Recombinant Pelagibacter ubique ATP synthase subunit b (atpF)
Description
Role in Proton Translocation
Subunit b is part of the peripheral stalk that links the F₀ proton channel (subunits a and c) to the F₁ catalytic domain (subunits α, β, γ, δ, ε). In bacterial ATP synthases, subunit b stabilizes the complex and facilitates rotational energy coupling during ATP synthesis or hydrolysis .
Homology and Sequence Features
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Domain Architecture: Subunit b contains hydrophobic transmembrane regions and a soluble domain that interacts with F₁ subunits .
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Interaction Network: P. ubique atpF likely interacts with subunits a (atpB), c (atpE), and F₁ components (α, β, γ) based on conserved bacterial ATP synthase organization .
Interaction Network and Functional Partners
Genomic Organization
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Gene Cluster: The atpF gene is part of the atpBEFHAGDC operon in P. ubique, as observed in other α-proteobacteria .
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Regulatory Features: SAM riboswitches upstream of atpF homologs in P. ubique suggest transcriptional regulation under sulfur limitation, though direct evidence for atpF is lacking .
Metabolic Linkages
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Energy Production: P. ubique relies on ATP synthase for energy generation, particularly during nutrient-limited conditions, where oxidative stress proteins (e.g., OsmC) are upregulated .
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One-Carbon Metabolism: P. ubique oxidizes methylated compounds (e.g., DMSP) to produce ATP, indicating a connection between substrate oxidation and ATP synthase activity .
Recombinant Production and Experimental Challenges
While no direct reports of P. ubique atpF recombinant expression exist, studies on homologous α-proteobacterial ATP synthases provide a framework:
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Cloning and Expression: Subunits from Rhodobacter capsulatus and Paracoccus denitrificans have been expressed in E. coli using pET plasmids, suggesting feasibility for P. ubique atpF .
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Purification: Solubility issues may require fusion tags (e.g., His-tag) or co-expression with chaperones .
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Functional Assays: ATP hydrolysis or proton translocation assays would validate subunit b’s role in the intact enzyme .
Comparative Analysis with Model Organisms
Key Research Gaps and Future Directions
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Structural Resolution: Cryo-EM studies of P. ubique ATP synthase are needed to confirm subunit b’s conformational states during catalysis .
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Functional Validation: Recombinant atpF should be tested for binding to F₀ (subunits a/c) and F₁ (subunits α/β/γ) using co-affinity chromatography .
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Ecological Relevance: Investigate how subunit b’s activity supports P. ubique’s survival in low-nutrient marine environments .
Product Specs
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Target Background
KEGG: pub:SAR11_0118
STRING: 335992.SAR11_0118
Q&A
How does ATP synthase subunit b function in Pelagibacter ubique?
ATP synthase subunit b serves as a critical component of the peripheral stalk in the F₀ sector of the ATP synthase complex. Its primary function is to connect the membrane-embedded F₀ sector with the catalytic F₁ sector, thereby helping maintain the structural integrity required for ATP synthesis . In Pelagibacter ubique, this function is particularly important because this organism has evolved streamlined metabolic systems through selection pressure .
Unlike the more conserved F₁ moiety components, the F₀ subcomplex (including subunit b) shows greater variation across species . In Pelagibacter ubique, the ATP synthase complex is essential for energy metabolism and adaptation to nutrient-limited marine environments . Studies suggest that when subjected to energy limitation, P. ubique may utilize light through proteorhodopsin to supplement its energy needs, affecting ATP production pathways that involve ATP synthase .
What are the optimal storage and handling conditions for recombinant Pelagibacter ubique atpF protein?
For optimal storage and handling of recombinant Pelagibacter ubique ATP synthase subunit b:
| Parameter | Recommended Condition | Notes |
|---|---|---|
| Long-term storage | -20°C to -80°C | Aliquoting necessary for multiple use |
| Short-term storage | 4°C | Working aliquots stable for up to one week |
| Storage buffer | Tris/PBS-based buffer with 50% glycerol, pH 8.0 | Some preparations include 6% Trehalose |
| Form | Typically lyophilized powder | Requires reconstitution before use |
| Reconstitution | Deionized sterile water (0.1-1.0 mg/mL) | Addition of 5-50% glycerol recommended for stability |
| Critical handling note | Avoid repeated freeze-thaw cycles | Significantly reduces protein activity |
When reconstituting the protein, it is recommended to briefly centrifuge the vial prior to opening to bring contents to the bottom . The high glycerol concentration (typically 50%) helps maintain protein stability during freeze-thaw processes, though minimizing such cycles is strongly advised .
