Recombinant Mannheimia succiniciproducens ATP synthase subunit b (atpF), partial
Description
About Mannheimia succiniciproducens
Mannheimia succiniciproducens is a Gram-negative, facultative anaerobic bacterium that can efficiently produce succinic acid from various carbon sources . This bacterium has garnered considerable attention in industrial biotechnology due to its ability to produce succinic acid, a valuable building block chemical with applications in producing polymers, resins, and pharmaceuticals .
ATP Synthase and Subunit b
ATP synthase, also known as F-ATPase, is a ubiquitous enzyme complex found in the membranes of bacteria, mitochondria, and chloroplasts . It harnesses the energy from a proton gradient across the membrane to synthesize ATP from adenosine diphosphate (ADP) and inorganic phosphate . ATP synthase consists of two main functional domains: F0 and F1 . The F0 domain is embedded in the membrane and acts as a proton channel, while the F1 domain is located in the cytoplasm (or matrix in mitochondria and stroma in chloroplasts) and carries out ATP synthesis .
The subunit b (atpF) is a component of the F0 domain, which is critical for the enzyme's function . It forms part of the stator that connects the F1 and F0 domains, providing structural support and facilitating the transfer of torque generated by proton flow to the F1 domain, which drives ATP synthesis.
Recombinant Form
The term "recombinant" indicates that the atpF subunit has been produced using genetic engineering techniques. Specifically, the gene encoding the atpF subunit from Mannheimia succiniciproducens is likely cloned and expressed in a different host organism (e.g., E. coli) to produce the protein in large quantities for research or industrial purposes.
Significance of Studying the atpF Subunit
Characterization of the recombinant Mannheimia succiniciproducens ATP synthase subunit b (atpF), partial, may be carried out:
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To understand the structure-function relationship of ATP synthase in this bacterium.
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To investigate the role of specific amino acid residues in proton translocation and ATP synthesis.
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To explore the potential for engineering ATP synthase to improve its efficiency or alter its substrate specificity.
Research Findings
Research findings related to Mannheimia succiniciproducens have revealed the following:
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Efficient Sucrose Utilization: M. succiniciproducens can efficiently utilize sucrose as a carbon source for succinic acid production . The bacterium employs a sucrose phosphotransferase system (PTS), sucrose-6-phosphate hydrolase, and a fructose PTS for sucrose transport and utilization .
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Metabolic Engineering for Enhanced Succinic Acid Production: Systems metabolic engineering has been applied to M. succiniciproducens to improve its ability to utilize formic acid (FA) as a secondary substrate for succinic acid production .
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PCK Activity and Succinic Acid Biosynthesis: Phosphoenolpyruvate carboxykinase (PCK) activity is closely related to succinic acid biosynthesis in M. succiniciproducens . Disruption of genes encoding phosphoenolpyruvate carboxylase (PPC) or malic enzyme (MAE) did not significantly affect succinic acid productivity or cell growth, whereas PCK plays a crucial role in CO2 fixation and cell growth .
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Cytochrome P450 Monooxygenases (CYPs): Studies on cytochrome P450 monooxygenases (CYPs) in plants have used bioinformatics and in vivo functional analysis to identify key amino acid residues that affect the catalytic activity and substrate specificity of CYP716A subfamily enzymes involved in triterpenoid biosynthesis .
Product Specs
Note: While we will prioritize shipping the format currently in stock, please specify any format requirements in your order notes for customized preparation.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The tag type is determined during production. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
KEGG: msu:MS2350
STRING: 221988.MS2350
Q&A
What is the genomic context of the atpF gene in Mannheimia succiniciproducens?
The atpF gene in Mannheimia succiniciproducens encodes the ATP synthase subunit b, which forms part of the peripheral stalk of the ATP synthase complex. This gene exists within the complete genome sequence of M. succiniciproducens, which has been fully characterized through genome-scale metabolic-flux analysis . The peripheral stalk, comprising subunit b, plays a crucial structural role in connecting the F₁ and F₀ portions of the ATP synthase complex, thereby maintaining the structural integrity necessary for ATP synthesis function . Unlike some other bacterial species, M. succiniciproducens maintains a compact genomic organization that reflects its specialized metabolism focused on succinic acid production under anaerobic conditions in the presence of CO₂ .
How does ATP synthase subunit b contribute to energy metabolism in Mannheimia succiniciproducens?
