Recombinant Agrobacterium tumefaciens ATP synthase subunit b/b' (atpG)

What is ATP synthase and what role does it play in Agrobacterium tumefaciens?

ATP synthase is a multi-subunit enzyme complex that catalyzes ATP synthesis by utilizing the electrochemical gradient of protons across membranes during oxidative phosphorylation. In Agrobacterium tumefaciens, as in other bacteria, ATP synthase is embedded in the cytoplasmic membrane and plays a crucial role in energy metabolism. The complex consists of two main sectors: the membrane-embedded Fo sector and the catalytic F1 sector. The enzyme not only synthesizes ATP but also contributes to membrane architecture and potentially to pathogenicity mechanisms in this plant pathogen. ATP synthase contains subunits that work together to convert the energy of proton movement into chemical energy stored in ATP bonds, which is essential for all cellular functions .

What are the subunits b/b' (atpG) and what functions do they serve in ATP synthase?

The b and b' subunits, with b' encoded by the atpG gene, are integral components of the peripheral stalk of ATP synthase. These subunits serve as a critical structural element that connects the membrane-embedded Fo sector to the catalytic F1 sector. Recent research has demonstrated that these peripheral stalk subunits are essential for the proper biogenesis and function of ATP synthase. Studies in the green alga Chlamydomonas reinhardtii have shown that ATPG (b' subunit) is absolutely required for ATP synthase function and accumulation, as knockout mutants completely prevent ATP synthase assembly . The peripheral stalk functions as a stator, holding the catalytic subunits in place while allowing rotation of other components during the catalytic cycle. Without functional b/b' subunits, the complex cannot assemble properly, resulting in complete loss of ATP synthase activity .

What is the genetic organization of ATP synthase genes in Agrobacterium tumefaciens?

In Agrobacterium tumefaciens, ATP synthase genes are organized in operons similar to other bacteria, though with some specific characteristics. The atpG gene encodes the b' subunit of the peripheral stalk. Recent studies have identified that mutations in the peripheral stalk subunits severely impact the assembly and function of the entire ATP synthase complex. In research with other organisms like Chlamydomonas reinhardtii, it was found that mutations in peripheral stalk genes can completely prevent ATP synthase accumulation, suggesting a similar essential role in A. tumefaciens . The genetic organization includes genes encoding both the Fo sector (membrane-embedded) and F1 sector (catalytic) components. This organization facilitates coordinated expression of the subunits, which is crucial for the proper assembly of the complex multiprotein enzyme. The atpA gene, encoding the alpha subunit, has been characterized and is available as a recombinant protein for research purposes .

What purification strategies yield the highest purity and activity for recombinant ATP synthase subunits?

Purification of recombinant ATP synthase subunits generally involves a multi-step approach to achieve both high purity and biological activity. Based on successful purification strategies for other ATP synthase components, the following protocol is recommended:

• Affinity chromatography: For His-tagged constructs (similar to the approach used for ATP5F1B), immobilized metal affinity chromatography (IMAC) provides an excellent first purification step, typically achieving >80% purity .

• Ion exchange chromatography: This second step separates proteins based on charge differences, further increasing purity.

• Size exclusion chromatography: As a final polishing step, this technique separates remaining contaminants based on size differences.

For ATP synthase subunits from A. tumefaciens, maintaining the native conformation during purification is critical for subsequent functional studies. Therefore, optimized buffer systems containing stabilizing agents such as glycerol and appropriate salt concentrations are essential. When purifying membrane-associated subunits, the inclusion of mild detergents during initial extraction steps helps solubilize the protein while maintaining native structure. Using this approach, recombinant ATP synthase subunits with purity exceeding 90% can be achieved, as demonstrated with recombinant human ATP5F1B protein .

How can researchers verify the structural integrity of purified recombinant ATP synthase subunits?

