Recombinant Azorhizobium caulinodans ATP synthase subunit b/b' (atpG)

What is the genomic context of the atpG gene in Azorhizobium caulinodans?

The atpG gene in A. caulinodans is part of the ATP synthase operon, which is essential for energy production in this organism. A. caulinodans has a genome size of approximately 5.4 Mb, making it one of the smallest among sequenced rhizobia . Within this compact genome, genes involved in energy metabolism, including the ATP synthase complex, represent a significant portion of the genome (303 genes) . Based on transcriptomic analyses, many energy metabolism genes show differential expression patterns between free-living and symbiotic states, indicating their importance in adaptation to different environmental conditions .

What structural features characterize the recombinant atpG protein from A. caulinodans?

The ATP synthase b/b' subunit (atpG) in A. caulinodans functions as part of the peripheral stalk of the ATP synthase complex, connecting the F1 catalytic domain to the membrane-embedded F0 domain. The protein typically features an N-terminal transmembrane helix anchored in the membrane, followed by a predominantly alpha-helical structure that extends into the cytoplasm. In recombinant expression systems, researchers often exclude the transmembrane domain to improve solubility while maintaining the structural integrity of the cytoplasmic portion essential for functional studies. Sequence alignment with other bacterial b/b' subunits reveals conserved regions critical for interaction with other ATP synthase components, particularly the delta subunit of the F1 domain.

What are the optimal expression systems for producing recombinant A. caulinodans atpG protein?

For recombinant expression of A. caulinodans atpG, E. coli-based expression systems (particularly BL21(DE3) or its derivatives) are commonly employed. The gene sequence should be codon-optimized for E. coli expression and cloned into vectors containing strong inducible promoters such as T7 (pET series) or tac promoters. Expression conditions require careful optimization with the following recommended parameters:

• Induction at mid-log phase (OD600 of 0.6-0.8)

• IPTG concentration of 0.1-0.5 mM (lower concentrations often yield more soluble protein)

• Post-induction growth at lower temperatures (16-20°C) for 12-16 hours to maximize proper folding

For challenging expressions, specialized strains like Rosetta (providing rare tRNAs) or Arctic Express (containing cold-adapted chaperonins) may improve yields. Testing multiple fusion tags (His6, MBP, SUMO) is advisable to determine which provides optimal solubility and purification efficiency.

What purification strategy yields the highest purity recombinant atpG protein suitable for structural studies?

A multi-step purification approach is essential for obtaining high-purity recombinant atpG protein:

• Initial capture: Affinity chromatography using Ni-NTA for His-tagged constructs or amylose resin for MBP-fusion proteins

• Tag removal: Specific protease treatment (TEV, PreScission, or SUMO protease) followed by reverse affinity chromatography

• Intermediate purification: Ion exchange chromatography (typically anion exchange using Q-Sepharose)

• Polishing step: Size exclusion chromatography using Superdex 75 or 200 columns

Buffer optimization is critical throughout the process. A typical buffer composition includes:

• 20-50 mM Tris-HCl or HEPES (pH 7.5-8.0)

• 100-300 mM NaCl

• 5-10% glycerol to improve stability

• 1-5 mM DTT or 0.5-1 mM TCEP as reducing agents

For structural studies, conducting dynamic light scattering (DLS) after the final purification step is recommended to confirm sample monodispersity before proceeding to crystallization trials or cryo-EM sample preparation.

How can researchers overcome solubility challenges when expressing recombinant atpG protein?

The b/b' subunit of ATP synthase contains hydrophobic regions that can lead to aggregation during recombinant expression. Several strategies can address this challenge:

• Construct optimization: Create truncated constructs excluding the N-terminal transmembrane domain (typically the first 20-30 amino acids) while preserving the cytoplasmic domain

• Solubility-enhancing fusion partners: Utilize MBP, SUMO, or Trx fusion tags, which often improve folding and solubility

• Co-expression strategies: Co-express with interaction partners from the ATP synthase complex, particularly the delta subunit, to stabilize the protein

• Buffer optimization: Include mild detergents (0.05% DDM, 0.5-1% CHAPS, or 0.5-1% OG) in lysis and purification buffers

• Refolding protocols: If inclusion bodies form, develop a denaturation and refolding protocol using 8M urea or 6M guanidine-HCl with gradual dialysis to remove the denaturant

Systematic testing of these approaches, often in parallel, is necessary to determine the optimal conditions for obtaining soluble, functional atpG protein.