ATP synthase subunit b (atpF) serves as a critical component of the peripheral stalk in the ATP synthase complex, facilitating the coupling of proton translocation to ATP synthesis. In M. succiniciproducens, this process is particularly important during anaerobic growth conditions when the bacterium produces succinic acid as a major fermentation product . The ATP synthase complex enables energy conservation through oxidative phosphorylation, with subunit b specifically helping to anchor the catalytic F₁ portion to the membrane-embedded F₀ portion . Knockout studies of related ATP synthase components have demonstrated that disruption of this complex severely impacts the organism's ability to generate ATP efficiently and significantly alters metabolic flux distributions, particularly affecting anaerobic fermentation pathways that lead to succinic acid production .
What is the relationship between ATP synthase function and succinic acid production in M. succiniciproducens?
M. succiniciproducens MBEL55E was isolated from the rumens of Korean cows and produces significant quantities of succinic acid under anaerobic conditions in the presence of CO₂ . The relationship between ATP synthase and succinic acid production involves several interconnected metabolic pathways:
| Metabolic Element | Relationship to Succinic Acid Production | Impact of ATP Synthase Function |
|---|---|---|
| PEP Carboxylation | Major CO₂-fixing step with direct relationship to succinic acid flux | ATP consumption/generation affects carbon flux distribution |
| Tricarboxylic Acid Cycle | Operates as a branched pathway under anaerobic conditions | ATP levels influence regulatory mechanisms |
| Electron Transport Chain | Provides reduced cofactors for reductive steps | ATP synthase couples this process to energy conservation |
| By-product Formation | Competes with succinic acid pathway for carbon flux | ATP availability influences metabolic branch points |
The ATP synthase complex, including the atpF subunit, plays a crucial role in maintaining the energetic state of the cell, which in turn affects the distribution of carbon flux through these pathways . Disruptions in ATP synthase function can therefore indirectly affect succinic acid production by altering the cell's energy balance and redox state.
What are the optimal expression systems for producing recombinant M. succiniciproducens ATP synthase subunit b?
Recombinant M. succiniciproducens ATP synthase subunit b (atpF) can be effectively expressed in several host systems, each with distinct advantages for different research applications:
| Expression Host | Advantages | Considerations | Typical Yield |
|---|---|---|---|
| E. coli | Rapid growth, high expression levels, well-established protocols | May require codon optimization, potential inclusion body formation | 10-20 mg/L culture |
| Yeast | Post-translational modifications, lower endotoxin | Longer expression time, more complex media requirements | 5-15 mg/L culture |
| Baculovirus | Superior folding for complex proteins, higher solubility | Technical complexity, higher cost, longer timeframe | 8-18 mg/L culture |
| Mammalian Cell | Closest to native conditions for functional studies | Highest cost, longest production time, technical expertise required | 2-10 mg/L culture |
All expression systems can achieve greater than 85% purity as determined by SDS-PAGE for the recombinant protein . The choice of expression system should be guided by the specific requirements of downstream applications, particularly whether structural or functional studies are prioritized. For structural studies requiring large protein quantities, E. coli systems typically offer the best yield-to-effort ratio, while functional studies may benefit from expression in systems that better preserve native protein conformation and activity.
How can fluorescence-based methods be adapted to study ATP synthase function in M. succiniciproducens?
Fluorescence-based methods offer significant advantages over radioactive assays for studying ATP synthase function. One particularly valuable approach utilizes Magnesium Green (MgGr™), a Mg²⁺-sensitive fluorescent dye that can detect changes in ATP and ADP concentrations due to their different binding affinities for Mg²⁺ . This method can be adapted for M. succiniciproducens ATP synthase studies as follows:
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Liposome Reconstitution: Purified recombinant ATP synthase components, including subunit b (atpF), can be reconstituted into unilamellar liposomes to create a controlled experimental system.
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Fluorescence Measurement Protocol:
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Load liposomes with MgGr™ and establish baseline fluorescence
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Introduce ADP to initiate exchange
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Monitor fluorescence changes that correspond to ADP/ATP exchange rates
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Calculate exchange rates based on calibration curves
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Validation Controls:
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Include ATP synthase-specific inhibitors (e.g., bongkrekic acid, carboxyatractyloside)
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Prepare liposomes with varying ATP synthase content to establish concentration dependence
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Compare to traditional radioisotope methods for validation
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What advanced approaches can be used to study interactions between atpF and other ATP synthase subunits?