What purification methods are most effective for isolating recombinant Pelagibacter ubique atpF protein?
Recombinant P. ubique ATP synthase subunit b is typically expressed with a His-tag and purified using affinity chromatography methods . The most effective purification protocol involves:
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Expression system selection: E. coli is the preferred heterologous expression system for recombinant P. ubique proteins .
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Affinity purification: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resins is most effective for His-tagged proteins, with elution performed using imidazole gradients.
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Quality assessment: SDS-PAGE analysis confirms purity (typically >90% as determined by densitometry) .
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Buffer exchange: Dialysis or gel filtration into a storage buffer containing Tris/PBS with high glycerol content (50%) stabilizes the protein .
The challenging nature of working with membrane-associated proteins like atpF often necessitates optimization of detergent conditions during purification. Researchers should consider that P. ubique proteins may require special handling due to the organism's adaptation to oligotrophic marine environments .
How does Pelagibacter ubique atpF differ structurally and functionally from homologous proteins in other organisms?
Pelagibacter ubique ATP synthase subunit b shows significant evolutionary divergence compared to homologous proteins in other organisms:
Functionally, while the core role of connecting F₀ and F₁ sectors is conserved, the regulatory mechanisms appear to differ. Studies of the T. brucei ATP synthase complex reveal that significantly diverged subunit b proteins can maintain functional ATP synthase complexes despite structural differences . The P. ubique atpF lacks certain regulatory domains found in other organisms, suggesting a simplified regulatory mechanism consistent with its streamlined metabolism .
How can recombinant Pelagibacter ubique atpF be utilized to study energy metabolism in oligotrophic marine environments?
Recombinant P. ubique atpF provides a valuable tool for investigating energy metabolism adaptations in the world's most abundant marine bacteria. Advanced applications include:
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Structure-function relationship studies: Using site-directed mutagenesis of the recombinant protein to determine critical residues for function in low-energy environments. This approach can reveal how P. ubique has adapted its ATP synthase for efficient operation in oligotrophic conditions .
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Reconstitution experiments: Incorporating purified recombinant atpF into liposomes or nanodiscs to study its functional properties in controlled membrane environments. This can help determine how the streamlined structure affects proton translocation efficiency and ATP production rates .
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Protein-protein interaction mapping: Using tagged recombinant atpF to identify interaction partners through pull-down assays or cross-linking studies. This approach has revealed that divergent forms of subunit b can maintain functional associations with other ATP synthase components despite structural differences .
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Ecological simulation experiments: Employing recombinant atpF in experiments that simulate changing ocean conditions (nutrient limitation, acidification) to understand how energy metabolism in dominant marine bacteria may respond to environmental changes .
The methodological challenge lies in maintaining the native conformation of the protein during experimental manipulations, as membrane proteins often require specific lipid environments for proper folding and function.
What techniques are most effective for studying the integration of atpF into the ATP synthase complex?
Investigating the assembly and integration of atpF into functional ATP synthase complexes requires specialized techniques:
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Blue Native PAGE (BN-PAGE): This technique has successfully revealed the impact of subunit depletion on ATP synthase assembly in related systems. For example, knockdown of a putative subunit b analog in Trypanosoma brucei showed that "the levels of F₁F₀ dimer and monomer were steadily decreased upon depletion to less than around 40% of the levels seen in uninduced cells" . This approach can be adapted to study P. ubique ATP synthase assembly.
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In vitro reconstitution: Recombinant atpF can be combined with other purified ATP synthase subunits to study complex assembly in controlled conditions. This approach has revealed that "knockdown of Tb927.8.3070 preferentially reduces the abundance of F₀ ATP synthase subunits but not F₁ subunits" , demonstrating the importance of proper subunit b integration for F₀ assembly.
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Cryo-electron microscopy: Advanced structural studies using cryo-EM can reveal the precise positioning and interactions of atpF within the ATP synthase complex. Recent structural work on trypanosomal ATP synthase demonstrates the power of this approach for understanding divergent ATP synthase architectures .
