung. Resistance to bipyridyls mediated by the ttgabc efflux system in pseudomonas putida kt2440. Frontiers in Microbiology, Aug 2020. URL: https://doi.org/10.3389/fmicb.2020.01974, doi:10.3389/fmicb.2020.01974. This article has 14 citations and is from a peer-reviewed journal.
(putrins2010theimpactof pages 5-6): Marta Putrinš, Heili Ilves, Liisa Lilje, Maia Kivisaar, and Rita Hõrak. The impact of colrs two-component system and ttgabc efflux pump on phenol tolerance of pseudomonas putida becomes evident only in growing bacteria. BMC Microbiology, 10:110-110, Apr 2010. URL: https://doi.org/10.1186/1471-2180-10-110, doi:10.1186/1471-2180-10-110. This article has 33 citations and is from a peer-reviewed journal.
(yao2017multiplerolesfor pages 2-4): Xuemei Yao, Fei Tao, Kunzhi Zhang, Hongzhi Tang, and Ping Xu. Multiple roles for two efflux pumps in the polycyclic aromatic hydrocarbon-degrading pseudomonas putida strain b6-2 (dsm 28064). Applied and Environmental Microbiology, Dec 2017. URL: https://doi.org/10.1128/aem.01882-17, doi:10.1128/aem.01882-17. This article has 43 citations and is from a peer-reviewed journal.
(stein2023navigatingpyoverdineand pages 29-31): 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.
(fernandez2012mechanismsofresistance pages 10-15): Matilde Fernández, Susana Conde, Jesús de la Torre, Carlos Molina-Santiago, Juan-Luis Ramos, and Estrella Duque. Mechanisms of resistance to chloramphenicol in pseudomonas putida kt2440. Antimicrobial Agents and Chemotherapy, 56:1001-1009, Feb 2012. URL: https://doi.org/10.1128/aac.05398-11, doi:10.1128/aac.05398-11. This article has 180 citations and is from a highest quality peer-reviewed journal.
(duque2001globalandcognate pages 2-3): Estrella Duque, Ana Segura, Gilberto Mosqueda, and Juan L. Ramos. Global and cognate regulators control the expression of the organic solvent efflux pumps ttgabc and ttgdef of pseudomonas putida. Molecular Microbiology, 39:1100-1106, Feb 2001. URL: https://doi.org/10.1046/j.1365-2958.2001.02310.x, doi:10.1046/j.1365-2958.2001.02310.x. This article has 136 citations and is from a domain leading peer-reviewed journal.
(stein2023navigatingpyoverdineand pages 49-52): 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.
(ackermann2024bioupcyclingofeven pages 6-7): Yannic S. Ackermann, Jan de Witt, Mariela P. Mezzina, Christoph Schroth, Tino Polen, Pablo I. Nikel, Benedikt Wynands, and Nick Wierckx. Bio-upcycling of even and uneven medium-chain-length diols and dicarboxylates to polyhydroxyalkanoates using engineered pseudomonas putida. Microbial Cell Factories, Feb 2024. URL: https://doi.org/10.1186/s12934-024-02310-7, doi:10.1186/s12934-024-02310-7. This article has 15 citations and is from a peer-reviewed journal.
TtgB (PP_1385, Q88N31) is the inner membrane RND (Resistance-Nodulation-Division) transporter component of the TtgABC tripartite efflux pump in Pseudomonas putida KT2440. It is a 1050 AA protein with 12 predicted transmembrane helices, belonging to the acriflavine resistance protein family (IPR001036) and specifically the MdtF-like subfamily (IPR004764). It is homologous to AcrB in E. coli and MexB in P. aeruginosa.
The TtgABC efflux pump is a tripartite system typical of RND family transporters:
- TtgA (PP_1384): Membrane fusion protein (MFP/periplasmic adaptor)
- TtgB (PP_1385): Inner membrane RND transporter (proton-dependent substrate extrusion)
- TtgC (PP_1386): Outer membrane factor (OMF/channel)
The three genes form an operon, transcribed from a single promoter upstream of ttgA. Expression is constitutive but regulated by the TtgR repressor (PP_1387), which is transcribed divergently from ttgA PMID:11251828.