Studying subunit interactions within the ATP synthase complex requires specialized techniques that can capture both static and dynamic protein-protein interactions:
| Technique | Application to atpF Research | Technical Considerations |
|---|---|---|
| Cross-linking MS | Identifies interaction interfaces between atpF and other subunits | Requires optimization of cross-linking conditions for peripheral stalk proteins |
| Surface Plasmon Resonance | Quantifies binding kinetics between purified subunits | Needs careful immobilization strategy to preserve protein orientation |
| FRET Analysis | Measures distances between fluorescently labeled subunits | Requires strategic placement of fluorophores to avoid functional interference |
| Cryo-EM | Provides structural context of atpF within the entire ATP synthase complex | Resource-intensive but yields high-resolution spatial information |
| Hydrogen-Deuterium Exchange MS | Maps dynamic interaction surfaces and conformational changes | Specialized equipment and expertise required for data interpretation |
These techniques have revealed that peripheral stalk subunits like atpF (subunit b) and ATPG (subunit b') form critical structural elements that prevent rotation of the catalytic portions against the membrane portions of the ATP synthase complex . Disruption of these interactions through mutations can prevent proper ATP synthase assembly and function, as demonstrated in frame-shift mutations of atpF that completely prevent ATP synthase accumulation .
How should researchers interpret apparent contradictions in experimental data regarding atpF function?
When analyzing experimental data on M. succiniciproducens atpF function, researchers frequently encounter contradictory results that require careful interpretation. These contradictions typically fall into several categories:
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Internal to the experimental system: Species-specific differences, genetic background variations, or dosage effects may explain contradictory findings .
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External to the experimental system: Environmental conditions like growth phase, temperature, or media composition can significantly influence ATP synthase expression and function .
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Endogenous/exogenous factors: Interactions with other cellular components or introduced reagents may alter expected outcomes .
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Known controversies: Some aspects of ATP synthase function remain actively debated in the literature .
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Literature contradictions: Methodological differences between studies often explain apparently conflicting results .
When encountering contradictory data, researchers should systematically examine experimental variables including:
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Growth conditions: M. succiniciproducens is capnophilic, requiring specific CO₂ concentrations for optimal growth and metabolism
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Genetic context: Frame-shift mutations in atpF prevent ATP synthase accumulation, while point mutations may produce more subtle phenotypes
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Purification methods: Protein stability and activity can be significantly affected by purification protocols
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Assay conditions: ATP synthase activity is highly sensitive to pH, ion concentrations, and temperature
By carefully controlling and documenting these variables, researchers can often resolve apparent contradictions and contribute to a more nuanced understanding of atpF function.
What statistical approaches are most appropriate for analyzing ATP synthase activity data?
Statistical analysis of ATP synthase activity data requires approaches that can account for the complex, multivariate nature of enzymatic reactions:
| Statistical Method | Application to ATP Synthase Research | Advantages |
|---|---|---|
| Michaelis-Menten Kinetics | Determining Km and Vmax for ATP synthesis/hydrolysis | Provides fundamental enzymatic parameters |
| Multiple Linear Regression | Identifying factors affecting enzyme activity | Accounts for multiple experimental variables |
| Principal Component Analysis | Reducing dimensionality of complex datasets | Identifies major sources of variation |
| Hierarchical Clustering | Grouping similar experimental conditions | Reveals patterns across multiple experiments |
| Bayesian Statistical Methods | Incorporating prior knowledge into analysis | Particularly useful when sample sizes are limited |
When analyzing fluorescence-based ADP/ATP exchange data, researchers should implement appropriate controls to account for background fluorescence and non-specific binding . Time-series data should be fitted to appropriate kinetic models, with careful attention to initial rates versus steady-state measurements. For comparative studies examining the effects of mutations or inhibitors on atpF function, ANOVA with post-hoc tests provides statistical rigor when comparing multiple experimental conditions.
How can researchers integrate data from proteomics, genomics, and functional studies of M. succiniciproducens ATP synthase?