What methods are most effective for assessing the functionality of recombinant atpG protein?

Multiple complementary approaches can verify the functionality of purified recombinant atpG:

• Binding assays: Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to measure binding affinity with other ATP synthase subunits, particularly the delta and alpha subunits

• Structure verification: Circular dichroism (CD) spectroscopy to confirm proper secondary structure (predominantly alpha-helical)

• Reconstitution experiments: In vitro reconstitution with other purified subunits to assess complex formation, analyzed by size exclusion chromatography or native PAGE

• Complementation studies: Genetic complementation in atpG-deficient bacterial strains to restore ATP synthesis function

• In vitro ATP synthesis assays: If reconstituted into proteoliposomes with other ATP synthase components, measure ATP synthesis driven by artificially generated proton gradients

For quantitative assessment, comparing the kinetic parameters and binding affinities with those of the wild-type protein provides valuable information about the functional integrity of the recombinant protein.

How does atpG expression correlate with nitrogen fixation capacity in A. caulinodans?

ATP production is critically important for the energy-intensive process of nitrogen fixation. In A. caulinodans, nitrogen fixation occurs both in the free-living state and during symbiosis with Sesbania rostrata . The energy requirements differ significantly between these states, suggesting potential regulatory differences in ATP synthase components.

Transcriptomic analyses of A. caulinodans under different conditions have shown significant changes in the expression patterns of energy metabolism genes between free-living cells and bacteroids . While specific data on atpG expression wasn't provided in the search results, the differential expression of genes involved in energy metabolism suggests that ATP synthase regulation is likely important for adapting to the high energy demands of nitrogen fixation.

The efficiency of nitrogen fixation, measured by acetylene reduction assays, could be correlated with ATP synthase activity and specifically atpG expression levels to establish the relationship between energy production capacity and nitrogen fixation performance.

How does atpG function integrate with nitrogen fixation and ammonia assimilation in A. caulinodans?

ATP synthase, including the atpG subunit, plays a crucial role in providing energy for nitrogen fixation in A. caulinodans. The integration between energy production and nitrogen metabolism involves several interconnected systems:

• Energy supply for nitrogenase: The ATP produced by ATP synthase fuels the energy-intensive nitrogen fixation process. Nitrogenase requires approximately 16 ATP molecules to reduce one N₂ molecule to two NH₃ molecules.

• Energy for ammonia assimilation: After nitrogen fixation, A. caulinodans assimilates the produced ammonia primarily through the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway, which also requires ATP.

• Regulation coordination: The expression and activity of ATP synthase components appear to be coordinated with nitrogen metabolism. In engineered strains where ammonia assimilation is impaired (such as in PII-deficient mutants), the energy generated can be redirected, resulting in ammonia excretion .

Transcriptomic data shows that under nitrogen-fixing conditions, both nitrogen fixation genes and energy metabolism genes undergo significant expression changes , suggesting coordinated regulation to balance energy production with nitrogen fixation demands.

What is the relationship between ATP synthase activity and ammonia excretion in engineered A. caulinodans strains?

In PII-deficient mutants (ΔglnB ΔglnK) or strains with modified glutamine synthetase adenylylation control, A. caulinodans can be engineered to excrete fixed nitrogen as ammonia . This occurs because:

• When glutamine synthetase (GS) activity is impaired through modified regulation, ammonia assimilation is reduced

• Continued ATP production by ATP synthase supports ongoing nitrogen fixation

• With reduced assimilation capacity but maintained fixation, excess ammonia is excreted

This relationship demonstrates that ATP synthase activity (including the atpG component) is a critical determinant of nitrogen fixation capacity, and when uncoupled from assimilation through genetic engineering, can support ammonia excretion - a trait of interest for agricultural applications .