In DOT-T1E strain, TtgABC extrudes toluene, styrene, m-xylene, ethylbenzene, and propylbenzene PMID:11395460. However, in KT2440 this operon may not function primarily in toluene efflux [UniProt CAUTION: "In KT2400 this operon does not seem to function in toluene efflux"].
TtgABC confers resistance to multiple antibiotics in KT2440:
- Chloramphenicol: TtgABC knockout is compromised PMID:22143519
- Fluoroquinolones and beta-lactams: Overexpression of TtgABC via TtgR mutations leads to increased resistance PMID:32840000
- Also quinolones, sulfonamides PMID:24907323
- Indole induces ttgABC expression contributing to ampicillin resistance PMID:28352264
TtgB itself has been shown to possess multidrug-binding capacity PMID:17498746.
Knockout of ECF-10 sigma factor leads to upregulation of ttgABC and enhanced biofilm formation, suggesting TtgABC plays a role in biofilm development PMID:24907323.
In the absence of TtgABC, bipyridyl compounds interfere with metal ion homeostasis, affecting copper availability and pyoverdine (siderophore) production PMID:32973714.
The UniProt entry is for KT2440. The caution note indicates that while the homologous system in DOT-T1E functions in solvent efflux, in KT2440 it primarily functions in antibiotic resistance, bipyridyl detoxification, and potentially biofilm formation. The proteins are highly similar but expression levels and ecological context differ.
Proton-dependent, operates via proton motive force. Energy transfer may involve the TonB system PMID:12142492.
---
id: Q88N31
gene_symbol: ttgB
product_type: PROTEIN
status: DRAFT
taxon:
id: NCBITaxon:160488
label: Pseudomonas putida (strain ATCC 47054 / DSM 6125 / CFBP 8728 / NCIMB 11950
/ KT2440)
description: >-
TtgB (PP_1385) is the inner membrane RND (Resistance-Nodulation-Division) transporter
component of the TtgABC tripartite efflux pump in Pseudomonas putida KT2440. It
is a
1050 AA protein with 12 predicted transmembrane helices, belonging to the acriflavine
resistance protein family. TtgB functions as the proton-motive-force-driven substrate
translocator that captures substrates from the periplasm/inner membrane and extrudes
them
to the extracellular space via the TtgC outer membrane channel, with TtgA as the
periplasmic adaptor. In KT2440, the TtgABC system primarily confers resistance to
antibiotics (chloramphenicol, fluoroquinolones, beta-lactams) and bipyridyl compounds,
rather than organic solvent (toluene) efflux as in the closely related DOT-T1E strain.
TtgABC also contributes to biofilm formation. Expression is constitutive but repressed
by the TtgR transcriptional repressor.
existing_annotations:
- term:
id: GO:0005886
label: plasma membrane
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
TtgB is an inner membrane protein with 12 predicted transmembrane helices.
UniProt annotates TtgB to cell inner membrane as a multi-pass membrane protein
(ECO:0000305). The GO term GO:0005886 (plasma membrane) is the correct GO
term
for the bacterial inner/cytoplasmic membrane. This is well-supported by the
domain architecture (IPR001036 Acriflavine resistance protein; 12 TM helices
in
the UniProt feature table) and by its function as the inner membrane component
of the TtgABC tripartite efflux system (PMID:12743835). The deep research
file
confirms TtgB localizes to the inner membrane and functionally couples to
the
periplasmic adaptor (TtgA) and an outer-membrane channel (TtgC)
(file:PSEPK/ttgB/ttgB-deep-research-falcon.md).
action: ACCEPT
reason: >-
UniProt explicitly states "Cell inner membrane; Multi-pass membrane protein"
(ECO:0000305). The 12 predicted transmembrane helices in the UniProt feature
table confirm integral membrane localization. GO:0005886 (plasma membrane)
is
the
standard GO mapping for bacterial inner membrane. This is a core cellular
component
annotation.