Verification of structural integrity for purified recombinant ATP synthase subunits requires a multi-faceted approach. SDS-PAGE analysis provides initial confirmation of protein size and purity, as demonstrated with recombinant human ATP5F1B, which showed the expected molecular weight of approximately 54 kDa and >90% purity . For more detailed structural assessment, circular dichroism (CD) spectroscopy can evaluate secondary structure content, particularly important for peripheral stalk subunits which contain significant α-helical regions. Mass spectrometry is critical for confirming the exact mass and potential post-translational modifications. Additionally, thermal shift assays can assess protein stability and proper folding. For functional verification, ATP hydrolysis assays provide essential information about catalytic capacity. Beyond these techniques, advanced structural methods like X-ray crystallography or cryo-electron microscopy may be employed for detailed structural analysis, though these require specialized expertise and equipment. Ultimately, the biological activity of the recombinant protein in reconstitution experiments provides the most relevant verification of structural integrity in the context of ATP synthase function .

What techniques are available for assessing ATP synthase assembly and function in vivo?

Multiple complementary approaches can be employed to assess ATP synthase assembly and function in living systems:

• Genetic disruption studies: CRISPR-Cas9 gene editing has proven effective for creating knockout mutations in ATP synthase genes. In Chlamydomonas reinhardtii, knockout ATPG mutants completely prevented ATP synthase function and accumulation, confirming the essential role of this subunit .

• Growth phenotype analysis: Organisms with impaired ATP synthase function typically display distinct growth defects, especially under conditions requiring efficient energy metabolism.

• Fluorescence microscopy: Using fluorescently tagged subunits allows visualization of ATP synthase localization and assembly in vivo.

• Membrane potential measurements: Since ATP synthase function is linked to membrane potential, fluorescent probes can indirectly assess activity.

• Biochemical isolation of complexes: Blue native PAGE combined with western blotting can visualize intact ATP synthase complexes and subcomplexes from cellular extracts.

Studies in C. reinhardtii demonstrated that crossing ATP synthase mutants with protease mutants (like ftsh1-1) helps identify degradation pathways for unassembled subunits, revealing AtpH as an FTSH substrate . This approach can be adapted to study A. tumefaciens ATP synthase assembly dynamics. Additionally, Southern blot analysis can verify genetic modifications, as demonstrated in transformation studies with efficiencies of up to 60 transformants per 10^6 cells .

How can researchers measure ATP synthase activity in purified or reconstituted systems?

ATP synthase activity in purified or reconstituted systems can be measured through several complementary approaches:

For recombinant ATP synthase subunits, researchers should first verify individual subunit function before attempting reconstitution of the complete complex. The catalytic β subunits, for example, can adopt different conformations and bind to Mg-ADP (βDP), Mg-ATP (βTP), or remain empty (βE) . This conformational flexibility is essential for the catalytic mechanism. When measuring activity of recombinant A. tumefaciens ATP synthase components, it's important to establish appropriate controls using known inhibitors like oligomycin or DCCD to confirm specificity of the observed activity. The electrochemical gradient of protons, which drives ATP synthesis in vivo, can be artificially established in reconstituted systems using pH jumps or potassium/valinomycin systems .

What approaches are used to study subunit interactions within ATP synthase complexes?

Understanding the interactions between ATP synthase subunits requires multiple complementary experimental approaches:

• Co-immunoprecipitation (Co-IP): This technique pulls down protein complexes using antibodies against one subunit and identifies interacting partners. For recombinant ATP synthase subunits, tag-based precipitation (such as His-tag pulldown) can be employed .

• Cross-linking mass spectrometry: Chemical cross-linkers can capture transient interactions between subunits, followed by mass spectrometric identification of cross-linked peptides, revealing proximity relationships.

• Surface plasmon resonance (SPR): This quantitative approach measures binding kinetics between purified subunits, providing affinity constants and interaction dynamics.

• Fluorescence resonance energy transfer (FRET): By labeling different subunits with donor and acceptor fluorophores, researchers can detect proximity in real-time in reconstituted systems.

• Yeast two-hybrid and bacterial two-hybrid systems: These genetic approaches can screen for potential interaction partners, though they may not capture membrane protein interactions effectively.

Research in C. reinhardtii revealed that peripheral stalk subunits b and b' (encoded by atpF and ATPG genes) are essential for ATP synthase biogenesis . When either subunit was absent, ATP synthase failed to accumulate, indicating their critical role in complex assembly. Mass spectrometry analysis showed minimal accumulation of functional ATP synthase in a knockdown ATPG mutant, while knockout mutants completely prevented ATP synthase function, demonstrating the utility of this approach for studying subunit dependencies .