Integration of multi-omics data provides comprehensive insights into atpF function that cannot be achieved through any single approach:
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Genomic Integration:
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Compare atpF sequence conservation across related bacterial species
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Identify regulatory elements controlling ATP synthase expression
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Map mutations that affect ATP synthase function to specific domains
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Proteomic Integration:
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Quantify ATP synthase subunit stoichiometry under different growth conditions
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Identify post-translational modifications affecting atpF function
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Map protein-protein interaction networks involving ATP synthase components
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Functional Data Integration:
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Correlate ATP synthesis rates with metabolic flux distributions
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Link ATP synthase activity to succinic acid production pathways
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Establish structure-function relationships for atpF domains
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Visualization and Computational Tools:
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Network analysis software to visualize integrated datasets
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Machine learning approaches to identify non-obvious patterns
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Metabolic modeling to predict effects of ATP synthase modifications
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What are common challenges in purifying recombinant M. succiniciproducens ATP synthase subunit b and how can they be addressed?
Purification of recombinant M. succiniciproducens ATP synthase subunit b presents several challenges that researchers must overcome to obtain functional protein:
| Challenge | Manifestation | Solution Approach |
|---|---|---|
| Protein Solubility | Formation of inclusion bodies | Use mild detergents; optimize expression temperature; consider fusion tags |
| Membrane Association | Difficult extraction | Test different detergent combinations; use sequential extraction methods |
| Structural Stability | Protein degradation during purification | Include protease inhibitors; minimize purification time; maintain consistent temperature |
| Co-purifying Contaminants | Bands on SDS-PAGE beyond target protein | Implement multi-step purification; consider size exclusion as final step |
| Function Preservation | Loss of activity post-purification | Validate with activity assays; optimize buffer conditions; consider lipid supplementation |
The standard for recombinant M. succiniciproducens ATP synthase subunit b purity is greater than or equal to 85% as determined by SDS-PAGE . Multiple expression systems can achieve this benchmark, including E. coli, yeast, baculovirus, and mammalian cell systems, each with their specific optimization requirements . For functional studies, maintaining native-like conditions throughout purification is critical, often necessitating the presence of phospholipids or mild detergents to preserve the structural integrity of this membrane-associated protein.
How can genetic manipulation techniques be optimized for studying atpF function in M. succiniciproducens?
Genetic manipulation of M. succiniciproducens requires specialized approaches due to its unique physiology as a capnophilic bacterium:
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Gene Knockout Strategies:
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Homologous recombination approaches have successfully disrupted metabolic genes in M. succiniciproducens
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For atpF studies, complete gene deletion may be lethal, necessitating conditional knockout strategies
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Frame-shift mutations in atpF prevent ATP synthase function and accumulation, providing a useful approach for functional studies
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Expression Optimization:
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Promoter selection is critical - constitutive vs. inducible systems offer different advantages
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Codon optimization may improve expression levels
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Translation efficiency can be enhanced through ribosome binding site optimization
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Site-Directed Mutagenesis:
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Strategic mutations targeting conserved residues can provide structure-function insights
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Alanine-scanning mutagenesis of atpF can map functionally important regions
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Introduction of reporter tags must be carefully positioned to avoid disrupting function
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CRISPR-Cas9 Applications:
Gene knockout studies in M. succiniciproducens have already provided valuable insights into its anaerobic fermentative metabolism and have facilitated the development of metabolically engineered strains for enhanced succinic acid production . Similar approaches targeting atpF can help elucidate its specific role in energy conservation and metabolic regulation.
What environmental factors affect ATP synthase expression and activity in M. succiniciproducens?
M. succiniciproducens, like other bacteria, modulates ATP synthase expression and activity in response to environmental conditions:
| Environmental Factor | Effect on ATP Synthase | Experimental Consideration |
|---|---|---|
| Oxygen Availability | Expression levels change between aerobic/anaerobic conditions | Maintain consistent anaerobic environment for reproducible results |
| CO₂ Concentration | Critical for M. succiniciproducens metabolism and affects ATP demand | Precisely control CO₂ levels during cultivation |
| Iron Availability | Iron deprivation affects respiratory chain components and ATP synthesis | Monitor iron levels; consider iron chelators as experimental variables |
| Temperature | Affects protein folding, membrane fluidity, and enzymatic rates | Maintain strict temperature control; consider temperature as an experimental variable |
| pH | Influences proton gradient and ATP synthase function | Buffer systems must maintain stable pH during experiments |
Studies in related organisms have demonstrated that virulence factor transcription, including genes encoding key metabolic enzymes, is environmentally regulated with specific roles for iron deprivation and temperature change . In M. succiniciproducens, these environmental factors likely influence ATP synthase expression through similar regulatory mechanisms. Understanding these environmental effects is crucial for designing experiments that yield reproducible results and for interpreting data in the context of the organism's natural physiology.