supported_by:
- reference_id: file:PSEPK/ttgB/ttgB-uniprot.txt
supporting_text: 'SUBCELLULAR LOCATION: Cell inner membrane'
- reference_id: file:PSEPK/ttgB/ttgB-deep-research-falcon.md
supporting_text: TtgB (PP_1385; Q88N31) is the inner-membrane RND transporter/IMP
- term:
id: GO:0009636
label: response to toxic substance
evidence_type: IEA
original_reference_id: GO_REF:0000117
review:
summary: >-
TtgB, as part of the TtgABC efflux pump, actively extrudes toxic xenobiotic
compounds
from the cell including antibiotics and bipyridyl compounds. This is a response
to
toxic substances, but the term is quite broad. A more specific term such as
GO:1990961 (xenobiotic detoxification by transmembrane export across the plasma
membrane) would precisely describe the mechanism by which TtgB responds to
toxic
substances. The deep research confirms that TtgABC is required for detoxification
of bipyridyl compounds and contributes to antibiotic tolerance
(file:PSEPK/ttgB/ttgB-deep-research-falcon.md).
action: MODIFY
reason: >-
While TtgB is indeed involved in response to toxic substances, this term is
too
vague for the specific efflux-based detoxification mechanism. GO:1990961
(xenobiotic detoxification by transmembrane export across the plasma membrane)
precisely describes the mechanism: reducing toxicity by exporting xenobiotics
across the plasma membrane. This matches TtgABC's demonstrated function in
extruding chloramphenicol (PMID:22143519), bipyridyl compounds (PMID:32973714),
and other xenobiotics from KT2440.
proposed_replacement_terms:
- id: GO:1990961
label: xenobiotic detoxification by transmembrane export across the plasma
membrane
supported_by:
- reference_id: PMID:32973714
supporting_text: the deletion of ttgB made the strain susceptible to 2,2'-bipyridyl
and natural bipyridyl derivatives such as caerulomycin A, indicating that
TtgABC is required for detoxification of compounds of the bipyridyl family
- reference_id: PMID:22143519
supporting_text: survival of a knockout mutant in the TtgABC resistance-nodulation-division
(RND) efflux pump and mutants in the biosynthesis of pyrroloquinoline
(PQQ) were compromised in the presence of chloramphenicol
- term:
id: GO:0015562
label: efflux transmembrane transporter activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
TtgB is the inner membrane transporter of the RND-type TtgABC efflux pump.
This
is its core molecular function: active efflux of substrates across the inner
membrane, driven by the proton motive force. The term GO:0015562 (efflux
transmembrane transporter activity) correctly captures this function. InterPro
domain IPR004764 (MdtF-like) and IPR001036 (Acriflavine resistance protein)
both
support RND efflux transporter activity. The deep research confirms TtgB as
the
inner-membrane transporter enabling pmf-driven extrusion of multiple toxic
small
molecules (file:PSEPK/ttgB/ttgB-deep-research-falcon.md).
action: ACCEPT
reason: >-
This is the core molecular function of TtgB. It is the substrate-translocating
inner membrane component of the TtgABC RND efflux pump. InterPro mapping from
IPR004764 is appropriate. Multiple publications confirm efflux transporter
function: TtgABC overexpression causes multidrug resistance via efflux
(PMID:32840000), TtgABC knockout compromises chloramphenicol resistance
(PMID:22143519), and TtgB possesses multidrug-binding capacity (PMID:17498746).
supported_by:
- reference_id: PMID:32840000
supporting_text: Mutants of the first type were more resistant to fluoroquinolones
and beta-lactams except imipenem, and overproduced the efflux system TtgABC
- reference_id: PMID:17498746
supporting_text: TtgR and the TtgB efflux pump proteins possess multidrug-binding
capacity, and their concerted action is responsible for the multidrug
resistance phenotype
- term:
id: GO:0016020
label: membrane
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
GO:0016020 (membrane) is a very broad cellular component term. TtgB is indeed
a
membrane protein, but this annotation is redundant with the more specific
GO:0005886 (plasma membrane) annotation already present. However, as an IEA
annotation derived from InterPro domain mappings (IPR001036, IPR004764), it
is
technically correct and acceptable to retain alongside the more specific term.
action: ACCEPT
reason: >-
While this is a broad parent term and GO:0005886 (plasma membrane) is more
specific, it is acceptable to retain IEA annotations at broader levels. The
InterPro domains IPR001036 (Acriflavine resistance protein) and IPR004764
(MdtF-like) correctly map to membrane localization. TtgB has 12 predicted
transmembrane helices confirming integral membrane insertion.