How can Agrobacterium tumefaciens-mediated transformation be optimized for studying ATP synthase genes?

Agrobacterium tumefaciens-mediated transformation (ATMT) can be effectively optimized for studying ATP synthase genes by carefully controlling several key parameters:

• Acetosyringone treatment: This compound is critical for inducing the virulence (vir) genes in A. tumefaciens. Research has shown that acetosyringone is essential for successful transformation, as colonies were only obtained when it was included in the co-cultivation medium .

• Co-cultivation conditions: The efficiency of ATMT is significantly affected by temperature and duration. Optimal results have been achieved with a 48-hour co-cultivation period at 23°C .

• Strain selection: The C58C1 strain of A. tumefaciens has been successfully used for transformation studies and could be adapted for ATP synthase research .

• Recipient preparation: For fungal recipients, using intact cells and spores rather than protoplasts has been effective, suggesting that similar approaches could work for other organisms when studying ATP synthase genes .

• Vector design: Including appropriate selection markers (such as hygromycin resistance) is essential for identifying transformants. The integration of marker genes into the genome can be verified by PCR amplification and sequencing .

ATMT has demonstrated transformation efficiencies of up to 60 transformants per 10^6 conidia in fungi , and with optimization, similar efficiencies could be achieved when targeting ATP synthase genes. The resulting transformants have been shown to be mitotically stable, with random integration of the transferred DNA as single copies into the recipient genome . This approach could be valuable for creating ATP synthase mutants to study the function of specific subunits like atpG.

What CRISPR-Cas9 strategies are most effective for creating ATP synthase subunit mutations?

CRISPR-Cas9 gene editing has emerged as a powerful tool for studying ATP synthase biology, with several strategies proving particularly effective:

• Knockout approaches: Complete knockout of ATP synthase genes using CRISPR-Cas9 has provided definitive evidence about subunit function. In Chlamydomonas reinhardtii, knockout ATPG mutants completely prevented ATP synthase function and accumulation, in contrast to knockdown mutants which allowed small amounts of functional complex to form .

• Guide RNA design: For ATP synthase subunit genes, guide RNAs targeting early exons generally produce more predictable loss-of-function phenotypes. Multiple guide RNAs targeting different regions of the same gene can increase editing efficiency.

• Homology-directed repair (HDR): This approach allows precise modifications, such as introducing point mutations or tagged versions of ATP synthase subunits for localization studies.

• Inducible CRISPR systems: These are particularly valuable for studying essential genes like ATP synthase components, allowing conditional knockout or knockdown.

• Multiplex editing: Targeting multiple ATP synthase subunits simultaneously can reveal functional relationships and compensatory mechanisms.

For verification of edits, researchers should employ multiple methods including PCR, sequencing, and functional assays. In C. reinhardtii, mass spectrometry confirmed the absence of target proteins in knockout lines and reduced abundance in knockdown lines . When designing CRISPR strategies for ATP synthase genes, researchers should consider the potential for pleiotropic effects due to complete loss of ATP synthase function, which may necessitate conditional or tissue-specific approaches depending on the experimental goals.

How can insertional mutagenesis be applied to study ATP synthase function?

Insertional mutagenesis provides a powerful approach for studying ATP synthase function, with several key considerations for implementation:

• Transposon-based strategies: Transposon insertions, such as those observed in the 3'UTR of ATPG in Chlamydomonas, can create knockdown rather than knockout phenotypes, allowing the study of hypomorphic effects . This approach revealed that small amounts of functional ATP synthase can still accumulate in knockdown mutants.

• T-DNA insertion approaches: Agrobacterium tumefaciens-mediated transformation (ATMT) has been successfully used for insertional mutagenesis with transformation efficiencies of up to 60 transformants per 10^6 conidia in fungi . This system generates a large number of stable transformants with random integration of the T-DNA as single copies into the genome.

• Selection strategies: Using selectable markers (like hygromycin resistance) allows identification of transformants. The integration of the marker gene can be verified by PCR amplification and sequencing .

• Screening methods: High-throughput screening for ATP synthase mutants can utilize phenotypes such as growth defects under specific conditions (like high light sensitivity in photosynthetic organisms) .

• Mapping insertion sites: Southern blot analysis can confirm the random integration pattern and copy number of inserted DNA .