The table below summarizes the relationship between ATP synthase, nitrogen fixation, and ammonia fate in different A. caulinodans strains:

How can researchers manipulate atpG expression to influence energy production for nitrogen fixation studies?

Researchers can employ several strategies to modulate atpG expression and consequently ATP synthase activity for nitrogen fixation studies:

• Promoter replacement: Replacing the native atpG promoter with inducible or constitutive promoters of varying strengths can alter expression levels. This approach allows for titration of ATP synthase activity and observation of its effects on nitrogen fixation.

• Post-translational regulation: Creating modified versions of atpG with altered regulatory sites can influence the assembly or activity of ATP synthase. This might include modifications to interaction domains or phosphorylation sites.

• Co-expression modifications: Altering the stoichiometry of ATP synthase components by overexpressing or underexpressing specific subunits can affect the formation of functional complexes.

• Integration with nitrogen regulation circuits: Engineering expression systems where atpG is under control of nitrogen-responsive regulatory elements can create feedback loops between energy production and nitrogen status. The example of placing uAT (unidirectional adenylyltransferase) expression under control of NifA and subsequently rhizopine control demonstrates the feasibility of such approaches for nitrogen metabolism components .

For experimental design, these modifications should be paired with comprehensive phenotypic assays measuring ATP production, nitrogen fixation rates, and ammonia excretion to understand the resulting metabolic changes.

How can recombinant atpG be used to study ATP synthase assembly in A. caulinodans?

Recombinant atpG can serve as a powerful tool for investigating ATP synthase assembly in A. caulinodans through several sophisticated approaches:

• Fluorescent protein fusions: Creating atpG-GFP fusion proteins allows real-time visualization of ATP synthase assembly and localization using confocal microscopy. This approach can reveal spatiotemporal dynamics during transitions between free-living and symbiotic states.

• Pull-down interaction studies: Using recombinant atpG as bait in pull-down assays followed by mass spectrometry can identify interaction partners and assembly intermediates. This reveals the assembly sequence and potential auxiliary factors involved.

• Crosslinking mass spectrometry: Employing chemical crosslinking followed by mass spectrometry (XL-MS) with purified recombinant atpG and its interaction partners can map specific interaction interfaces at amino acid resolution.

• In vitro reconstitution: Systematic assembly studies using purified components, including recombinant atpG, can establish the order and kinetics of complex formation, as well as the minimum components required for functional assemblies.

• Cryo-electron microscopy: Using recombinant components to reconstitute ATP synthase complexes at different assembly stages for structural determination by cryo-EM, revealing conformational changes during assembly.

These approaches can answer fundamental questions about ATP synthase assembly that may differ in A. caulinodans compared to other bacteria, particularly regarding adaptations related to its dual lifestyle as both free-living and symbiotic nitrogen-fixer.

What role might atpG play in the adaptation of A. caulinodans to different oxygen concentrations during nitrogen fixation?

A. caulinodans possesses the remarkable ability to fix nitrogen at relatively high oxygen concentrations (up to 12 μM) compared to most nitrogen-fixing bacteria. The ATP synthase complex, including atpG, likely plays a critical role in this adaptation through several mechanisms:

• Energy supply for respiratory protection: A. caulinodans employs respiratory protection mechanisms to shield nitrogenase from oxygen. These protection systems consume oxygen but require significant energy input, which is provided by ATP synthase.

• Structural adaptations: The b/b' subunit (atpG) might contain structural modifications that enhance ATP synthase performance under varying oxygen conditions, potentially through altered interactions with other subunits or changed sensitivity to the proton motive force.

• Regulatory integration: The expression and activity of ATP synthase components, including atpG, may be integrated with oxygen-sensing regulatory networks, allowing for coordinated response to changing oxygen levels.

Research approaches to investigate this role include:

• Comparative sequence and structural analysis of atpG across rhizobia with different oxygen tolerances

• Creating atpG variants and testing their functionality under varying oxygen concentrations

• Transcriptomic and proteomic analysis of ATP synthase components across oxygen gradients

• Metabolic flux analysis to quantify energy production and consumption under different oxygen regimes

Understanding these adaptations could provide insights applicable to engineering other nitrogen-fixing bacteria for improved performance in agricultural contexts.