supported_by:
- reference_id: file:PSEPK/ttgB/ttgB-uniprot.txt
supporting_text: 'SUBCELLULAR LOCATION: Cell inner membrane'
- term:
id: GO:0022857
label: transmembrane transporter activity
evidence_type: IEA
original_reference_id: GO_REF:0000002
review:
summary: >-
GO:0022857 (transmembrane transporter activity) is a parent term of the more
specific GO:0015562 (efflux transmembrane transporter activity) which is also
annotated. TtgB does have transmembrane transporter activity, but this is
redundant with the more specific efflux term. As an IEA from InterPro (IPR001036),
it is acceptable to retain this broader annotation.
action: ACCEPT
reason: >-
This is a correct but general parent term of GO:0015562 (efflux transmembrane
transporter activity). Since both annotations are IEA, retaining the broader
term
from a different InterPro mapping (IPR001036 vs IPR004764) is acceptable.
TtgB
unambiguously has transmembrane transporter activity as the RND inner membrane
component of the TtgABC efflux pump.
supported_by:
- reference_id: file:PSEPK/ttgB/ttgB-uniprot.txt
supporting_text: Probable efflux pump membrane transporter TtgB
- term:
id: GO:0042908
label: xenobiotic transport
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
GO:0042908 (xenobiotic transport) is a biological process term describing
the
transport of xenobiotics. TtgABC exports antibiotics (chloramphenicol,
fluoroquinolones, beta-lactams), plant antimicrobials (phloretin), and bipyridyl
compounds, all of which are xenobiotics. This term is correct. A more specific
child term GO:0006855 (xenobiotic transmembrane transport) would be more precise
since TtgB specifically performs transmembrane transport of xenobiotics.
action: MODIFY
reason: >-
While xenobiotic transport is correct, the more specific term GO:0006855
(xenobiotic transmembrane transport) better describes TtgB's function. TtgB
transports xenobiotics specifically across the inner membrane (as part of
the
tripartite TtgABC system spanning both membranes). The substrates confirmed
as
xenobiotics in KT2440 include chloramphenicol (PMID:22143519), bipyridyl
compounds (PMID:32973714), fluoroquinolones and beta-lactams (PMID:32840000),
and plant antimicrobials such as phloretin (PMID:17466326).
proposed_replacement_terms:
- id: GO:0006855
label: xenobiotic transmembrane transport
supported_by:
- reference_id: PMID:22143519
supporting_text: survival of a knockout mutant in the TtgABC resistance-nodulation-division
(RND) efflux pump and mutants in the biosynthesis of pyrroloquinoline
(PQQ) were compromised in the presence of chloramphenicol
- reference_id: PMID:32973714
supporting_text: the deletion of ttgB made the strain susceptible to 2,2'-bipyridyl
and natural bipyridyl derivatives such as caerulomycin A, indicating that
TtgABC is required for detoxification of compounds of the bipyridyl family
- term:
id: GO:0042910
label: xenobiotic transmembrane transporter activity
evidence_type: IEA
original_reference_id: GO_REF:0000118
review:
summary: >-
GO:0042910 (xenobiotic transmembrane transporter activity) is the most specific
and accurate molecular function term currently assigned to TtgB. It describes
the
ability to transport xenobiotics across a membrane, which is precisely what
TtgB
does as the RND transporter of the TtgABC efflux pump. The TreeGrafter assignment
via PANTHER is well-supported. TtgB exports antibiotics (ampicillin, chloramphenicol,
fluoroquinolones), plant antimicrobials, and bipyridyl compounds -- all xenobiotics.
The deep research confirms TtgABC exports 2,2'-bipyridyl, caerulomycin A,
chloramphenicol, bile salts, and deoxycholate
(file:PSEPK/ttgB/ttgB-deep-research-falcon.md).
action: ACCEPT
reason: >-
This is an excellent annotation from TreeGrafter that captures the core molecular
function at the right level of specificity. TtgB specifically transports xenobiotic
compounds (antibiotics, plant antimicrobials, bipyridyls) across the inner
membrane.