The ATMT system has been shown to produce transformants that show vigorous growth, indicating it can be an efficient tool for molecular manipulation . The frequency of homologous recombination has been found to be higher with ATMT than with conventional transformation methods, making it particularly valuable for targeted modifications of ATP synthase genes . This approach could be adapted to generate a library of A. tumefaciens ATP synthase mutants for comprehensive functional analysis.

How do peripheral stalk subunits like atpG coordinate ATP synthase biogenesis?

Peripheral stalk subunits, including atpG (encoding subunit b'), play a critical role in orchestrating ATP synthase biogenesis through multiple mechanisms:

• Assembly scaffolding: Research in Chlamydomonas reinhardtii has demonstrated that peripheral stalk subunits b and b' (encoded by atpF and ATPG) are absolutely essential for ATP synthase accumulation. Knockout ATPG mutants completely prevented ATP synthase function and assembly, while knockdown mutants showed minimal accumulation of functional complex .

• Stoichiometric regulation: The peripheral stalk helps maintain proper stoichiometry of ATP synthase components during assembly, preventing aggregation of unpaired subunits.

• Protection from degradation: In C. reinhardtii, crossing ATP synthase mutants with the ftsh1-1 mutant of the major thylakoid protease revealed that FTSH significantly contributes to the concerted accumulation of ATP synthase subunits . This suggests that peripheral stalk subunits may protect other components from premature degradation.

• Structural stability: The peripheral stalk functions as a rigid connection between the membrane-embedded Fo sector and the catalytic F1 sector, providing structural stability to the entire complex.

• Evolutionary coordination: Interestingly, peripheral stalk components have co-evolved with regulatory factors. For example, in C. reinhardtii, an octotricopeptide repeat (OPR) protein called MDE1 was identified as essential for stabilizing the chloroplast-encoded atpE mRNA, demonstrating a nucleus/chloroplast interplay that evolved relatively recently (approximately 300 million years ago) .

These findings highlight the multifaceted role of peripheral stalk subunits beyond their structural function, positioning them as central coordinators in the complex process of ATP synthase biogenesis.

What is the relationship between ATP synthase and membrane architecture?

ATP synthase plays a multifaceted role in membrane architecture that extends beyond its primary function in ATP production:

• Membrane curvature induction: ATP synthase, particularly its F1 sector which protrudes from the membrane, can induce local membrane curvature. Research has demonstrated that "ATP synthase is not only critical for ATP synthesis but is also critical for the architecture of the mitochondrial inner membrane" . This structural role likely applies across bacterial, chloroplast, and mitochondrial membranes.

• Organization into supercomplexes: ATP synthase often organizes into dimers and higher-order oligomers that form rows along membrane ridges, particularly in mitochondrial cristae. While less studied in bacteria like A. tumefaciens, similar principles may apply.

• Lipid microdomain association: ATP synthase has been found to associate with specific lipid microdomains, which may facilitate proton conduction and enzyme activity.

• Proton gradient maintenance: By regulating the proton flux across membranes, ATP synthase helps maintain the electrochemical gradient necessary for membrane potential-dependent processes.

• Interaction with other membrane complexes: ATP synthase coordinates with electron transport chain components to optimize energy conversion efficiency.

The unique structure of ATP synthase, with its membrane-embedded Fo sector connected to the catalytic F1 sector via peripheral stalks including the b and b' subunits, positions it as both a membrane architect and a functional enzyme. In recombinant studies, preserving these membrane interactions is particularly challenging but essential for understanding the full biological context of ATP synthase function .

What strategies can address low expression of recombinant ATP synthase subunits?

Researchers facing challenges with low expression of recombinant ATP synthase subunits can implement several targeted strategies:

• Optimization of expression systems: While E. coli is commonly used, alternative expression hosts may be more suitable for ATP synthase subunits. For instance, human ATP synthase subunit beta (ATP5F1B) has been successfully expressed in yeast with high purity (>90%) . For A. tumefaciens subunits, consider testing multiple expression hosts.

• Codon optimization: Adapting the coding sequence to the preferred codon usage of the expression host can significantly improve translation efficiency and protein yield.

• Expression temperature modulation: Lowering the expression temperature (16-20°C) often enhances proper folding of complex proteins like ATP synthase subunits.

