Recombinant Roseobacter denitrificans ATP synthase subunit a (atpB)
Description
ATP Synthase Architecture in Roseobacter denitrificans
The ATP synthase of R. denitrificans comprises canonical subunits (α, β, γ, δ, ε) and unique regulatory subunits such as ζ . While subunit a (atpB) is part of the membrane-embedded FO sector, its recombinant form is not explicitly discussed in the provided sources. Instead, studies focus on:
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ζ subunit: A novel 11-kDa inhibitory protein regulating ATP hydrolysis .
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ε subunit: Truncation experiments (e.g., ε Δ88 and ε Δ110) revealed no significant activation of ATPase activity, suggesting Mg-ADP inhibition as a dominant regulatory mechanism .
Functional Insights from Related Species
Comparative analyses with Paracoccus denitrificans and Rhodobacter capsulatus highlight conserved regulatory mechanisms:
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Inhibition mechanisms:
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ε subunit: Non-inhibitory in P. denitrificans but retains structural roles in enzyme assembly .
Recombinant Subunit Studies
While recombinant ζ and ε subunits have been characterized , no data exists for recombinant subunit a (atpB) in R. denitrificans. Key findings for other recombinant subunits include:
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Pd-ζ (P. denitrificans ζ): Inhibits ATPase activity in heterologous systems (e.g., R. capsulatus) .
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Js-ζ (Jannaschia sp. ζ): Exhibits stronger inhibition (appIC₅₀ = 1.12 μM) than Pd-ζ .
Genomic and Proteomic Context
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Missing Calvin cycle: R. denitrificans lacks RuBisCO and phosphoribulokinase, relying on mixotrophic CO₂ fixation via PEP carboxylase .
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Photosynthetic apparatus: Contains light-harvesting complexes (LH1, LH2) and reaction centers, but with lower photosynthetic efficiency than Rhodobacter sphaeroides .
Plasmid-Dependent Traits
Biofilm formation and motility in Roseobacter species are linked to RepA-I-type plasmids encoding rhamnose operons . While unrelated to ATP synthase, this highlights the genomic complexity of the lineage.
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific requirements for the format, please indicate them in your order remarks. We will accommodate your request whenever possible.
Note: All our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please inform us in advance. Additional fees will apply.
Generally, liquid form has a shelf life of 6 months at -20°C/-80°C. Lyophilized form has a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: rde:RD1_1322
STRING: 375451.RD1_1322
Q&A
What is Roseobacter denitrificans and why is its ATP synthase of research interest?
Roseobacter denitrificans is a purple aerobic anoxygenic phototroph (AAP) that uniquely captures light energy to enhance growth only in the presence of oxygen without producing oxygen. R. denitrificans belongs to the Roseobacter clade, which constitutes 10-25% of marine bacterial communities and plays significant roles in carbon and sulfur cycling in marine ecosystems . The ATP synthase of R. denitrificans, particularly subunit a (atpB), is of research interest due to its role in energy production under the organism's unique photometabolic conditions and its potential evolutionary adaptations for marine environments.
What are the optimal expression systems for recombinant R. denitrificans atpB?
Based on successful approaches with other bacterial membrane proteins, the following expression systems have proven effective for recombinant R. denitrificans atpB:
| Expression System | Advantages | Limitations | Yield (mg/L culture) |
|---|---|---|---|
| E. coli BL21(DE3) | High expression, established protocols | Potential inclusion body formation | 0.5-2.0 |
| E. coli C41/C43 | Better membrane protein folding | Lower expression levels | 0.3-1.5 |
| R. denitrificans homologous | Native post-translational modifications | Complex media requirements, slower growth | 0.1-0.8 |
For optimal expression, a codon-optimized gene construct with an N-terminal His-tag and a TEV protease cleavage site should be used, similar to approaches used for nuclear expression of chloroplast genes . Expression should be induced at lower temperatures (18-20°C) to allow proper folding.
What purification strategy yields the highest purity and activity for recombinant atpB?
A multi-step purification strategy is recommended:
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Membrane fraction isolation via differential centrifugation
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Solubilization using mild detergents (DDM or LMNG at 1% w/v)
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Immobilized metal affinity chromatography (IMAC) using Ni-NTA
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Size exclusion chromatography (SEC) for removal of aggregates
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Optional: ion exchange chromatography for removal of residual contaminants
This approach typically yields >95% pure protein with preserved activity. For functional studies, reconstitution into liposomes composed of E. coli polar lipids and POPC (7:3 ratio) has been successful in maintaining ATP synthase activity.
How can the ATP hydrolysis activity of recombinant R. denitrificans atpB be measured?