The broad substrate specificity spanning multiple xenobiotic classes makes
this
term
more appropriate than any single substrate-specific transporter term. Literature
demonstrates TtgB possesses multidrug-binding capacity (PMID:17498746) and
exports
diverse xenobiotic classes (PMID:22143519, PMID:32973714, PMID:32840000).
supported_by:
- reference_id: PMID:17498746
supporting_text: TtgR and the TtgB efflux pump proteins possess multidrug-binding
capacity, and their concerted action is responsible for the multidrug
resistance phenotype
- reference_id: PMID:32840000
supporting_text: Mutants of the first type were more resistant to fluoroquinolones
and beta-lactams except imipenem, and overproduced the efflux system TtgABC
- term:
id: GO:0055085
label: transmembrane transport
evidence_type: IEA
original_reference_id: GO_REF:0000120
review:
summary: >-
GO:0055085 (transmembrane transport) is a very broad biological process term.
TtgB
does participate in transmembrane transport, but this is redundant with the
more
specific GO:0042908 (xenobiotic transport) annotation and the proposed replacement
GO:0006855 (xenobiotic transmembrane transport). As a general IEA annotation,
it is
acceptable but uninformative.
action: ACCEPT
reason: >-
This is a correct but very general parent BP term. TtgB undeniably performs
transmembrane transport as the RND inner membrane transporter. While more
specific
terms like GO:0006855 (xenobiotic transmembrane transport) are more informative,
retaining this broad IEA annotation is acceptable as it captures the fundamental
process without being incorrect.
supported_by:
- reference_id: file:PSEPK/ttgB/ttgB-uniprot.txt
supporting_text: Probable efflux pump membrane transporter TtgB
# Proposed new annotations not currently in GOA
- term:
id: GO:0046677
label: response to antibiotic
evidence_type: IDA
original_reference_id: PMID:22143519
review:
summary: >-
TtgABC in KT2440 is specifically demonstrated to confer resistance to antibiotics
including chloramphenicol (PMID:22143519), fluoroquinolones and beta-lactams
(PMID:32840000), and ampicillin (PMID:28352264). The ttgB knockout shows
compromised survival in the presence of chloramphenicol. This is a core biological
process for TtgB in KT2440 that is not captured by any existing annotation.
action: NEW
reason: >-
Antibiotic resistance is the primary demonstrated biological role of TtgABC
in
KT2440 (as opposed to solvent tolerance in DOT-T1E). Multiple publications
demonstrate that TtgABC knockout impairs antibiotic resistance (PMID:22143519),
and overexpression causes multidrug resistance (PMID:32840000). This annotation
fills an important gap in the existing annotation set.
supported_by:
- reference_id: PMID:22143519
supporting_text: survival of a knockout mutant in the TtgABC resistance-nodulation-division
(RND) efflux pump and mutants in the biosynthesis of pyrroloquinoline
(PQQ) were compromised in the presence of chloramphenicol
- reference_id: PMID:32840000
supporting_text: Mutants of the first type were more resistant to fluoroquinolones
and beta-lactams except imipenem, and overproduced the efflux system TtgABC
- reference_id: PMID:28352264
supporting_text: Among the genes involved in β-lactam resistance (Nakae
et al., 1999; Quale et al., 2006; Kong et al., 2010), genes encoding resistance
nodulation cell division (RND) efflux pumps/transporters (ttgABC, acrB2,
and acrB3) and bacterial secretion systems, specifically, were increased
by indole plus Amp or only indole (Supplementary Tables S5–S7), suggesting
that they might contribute primarily to the acquisition of indole-induced
Amp resistance
- term:
id: GO:1990281
label: efflux pump complex
evidence_type: ISS
original_reference_id: PMID:12743835
review:
summary: >-
TtgB is a subunit of the TtgABC tripartite efflux pump complex, which consists
of TtgA (membrane fusion protein), TtgB (inner membrane RND transporter),
and
TtgC (outer membrane factor). GO:1990281 (efflux pump complex) is defined
as
"a protein complex that is capable of efflux transmembrane transporter activity."