• Fusion tags selection: Beyond the commonly used 6xHis-tag , alternative tags such as MBP (maltose-binding protein) or SUMO can enhance solubility of challenging proteins.

• Co-expression with chaperones: Co-expressing molecular chaperones like GroEL/GroES can facilitate proper folding of ATP synthase subunits.

• Expression of truncated constructs: For peripheral stalk subunits like b/b' (atpG), expressing defined structural domains rather than full-length proteins may improve yields.

• Media and induction optimization: Rich auto-induction media often provides higher yields than standard IPTG induction for challenging proteins.

When optimizing expression conditions, it's essential to verify not just the quantity but also the quality of the expressed protein. SDS-PAGE analysis should confirm the expected molecular weight (as demonstrated for ATP5F1B which showed approximately 54 kDa) , while functional assays must verify biological activity of the recombinant protein.

How can researchers troubleshoot ATP synthase activity assays?

Troubleshooting ATP synthase activity assays requires a systematic approach to identify and resolve common issues:

When working with recombinant ATP synthase subunits, it's important to understand that the catalytic β subunits can adopt different conformations (βDP, βTP, or βE) , which affects activity measurements. Additionally, ATP synthase requires the electrochemical gradient of protons across membranes for ATP synthesis , so reconstitution systems must properly establish this gradient.

For peripheral stalk subunits like those encoded by atpF and ATPG, direct activity measurements may not be applicable since they don't possess catalytic activity themselves . Instead, their function is better assessed through reconstitution experiments or in vivo complementation studies. Mass spectrometry can be used to verify the presence and quantity of ATP synthase complexes in such experiments, as demonstrated in studies with C. reinhardtii where it confirmed minimal accumulation of functional ATP synthase in knockdown mutants .

What approaches help resolve aggregation issues with recombinant ATP synthase subunits?

Aggregation of recombinant ATP synthase subunits presents a significant challenge that can be addressed through multiple complementary approaches:

• Solubilization optimization: For membrane-associated subunits like those in the peripheral stalk, careful selection of detergents is critical. A stepwise screening approach starting with mild detergents (DDM, LMNG) at various concentrations can identify optimal solubilization conditions.

• Buffer composition refinement: Including stabilizing agents such as glycerol (10-20%), specific ions (particularly Mg²⁺), and appropriate salt concentrations can significantly reduce aggregation. Systematic buffer optimization should evaluate pH ranges typically between 7.0-8.5.

• Co-expression strategies: Expressing multiple interacting subunits simultaneously can improve stability by allowing proper complex formation. For peripheral stalk subunits like b and b' (encoded by atpF and ATPG), co-expression may be particularly beneficial given their demonstrated interdependence in ATP synthase biogenesis .

• Refolding approaches: For subunits expressed as inclusion bodies, step-wise dialysis with decreasing denaturant concentrations, potentially coupled with molecular chaperones, can recover properly folded protein.

• Fusion partner selection: Beyond improving expression, fusion partners like MBP can enhance solubility. When using His-tags (as with ATP5F1B ), optimizing tag position (N- versus C-terminal) can significantly impact solubility.

• Size exclusion chromatography: As a final purification step, this technique not only increases purity but also allows selection of properly folded monomeric protein fractions versus aggregates.

When working with peripheral stalk subunits, it's important to recognize their structural role in connecting the membrane-embedded Fo sector to the catalytic F1 sector . This structural context may need to be partially reconstituted to achieve stable, non-aggregated protein preparations for detailed functional and structural studies.

What emerging technologies show promise for ATP synthase structural studies?

Several cutting-edge technologies are revolutionizing structural studies of ATP synthase and its components, including the peripheral stalk subunits:

• Cryo-electron microscopy advancements: Recent improvements in cryo-EM resolution now allow visualization of ATP synthase at near-atomic resolution without crystallization. This is particularly valuable for studying membrane proteins like ATP synthase that are challenging to crystallize. Time-resolved cryo-EM is emerging as a technique to capture different conformational states during the catalytic cycle.

• Integrative structural biology approaches: Combining multiple techniques including X-ray crystallography, cryo-EM, nuclear magnetic resonance (NMR), and mass spectrometry provides complementary structural information. Cross-linking mass spectrometry has become particularly valuable for mapping subunit interactions within the ATP synthase complex.