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SILAC-MS approaches: Stable isotope labeling with amino acids in cell culture combined with mass spectrometry has successfully identified changes in ATP synthase subunit abundance following depletion of specific components . This approach could be adapted to study P. ubique ATP synthase assembly dynamics.
How does the structure and function of Pelagibacter ubique atpF reflect evolutionary adaptation to marine environments?
The structure and function of P. ubique atpF reflects the evolutionary pressure of genome streamlining that characterizes the SAR11 clade:
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Genome minimization: The relatively compact 179-amino acid structure of P. ubique atpF represents evolutionary selection for genome reduction. As noted in research on the SAR11 clade, "Most interestingly, the evolutionary pressure for reduction of genome size" has shaped core metabolic systems including ATP synthase components.
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Metabolic efficiency: The streamlined structure likely represents optimization for function in low-nutrient environments. P. ubique thrives in oligotrophic ocean regions through "an increased dependence on organosulfur compounds produced by other members of the plankton community" , suggesting coordinated evolution of simplified but efficient metabolic systems.
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Environmental resilience: The structural features of atpF contribute to the remarkable ecological success of the organism. Research indicates that "One reason for the success of Pelagibacteraceae might be its ability to thrive under nutrient-limited conditions, like those in the open ocean, possibly due to adaptive genome streamlining" .
This evolutionary adaptation extends beyond individual proteins to entire metabolic pathways, where "many SAR11 metabolic systems have been reduced in complexity by streamlining selection, resulting in noncanonical pathways with fewer enzymatic reactions" .
What is the significance of ATP synthase function in the ecological dominance of the SAR11 clade?
The ATP synthase complex, including atpF, plays a crucial role in the ecological success of Pelagibacteraceae/SAR11 bacteria:
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Bioenergetic efficiency: The streamlined but functional ATP synthase of P. ubique enables efficient energy harvesting in low-nutrient environments. This is particularly important as "the SAR11 clade, is one of the most abundant bacterial clades in the world's oceans" , representing "approximately one quarter of all rRNA genes identified in clone libraries from marine environments" .
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Metabolic flexibility: Under energy limitation, P. ubique can employ alternative strategies that involve ATP synthase. Research demonstrates that "light causes dramatic changes in physiology and gene expression in Cand. P. ubique" , with evidence that proteorhodopsin-driven proton pumping can supplement ATP production via ATP synthase.
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Stress response integration: ATP production is central to cellular responses to various stressors. Studies show that P. ubique has evolved streamlined but effective responses to nitrogen limitation and sulfur limitation , both of which interface with energy metabolism.
The ATP synthase complex thus represents a crucial nexus in the cell's adaptations to challenging marine environments, contributing to the remarkable finding that SAR11 bacteria constitute "one third of the prokaryotic cells in the surface waters of the northwestern Sargasso Sea" .
What are the potential applications of studying atpF in understanding microbial adaptation to changing ocean conditions?
Research on P. ubique atpF has significant implications for understanding marine microbial responses to changing ocean conditions:
What methodological advances would enhance our understanding of ATP synthase function in uncultivated marine bacteria?
Several methodological advances could significantly improve research on ATP synthase in difficult-to-culture marine bacteria:
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Improved cultivation techniques: Development of specialized media and growth conditions that better replicate oligotrophic marine environments could enable laboratory studies of previously uncultivable bacteria with divergent ATP synthase structures.
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Single-cell approaches: Advances in single-cell proteomics and metabolomics could allow direct study of ATP synthase components and function in uncultivated bacteria from environmental samples.
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Metaproteomics with improved sensitivity: Enhanced methods for detecting and quantifying low-abundance membrane proteins in complex environmental samples would allow better characterization of ATP synthase diversity.
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Computational prediction tools: Advanced algorithms for predicting protein function from highly divergent sequences could help identify ATP synthase components in metagenomes that escape conventional homology searches, addressing challenges like those noted with trypanosomal ATP synthase where "no subunit b could be identified in T. brucei, the question of whether the trypanosomal version of the protein could be either further reduced in length or even be completely absent was raised" .
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Environmental transcriptomics: Improved methods for capturing and analyzing mRNA from environmental samples could reveal how ATP synthase expression varies across diverse conditions in natural settings.