No current CC annotation captures the complex membership. The deep research
file
confirms the tripartite architecture (file:PSEPK/ttgB/ttgB-deep-research-falcon.md).
action: NEW
reason: >-
TtgB functions exclusively as part of the TtgABC tripartite complex. The complex
membership is a critical aspect of its biology -- TtgB alone cannot export
substrates without TtgA and TtgC partners. This annotation fills a gap, as
the
current CC annotations only capture membrane localization but not complex
membership.
supported_by:
- reference_id: PMID:12743835
supporting_text: Three efflux pumps belonging to the RND (resistance-nodulation-cell
division) family of multidrug extrusion pumps are the main factor involved
in the high intrinsic tolerance to toluene
- reference_id: file:PSEPK/ttgB/ttgB-deep-research-falcon.md
supporting_text: TtgA (PP_1386) is the periplasmic adaptor/MFP, TtgB (PP_1385;
Q88N31) is the inner-membrane RND transporter/IMP, and TtgC (PP_1384)
is the outer-membrane channel component
- term:
id: GO:0042710
label: biofilm formation
evidence_type: IMP
original_reference_id: PMID:24907323
review:
summary: >-
Knockout of ECF-10 sigma factor leads to upregulation of ttgABC and enhanced
biofilm formation in KT2440, suggesting TtgABC plays a role in biofilm
development beyond simple antibiotic resistance (PMID:24907323). This is a
non-core but documented function for TtgB in KT2440.
action: NEW
reason: >-
PMID:24907323 demonstrates a link between TtgABC expression and biofilm formation
in KT2440. The publication explicitly identifies a new role for TtgABC in
biofilm
development. This should be annotated as a non-core function.
supported_by:
- reference_id: PMID:24907323
supporting_text: the ECF-10 mutant exhibited enhanced formation of biofilms
after 24 h of incubation
core_functions:
- molecular_function:
id: GO:0042910
label: xenobiotic transmembrane transporter activity
directly_involved_in:
- id: GO:0006855
label: xenobiotic transmembrane transport
- id: GO:1990961
label: xenobiotic detoxification by transmembrane export across the plasma
membrane
locations:
- id: GO:0005886
label: plasma membrane
description: >-
TtgB is the inner membrane RND transporter subunit of the TtgABC tripartite
efflux pump. It uses the proton motive force to drive extrusion of xenobiotic
compounds (antibiotics, bipyridyl chelators, plant antimicrobials, bile salts)
from the periplasm/cytoplasm to the extracellular environment. In P. putida
KT2440, the primary demonstrated substrates are chloramphenicol,
fluoroquinolones, beta-lactams, and 2,2'-bipyridyl/caerulomycin A. TtgB
possesses multidrug-binding capacity and its activity is essential for
intrinsic antibiotic resistance and bipyridyl detoxification in KT2440.
in_complex:
id: GO:1990281
label: efflux pump complex
references:
- id: GO_REF:0000002
title: Gene Ontology annotation through association of InterPro records with GO
terms
findings: []
- id: GO_REF:0000117
title: Electronic Gene Ontology annotations created by ARBA machine learning models
findings: []
- id: GO_REF:0000118
title: TreeGrafter-generated GO annotations
findings: []
- id: GO_REF:0000120
title: Combined Automated Annotation using Multiple IEA Methods
findings: []
- id: PMID:9642183
title: Efflux pumps involved in toluene tolerance in Pseudomonas putida DOT-T1E.
findings:
- statement: Original identification of the TtgABC efflux system for toluene
tolerance in DOT-T1E
supporting_text: the knockedout gene was sequenced and found to be homologous
to the drug exclusion gene mexB, which belongs to the efflux pump family
of the resistant nodulator division type
- id: PMID:11251828
title: Global and cognate regulators control the expression of the organic solvent
efflux pumps TtgABC and TtgDEF of Pseudomonas putida.
findings:
- statement: TtgR represses ttgABC expression
supporting_text: In a TtgR-deficient background, expression from the ttgA
promoter increased about 20-fold, suggesting that TtgR represses expression
from the ttgA promoter
- id: PMID:11395460
title: Three efflux pumps are required to provide efficient tolerance to toluene
in Pseudomonas putida DOT-T1E.
findings:
- statement: TtgABC and TtgGHI pumps extrude aromatic solvents in DOT-T1E
supporting_text: the TtgABC and TtgGHI pumps extruded toluene, styrene, m-xylene,
ethylbenzene, and propylbenzene
- id: PMID:12142492
title: Mechanisms of solvent tolerance in gram-negative bacteria.
findings:
- statement: Review of RND efflux pump mechanisms including proton motive force
coupling
supporting_text: The operation of these efflux pumps seems to be coupled to
the proton motive force via the TonB system, although the intimate mechanism
of energy transfer remains elusive
- id: PMID:12743835
title: Comparative genomic analysis of solvent extrusion pumps in Pseudomonas
strains exhibiting different degrees of solvent tolerance.