• Single-molecule techniques: Methods like single-molecule FRET and high-speed atomic force microscopy (HS-AFM) allow direct observation of ATP synthase dynamics and rotation in real-time, providing insights into the function of peripheral stalk subunits in the context of the rotating enzyme.

• In-cell structural biology: Emerging techniques for structural studies within intact cells, including cryo-electron tomography, will help place ATP synthase structures in their native membrane environment.

• AlphaFold and machine learning approaches: AI-based structure prediction tools are increasingly accurate for predicting protein structures and could help model ATP synthase components for which experimental structures are challenging to obtain.

These technologies could help resolve long-standing questions about how peripheral stalk subunits like those encoded by atpF and ATPG contribute to ATP synthase assembly and function. Studies in Chlamydomonas reinhardtii have already demonstrated that these components are essential for ATP synthase accumulation , and advanced structural techniques could reveal the molecular mechanisms underlying this dependency.

How might synthetic biology approaches advance ATP synthase research?

Synthetic biology offers transformative approaches for advancing ATP synthase research, particularly for studying peripheral stalk components like atpG:

• Designer ATP synthase variants: By systematically redesigning peripheral stalk subunits, researchers can probe structure-function relationships and potentially create ATP synthases with novel properties. This approach could build on findings from C. reinhardtii, where knockout ATPG mutants completely prevented ATP synthase accumulation .

• Minimal ATP synthase systems: Synthetic biology approaches can help determine the minimal components needed for functional ATP synthesis, potentially simplifying the complex for detailed mechanistic studies.

• Orthogonal expression systems: Developing systems that allow expression of artificial ATP synthase variants without interference from endogenous complexes could facilitate in vivo studies of modified subunits.

• Bio-hybrid energy systems: ATP synthase components could be incorporated into artificial membranes or coupled with light-harvesting systems to create bio-hybrid energy conversion devices.

• Cross-species chimeric complexes: Creating chimeric ATP synthases with components from different species (bacterial, chloroplast, mitochondrial) could reveal evolutionary adaptations and functional specializations.

• Biosensor development: Modified ATP synthase components could serve as biosensors for monitoring cellular energy status or detecting specific environmental conditions.

These approaches could build on established techniques like Agrobacterium tumefaciens-mediated transformation, which has demonstrated high efficiency (up to 60 transformants per 10^6 conidia) and could be adapted for introducing synthetic ATP synthase variants. The development of recombinant ATP synthase subunits with high purity (>90%) provides a foundation for incorporating these components into synthetic biological systems.

What are the potential applications of engineered ATP synthase components in biotechnology?

Engineered ATP synthase components, including modified peripheral stalk subunits, hold significant potential for diverse biotechnology applications:

• Bioenergy applications: Modified ATP synthases could enhance energy conversion efficiency in biofuel cells or artificial photosynthesis systems. Understanding the structural roles of peripheral stalk subunits in membrane architecture could inform the design of optimized energy-harvesting membranes.

• Nanomotor development: As one of nature's most efficient rotary motors, engineered ATP synthase could serve as the basis for molecular machines and nanomotors with applications in nanomedicine and smart materials.

• Biosensing platforms: The conformational changes in ATP synthase subunits, particularly the catalytic β subunits that can adopt different states (βDP, βTP, or βE) , could be exploited to develop sensitive biosensors for ATP/ADP ratios or proton gradients.

• Drug discovery platforms: Recombinant ATP synthase components could serve as targets for screening potential antimicrobials, particularly against pathogenic bacteria.

• Protein production enhancers: Understanding ATP synthase biogenesis could lead to improved cellular energy production systems for biotechnology applications requiring high ATP levels, such as recombinant protein production.

• Biomedical applications: Insights from bacterial ATP synthase research could inform therapeutic approaches targeting mitochondrial ATP synthase dysfunction in human diseases.

The availability of high-purity recombinant ATP synthase subunits (>90%) provides a foundation for these applications. Additionally, techniques like Agrobacterium tumefaciens-mediated transformation, which has shown high efficiency and stability , could be valuable for introducing engineered ATP synthase components into various organisms for biotechnological applications.