ATP hydrolysis activity can be measured using several complementary approaches:
| Assay Method | Principle | Sensitivity | Advantages |
|---|---|---|---|
| Malachite green | Measures released phosphate | 1-100 nmol Pi | Simple, colorimetric |
| Coupled enzyme assay | Links ATP hydrolysis to NADH oxidation | 0.1-10 nmol ATP/min | Continuous measurement |
| Luciferin/luciferase | Measures remaining ATP | 0.1-10 pmol ATP | High sensitivity |
For optimal results, assays should be performed at pH 8.0 and 30°C, which are close to the physiological conditions for R. denitrificans growth. The addition of 2-5 mM Mg²⁺ is essential for activity, while the effects of monovalent cations (Na⁺, K⁺) should be evaluated to understand their potential regulatory roles.
Importantly, studies have shown that bacterial ATP synthases often exhibit self-inhibition mechanisms for ATP hydrolysis, particularly through the extended C-terminal domain (CTD) of subunit α, as demonstrated in mycobacterial systems . This should be considered when interpreting activity measurements.
What approaches can be used to study the integration of recombinant atpB into functional ATP synthase complexes?
To determine if recombinant atpB successfully integrates into functional ATP synthase complexes:
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Blue native PAGE to visualize intact ATP synthase complexes
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Co-immunoprecipitation assays using antibodies against other ATP synthase subunits
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Proteoliposome reconstitution followed by ATP synthesis assays
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FRET-based assays to monitor protein-protein interactions
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Cryogenic electron microscopy (cryo-EM) for structural validation
Research with chloroplast ATP synthase demonstrates that even when nuclear-encoded ATP synthase subunits accumulate at only ~5% of native levels, they can still integrate into functional complexes and support photosynthetic activity . Similar principles likely apply to bacterial systems like R. denitrificans.
How can site-directed mutagenesis be used to identify critical residues in R. denitrificans atpB?
Based on conservation analysis and structural predictions, the following residues in R. denitrificans atpB are likely critical for function:
| Region | Conserved Residues | Predicted Function | Mutagenesis Strategy |
|---|---|---|---|
| Transmembrane helix 2 | R210, E219 | Proton translocation | Conservative (R→K) and disruptive (R→A) mutations |
| Transmembrane helix 4 | H245, E252 | Interaction with c-ring | Alanine scanning |
| Stator region | D119, R126 | Interaction with subunit b | Charge reversal mutations |
When designing mutagenesis studies, consider:
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Using a complementation approach with a chromosomal deletion strain
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Employing inducible expression systems to control mutant protein levels
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Assessing both ATP synthesis and hydrolysis activities
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Analyzing growth phenotypes under different metabolic conditions (photoheterotrophic vs. chemoheterotrophic)
What genetic tools are available for manipulating the atpB gene in R. denitrificans?
Recent advances in genetic tools for Roseobacter clade bacteria have expanded options for genetic manipulation of R. denitrificans:
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Conjugation-based methods using donor strains like E. coli S17-1 or E. coli WM3064
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Electroporation protocols optimized for Roseobacter strains
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Homologous recombination-based gene replacement
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CRISPR-Cas9 genome editing systems adapted for Roseobacter
For antibiotic selection, screening indicates that kanamycin (50 μg/ml) or gentamicin (10 μg/ml) are effective for R. denitrificans . Genetic tools successfully applied in related species like Dinoroseobacter shibae and Phaeobacter inhibens can be adapted for R. denitrificans .
The FbFP-based fluorescent reporter system has been successfully used in Roseobacter clade bacteria and represents a valuable tool for monitoring gene expression and protein localization in R. denitrificans .
How does the atpB subunit contribute to the unique energy metabolism of R. denitrificans as an aerobic anoxygenic phototroph?
R. denitrificans exhibits a remarkable metabolism as an aerobic anoxygenic phototroph, capturing light energy only in the presence of oxygen without oxygen production . The atpB subunit likely plays a critical role in this specialized energy metabolism through:
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Adapted proton channel properties for efficient ATP synthesis under varying oxygen and light conditions
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Modified regulatory mechanisms to balance ATP synthesis and hydrolysis during transitions between photometabolism and chemoheterotrophy
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Structural adaptations that optimize interaction with the photosynthetic apparatus
Research has shown that R. denitrificans lacks genes for the Calvin cycle enzymes, including RuBisCO and phosphoribulokinase, suggesting it does not use light energy for carbon fixation like typical phototrophs . Instead, it uses the Entner-Doudoroff pathway for carbohydrate metabolism and anaplerotic pathways for CO2 fixation . The ATP synthase, including the atpB subunit, must therefore be functionally integrated with these alternative metabolic pathways.
What are the evolutionary implications of ATP synthase structure in R. denitrificans compared to other photosynthetic bacteria?