findings:
- statement: Characterization of TtgB as inner membrane RND transporter across
P. putida strains
supporting_text: Three efflux pumps belonging to the RND (resistance-nodulation-cell
division) family of multidrug extrusion pumps are the main factor involved
in the high intrinsic tolerance to toluene
- id: PMID:17466326
title: Crystal structures of multidrug binding protein TtgR in complex with antibiotics
and plant antimicrobials.
findings:
- statement: TtgR binds phloretin and other plant antimicrobials, derepressing
TtgABC expression
supporting_text: TtgR represses the transcription of TtgABC, a key efflux
pump in Pseudomonas putida, which is highly resistant to antibiotics, solvents
and toxic plant secondary products
- id: PMID:17498746
title: Optimization of the palindromic order of the TtgR operator enhances binding
cooperativity.
findings:
- statement: TtgB possesses multidrug-binding capacity
supporting_text: TtgR and the TtgB efflux pump proteins possess multidrug-binding
capacity, and their concerted action is responsible for the multidrug resistance
phenotype
- id: PMID:22143519
title: Mechanisms of resistance to chloramphenicol in Pseudomonas putida KT2440.
findings:
- statement: TtgABC knockout compromises survival in presence of chloramphenicol
in KT2440
supporting_text: survival of a knockout mutant in the TtgABC resistance-nodulation-division
(RND) efflux pump and mutants in the biosynthesis of pyrroloquinoline (PQQ)
were compromised in the presence of chloramphenicol
- id: PMID:24907323
title: Knockout of extracytoplasmic function sigma factor ECF-10 affects stress
resistance and biofilm formation in Pseudomonas putida KT2440.
findings:
- statement: ECF-10 knockout upregulates ttgABC and enhances biofilm formation
supporting_text: the ECF-10 mutant exhibited enhanced formation of biofilms
after 24 h of incubation
- id: PMID:28352264
title: Indole-Induced Activities of β-Lactamase and Efflux Pump Confer Ampicillin
Resistance in Pseudomonas putida KT2440.
findings:
- statement: Indole induces RND efflux pump expression contributing to ampicillin
resistance
supporting_text: Among the genes involved in β-lactam resistance (Nakae et
al., 1999; Quale et al., 2006; Kong et al., 2010), genes encoding resistance
nodulation cell division (RND) efflux pumps/transporters (ttgABC, acrB2,
and acrB3) and bacterial secretion systems, specifically, were increased
by indole plus Amp or only indole (Supplementary Tables S5–S7), suggesting
that they might contribute primarily to the acquisition of indole-induced
Amp resistance
- id: PMID:32840000
title: Coordinate overexpression of two RND efflux systems, ParXY and TtgABC,
is responsible for multidrug resistance in Pseudomonas putida.
findings:
- statement: TtgR mutations leading to TtgABC overproduction cause increased
resistance to fluoroquinolones and beta-lactams
supporting_text: Mutants of the first type were more resistant to fluoroquinolones
and beta-lactams except imipenem, and overproduced the efflux system TtgABC
- id: PMID:32973714
title: Resistance to Bipyridyls Mediated by the TtgABC Efflux System in Pseudomonas
putida KT2440.
findings:
- statement: ttgB deletion makes KT2440 susceptible to bipyridyl compounds and
caerulomycin A
supporting_text: the deletion of ttgB made the strain susceptible to 2,2'-bipyridyl
and natural bipyridyl derivatives such as caerulomycin A, indicating that
TtgABC is required for detoxification of compounds of the bipyridyl family
- statement: Addition of copper restores growth of ttgB mutant
supporting_text: the addition of copper restores the growth of the ttgB mutant
and the production of pyoverdine
- id: file:PSEPK/ttgB/ttgB-deep-research-falcon.md
title: Deep research on ttgB in Pseudomonas putida KT2440
findings:
- statement: TtgB is the inner-membrane RND transporter of the TtgABC tripartite
efflux pump
supporting_text: TtgA (PP_1386) is the periplasmic adaptor/MFP, TtgB (PP_1385;
Q88N31) is the inner-membrane RND transporter/IMP, and TtgC (PP_1384) is
the outer-membrane channel component