How can structural studies of recombinant atpG contribute to understanding the energetics of nitrogen fixation in A. caulinodans?

Structural studies of recombinant atpG can provide critical insights into the energetics of nitrogen fixation in A. caulinodans:

• Structural determinants of efficiency: High-resolution structures of atpG and its interactions within the ATP synthase complex can reveal specific adaptations that may enhance ATP synthesis efficiency in A. caulinodans. This is particularly relevant given the high ATP demand for nitrogen fixation.

• Regulatory binding sites: Structural studies can identify potential regulatory binding sites on atpG that might respond to metabolic signals related to nitrogen status, allowing for integration of energy production with nitrogen metabolism.

• Conformational dynamics: Using techniques like hydrogen-deuterium exchange mass spectrometry (HDX-MS) or single-molecule FRET with recombinant atpG can reveal conformational changes during the catalytic cycle, potentially identifying unique features that support the energetics of nitrogen fixation.

• Comparative structural biology: Comparing the structure of A. caulinodans atpG with homologs from non-nitrogen-fixing bacteria can highlight adaptations specific to supporting nitrogen fixation.

• Structure-guided mutagenesis: Insights from structural studies enable targeted mutagenesis to test hypotheses about structure-function relationships, with phenotypic consequences for nitrogen fixation capacity.

The structural information obtained can guide rational engineering of ATP synthase to enhance energy production for nitrogen fixation, potentially supporting the development of improved biofertilizers or engineered nitrogen-fixing systems.

What are common pitfalls when working with recombinant A. caulinodans atpG protein and how can they be addressed?

Researchers commonly encounter several challenges when working with recombinant atpG protein:

• Expression toxicity: Overexpression of membrane-associated proteins like atpG can be toxic to host cells.
  Solution: Use tightly controlled expression systems, lower induction levels (0.1 mM IPTG or less), and consider expression hosts with enhanced membrane protein production capacity such as C41(DE3) or C43(DE3).

• Protein aggregation: The hydrophobic regions of atpG often lead to aggregation during expression or purification.
  Solution: Optimize solubilization conditions by testing different detergents (DDM, LDAO, or CHAPS) or employ fusion partners that enhance solubility.

• Loss of function during purification: The protein may lose its native conformation during purification steps.
  Solution: Include stabilizing agents like glycerol (10-15%) and specific lipids (0.1-0.5 mg/ml) that mimic the native membrane environment throughout the purification process.

• Heterogeneous protein populations: Purified samples may contain multiple conformational states or degradation products.
  Solution: Implement additional purification steps, particularly size exclusion chromatography, and use DLS to confirm sample homogeneity before functional or structural studies.

• Inconsistent activity measurements: Functional assays may yield variable results.
  Solution: Standardize protein handling conditions, include appropriate controls, and consider co-reconstitution with interacting partners to stabilize the functional state.

By anticipating these challenges and implementing mitigation strategies, researchers can significantly improve their success rates when working with this challenging but important protein.

How can researchers optimize conditions for crystallization or cryo-EM studies of recombinant atpG?

Obtaining high-quality structural data for atpG requires careful optimization:

• Sample preparation for crystallization:

  • Achieve protein concentrations of 5-15 mg/ml in a buffer with minimal components (typically 20 mM Tris or HEPES, 100-150 mM NaCl)

  • Perform stability screening using thermal shift assays to identify optimal buffer conditions

  • Consider adding stabilizing lipids or forming complexes with interaction partners

  • Use commercial crystallization screens designed for membrane proteins or membrane-associated proteins

  • Implement vapor diffusion techniques (hanging or sitting drop) with various precipitants

• Optimization for cryo-EM:

  • Ensure sample homogeneity through glycerol gradient ultracentrifugation or GraFix methods

  • Test multiple grid types (Quantifoil, C-flat) and hole sizes

  • Optimize protein concentration (typically 0.5-3 mg/ml for cryo-EM)