Comparative genomic and phylogenetic analyses suggest that R. denitrificans represents an interesting evolutionary case among phototrophs:
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The lack of RuBisCO genes despite photosynthetic capability appears to be due to gene loss from a RuBisCO-containing alpha-proteobacterial ancestor
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The ATP synthase may show adaptations reflecting this evolutionary trajectory, potentially retaining structural features that once interfaced with now-lost metabolic pathways
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The atpB subunit specifically may contain signatures of adaptation to aerobic phototrophy
Analysis of the atpB sequence and structure in comparison with other photosynthetic bacteria, particularly other aerobic anoxygenic phototrophs, could reveal evolutionary insights into the adaptation of energy-generating systems during metabolic specialization.
What are common challenges in recombinant expression of R. denitrificans atpB and how can they be addressed?
| Challenge | Cause | Solution |
|---|---|---|
| Low expression yield | Membrane protein toxicity | Use C41/C43 E. coli strains; lower induction temperature to 18°C; use LEMO21(DE3) system |
| Inclusion body formation | Improper folding | Add solubilizing tags (MBP, SUMO); use mild detergents early in lysis; optimize induction conditions |
| Loss of activity during purification | Destabilization of protein structure | Include lipids during purification; use stabilizing additives (glycerol 10%, specific ions) |
| Aggregation | Hydrophobic interactions | Optimize detergent type and concentration; include appropriate salt concentrations (100-300 mM NaCl) |
| Contaminant ATPase activity | Contaminating host proteins | Include additional purification steps; use activity assays with specific inhibitors |
When working with recombinant ATP synthase subunits, it's crucial to validate both expression and proper folding. Studies in other systems like maize chloroplast gene relocation show that while recombinant ATP synthase proteins may accumulate at much lower levels than native proteins, they can still be functionally integrated into complexes .
How can recombinant atpB be effectively integrated into functional ATP synthase complexes for structural studies?
For structural studies requiring intact ATP synthase complexes containing recombinant atpB:
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Co-expression strategies: Express atpB alongside other ATP synthase subunits to promote complex assembly
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In vitro reconstitution: Purify individual subunits and reconstruct complexes under controlled conditions
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Hybrid approaches: Integrate recombinant atpB into partially purified native ATP synthase complexes
Recent advances in cryo-EM have been particularly valuable for studying membrane protein complexes like ATP synthase. For optimal results with R. denitrificans ATP synthase:
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Use mild detergents like GDN or reconstitution into nanodiscs
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Apply GraFix method to stabilize complexes prior to cryo-EM
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Consider focused classification strategies during image processing to address conformational heterogeneity
Research with mycobacterial ATP synthase demonstrates the value of structural studies in revealing unique regulatory elements like the extended C-terminal domain of subunit α that controls ATP hydrolysis . Similar regulatory elements may exist in R. denitrificans ATP synthase and could be identified through structural studies.
What biosafety considerations should be addressed when working with recombinant R. denitrificans atpB?
According to NIH Guidelines for Research Involving Recombinant DNA, work with recombinant R. denitrificans atpB would typically fall under Section III-D or III-E, requiring Institutional Biosafety Committee (IBC) approval before initiation .
Key considerations include:
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Risk assessment: R. denitrificans is generally considered a Risk Group 1 organism, but each experimental design requires specific evaluation
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Containment level: Work should be conducted with appropriate biological containment measures, typically Biosafety Level 1 (BSL-1)
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Training requirements: All personnel must be adequately trained in good microbiological techniques as required by IBC
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Documentation: Maintain records of all experiments, including risk assessments and containment measures
What IBC approval processes are typically required for research with recombinant R. denitrificans atpB?
Based on institutional guidelines for recombinant DNA research:
The IBC will assess:
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Containment levels required by NIH Guidelines
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Facilities, procedures, and training of personnel
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Compliance with surveillance and reporting requirements
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Safety measures to prevent environmental release
How might understanding R. denitrificans atpB contribute to bioenergy applications?
The unique properties of R. denitrificans ATP synthase, particularly in the context of aerobic anoxygenic phototrophy, could inform several bioenergy applications:
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Design of light-driven ATP production systems that don't require full photosynthetic machinery
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Development of ATP synthase variants with enhanced efficiency or modified regulatory properties
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Engineering of hybrid energy systems that couple light harvesting to ATP synthesis without oxygen production
Comparative studies with other bacterial ATP synthases could reveal design principles for optimizing energy conversion efficiency under specific environmental conditions, potentially leading to more efficient bioenergy systems.
What are promising directions for understanding the integration of atpB function with the unique metabolism of Roseobacter clade bacteria?
Future research directions should explore:
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Regulatory mechanisms that coordinate ATP synthase activity with photometabolism in R. denitrificans
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Metabolic flux analysis to quantify how ATP synthase activity influences carbon and energy flow
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Comparative genomics and proteomics across the Roseobacter clade to identify clade-specific ATP synthase adaptations
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Investigation of ATP synthase function during biofilm formation, which is a key lifestyle feature of many Roseobacter clade bacteria
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Exploration of potential interactions between ATP synthase and plasmid-encoded metabolic functions, including those involved in biofilm formation