  • Test various blotting times and conditions

  • Consider adding specific nanobodies or antibody fragments to increase particle size and provide fiducial markers

  • Use computational approaches to handle conformational heterogeneity during image processing

• General optimization strategies:

  • Perform limited proteolysis to identify stable fragments that may crystallize more readily

  • Consider inserting crystallization chaperones like T4 lysozyme or BRIL into flexible loops

  • For cryo-EM, crosslink the protein with mild crosslinkers (0.05-0.1% glutaraldehyde) to stabilize the structure

Successful structure determination typically requires systematically exploring these variables while maintaining rigorous quality control of the protein sample throughout the optimization process.

What strategies can address inconsistent results in functional assays of recombinant atpG?

Functional assay inconsistencies for recombinant atpG can arise from multiple sources. Here are evidence-based strategies to improve reproducibility:

• Protein quality verification:

  • Implement multi-angle light scattering (MALS) to confirm the oligomeric state

  • Use thermal shift assays to assess protein stability across buffer conditions

  • Verify secondary structure through circular dichroism before functional testing

  • Employ negative stain EM to visualize sample homogeneity

• Assay stabilization:

  • Standardize protein:lipid ratios when reconstituting into liposomes or nanodiscs

  • Carefully control temperature throughout experiments (±0.5°C)

  • Include internal standards and calibration curves in each experimental set

  • Validate buffer components for interference with assay readouts

• Advanced troubleshooting:

  • For binding assays, test multiple immobilization strategies and regeneration conditions

  • For activity assays, systematically vary component concentrations to identify rate-limiting factors

  • Implement technical replicates within experiments and biological replicates across independent protein preparations

  • Consider time-dependent effects by measuring activity at multiple time points

• Data analysis improvements:

  • Apply statistical methods appropriate for the data distribution

  • Use control experiments to establish baseline variability

  • Consider Bayesian analysis approaches for complex datasets

  • Implement blinded analysis when possible to reduce experimenter bias

How might synthetic biology approaches using recombinant atpG enhance nitrogen fixation capacity in A. caulinodans?

Synthetic biology offers promising approaches to enhance nitrogen fixation through ATP synthase engineering:

These approaches could significantly enhance nitrogen fixation efficiency and potentially support the development of improved biofertilizers or engineered nitrogen-fixing systems for sustainable agriculture.

What insights can comparative analysis of atpG across different nitrogen-fixing bacteria provide?

Comparative analysis of atpG across nitrogen-fixing bacteria can reveal evolutionary adaptations that optimize energy production for nitrogen fixation:

• Sequence conservation patterns: Identifying highly conserved residues across nitrogen-fixing bacteria that differ from non-nitrogen-fixers can highlight functionally important adaptations specific to supporting nitrogen fixation.

• Structural variations: Comparing predicted or determined structures of atpG from diverse nitrogen-fixing bacteria can reveal structural adaptations, particularly in regions that interact with other ATP synthase subunits or might respond to regulatory signals.

• Expression regulation comparison: Analyzing promoter regions and regulatory elements of atpG genes across species can reveal how expression control has evolved in different nitrogen-fixing contexts, from free-living diazotrophs to obligate symbionts.

• Co-evolution patterns: Detecting co-evolutionary relationships between atpG and other genes involved in nitrogen fixation can identify functional interactions and regulatory networks that have been preserved across evolutionary lineages.

• Adaptation to oxygen sensitivity: Comparing atpG sequences from microaerobic, aerobic, and anaerobic nitrogen-fixers can reveal adaptations related to oxygen tolerance, which is particularly relevant given A. caulinodans' ability to fix nitrogen at higher oxygen concentrations than most nitrogen-fixers .

This comparative approach can guide rational engineering of atpG for improved nitrogen fixation and provide fundamental insights into the co-evolution of energy production and nitrogen fixation systems.

How might the unique features of A. caulinodans atpG contribute to its dual lifestyle as both free-living and symbiotic nitrogen-fixer?

A. caulinodans is remarkable for its ability to fix nitrogen in both free-living and symbiotic states, a versatility that likely requires specialized adaptations in its energy production systems:

• Regulatory flexibility: The atpG protein may contain unique regulatory features that allow rapid adaptation to the different energetic demands of free-living versus symbiotic states. Transcriptomic data shows significant expression changes in energy metabolism genes between these states .

• Structural adaptations: The structure of atpG may include specific domains or interaction surfaces that optimize ATP synthase function under the distinct physiological conditions encountered in soil versus plant nodules.

• Integration with oxygen adaptation: A. caulinodans can fix nitrogen at higher oxygen concentrations (up to 12 μM) than most nitrogen-fixers, suggesting its ATP synthase components, including atpG, may have adaptations for function across broader oxygen ranges.

• Metabolic integration: The atpG protein might contain features that facilitate integration with the different carbon metabolism pathways utilized in free-living versus symbiotic states. The transcriptomic data indicated significant changes in acetone metabolism and carbon source utilization between these states .

• Signaling responsiveness: Unique features in atpG may allow ATP synthase to respond to plant-derived signals, facilitating the transition between free-living and symbiotic energy production modes.

Understanding these adaptations could provide insights for engineering other nitrogen-fixing bacteria with enhanced environmental flexibility or for developing improved biofertilizers with broader application ranges.

How does atpG expression data integrate with broader metabolic networks in A. caulinodans?

Understanding atpG expression in the context of broader metabolic networks requires integrating multiple data types:

• Transcriptomic integration: Whole-genome microarray data revealed that genes involved in energy metabolism show significant expression changes between free-living and symbiotic states in A. caulinodans . As shown in the data table below, a substantial portion of energy metabolism genes (over 15% of the 303 total energy metabolism genes) show differential expression:

• Metabolic pathway context: ATP synthase functions within a broader energetic network involving electron transport chain components, TCA cycle enzymes, and carbon metabolism pathways. Transcriptomic data showed that central intermediary metabolism genes (71 increased, 18 decreased) and energy metabolism genes (49 increased, 45 decreased) had significant expression changes in bacteroids compared to free-living cells .

• Regulatory network integration: The expression of atpG likely responds to multiple regulatory inputs, including oxygen levels, carbon source availability, and nitrogen status. These regulatory connections can be mapped using comparative genomics and transcriptomic data.

• Flux balance analysis: Integrating expression data with metabolic models allows prediction of ATP production rates under different conditions and how these rates support nitrogen fixation demands.

This integrative approach provides a systems-level understanding of how ATP synthase activity is coordinated with other metabolic processes to support the energy-intensive process of nitrogen fixation in different environmental contexts.

How can metabolomic and fluxomic data enhance our understanding of atpG function in nitrogen fixation?

Integration of metabolomic and fluxomic data provides deeper insights into the role of atpG in nitrogen fixation:

• Metabolic fingerprinting: Comparative metabolomics between wild-type and atpG-modified strains can reveal how alterations in ATP synthase affect broader metabolite pools. Key signatures include:

  • Changes in adenylate energy charge (ATP:ADP:AMP ratios)

  • Accumulation or depletion of TCA cycle intermediates

  • Alterations in nitrogen assimilation metabolites (glutamate, glutamine)

  • Shifts in redox cofactor levels (NADH/NAD+, NADPH/NADP+)

• Metabolic flux analysis: Using isotope-labeled tracers (13C, 15N) to track carbon and nitrogen flow through central metabolism can quantify how atpG modifications affect:

  • ATP synthesis rates in vivo

  • Carbon flux distribution between energy generation and biosynthesis

  • Nitrogen fixation rates and efficiency (ATP consumed per N2 fixed)

  • Ammonia assimilation versus excretion patterns

• Integration with transcriptomics: Correlating metabolic flux changes with transcriptomic responses reveals regulatory adaptations that compensate for altered ATP synthase function.

• Temporal dynamics: Time-course metabolomic and fluxomic analyses during transitions (e.g., initiation of nitrogen fixation) can reveal the dynamic role of ATP synthase in supporting changing energy demands.