How does M. succiniciproducens ATP synthase subunit b compare to homologous proteins in other bacterial species?
Comparative analysis of ATP synthase subunit b across bacterial species reveals important evolutionary patterns and functional conservation:
| Species | atpF Similarity to M. succiniciproducens | Notable Structural/Functional Differences |
|---|---|---|
| Escherichia coli | Moderate sequence homology (~55-65%) | Well-characterized dimerization domain |
| Mannheimia haemolytica | High sequence homology (~80-90%) | Similar peripheral stalk organization |
| Pasteurella multocida | High sequence homology (~75-85%) | Comparable membrane association properties |
| Chlamydomonas reinhardtii | Low sequence homology (chloroplast atpF) | Requires nuclear-encoded ATPG for peripheral stalk formation |
| Mycobacterium tuberculosis | Low sequence homology (~30-40%) | Contains unique structural adaptations for specialized metabolism |
The peripheral stalk, including subunit b (atpF), plays a crucial structural role in all ATP synthases, connecting the F₁ and F₀ portions of the complex . In Chlamydomonas reinhardtii, peripheral stalk subunits b and b' are encoded by atpF and ATPG genes respectively, with mutations in either gene affecting ATP synthase function and accumulation . This fundamental architecture is conserved in M. succiniciproducens, though with species-specific adaptations that likely reflect its specialized metabolism focused on succinic acid production under anaerobic conditions .
What insights can comparative genomics provide about the evolution of ATP synthase in M. succiniciproducens?
Comparative genomics analyses offer valuable insights into the evolutionary history and functional adaptation of ATP synthase in M. succiniciproducens:
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Phylogenetic Context:
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M. succiniciproducens is phylogenetically related to Mannheimia haemolytica, an important pathogen in bovine respiratory disease
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The genomic organization of ATP synthase genes shows conservation patterns that reflect evolutionary selection pressures
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Horizontal gene transfer events may have contributed to metabolic specialization
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Gene Organization:
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Selection Pressures:
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Metabolic specialization for succinic acid production has likely shaped ATP synthase evolution
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Adaptation to the rumen environment (the natural habitat of M. succiniciproducens) imposes unique energetic challenges
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Conservation of critical functional domains amidst sequence divergence highlights essential structural elements
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Coevolution Patterns:
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Correlated mutations between interacting subunits preserve functional interfaces
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Coevolution of ATP synthase with other metabolic pathways ensures integrated cellular function
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Regulatory networks linking energy production to carbon metabolism show adaptive signatures
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The genome-scale metabolic modeling of M. succiniciproducens has provided a framework for understanding how its ATP synthase functions within a specialized metabolic network optimized for succinic acid production . Comparative analyses with related organisms continue to reveal how evolutionary processes have shaped this efficient biocatalyst.
How do mutations in atpF affect ATP synthase assembly and function across different organisms?
Mutations in atpF have profound effects on ATP synthase assembly and function across diverse organisms, providing valuable comparative insights:
| Mutation Type | Observed Effects | Comparative Context |
|---|---|---|
| Frame-shift Mutations | Prevent ATP synthase accumulation entirely | Consistent effect across bacterial species |
| Point Mutations in Dimerization Domain | Disrupt peripheral stalk formation | Similar phenotypes in diverse bacteria |
| Transmembrane Region Mutations | Alter membrane association | Effects depend on membrane composition |
| C-terminal Modifications | Disrupt interaction with F₁ portion | Conserved function across species |
| N-terminal Variations | Species-specific effects | Reflects evolutionary adaptation |
In Chlamydomonas reinhardtii, a frame-shift mutation in atpF completely prevents ATP synthase function and accumulation . Similarly, CRISPR-Cas9 knockout of ATPG (encoding the related peripheral stalk subunit b') fully prevents ATP synthase function . These findings highlight the essential structural role of peripheral stalk subunits in ATP synthase assembly and function. The high conservation of this effect across diverse organisms underscores the fundamental importance of these structural elements for ATP synthase function.
Studies crossing ATP synthase mutants with protease mutants have identified key quality control mechanisms that regulate the concerted accumulation of ATP synthase subunits . These proteolytic systems, such as the FTSH thylakoid protease in Chlamydomonas, contribute significantly to maintaining proper stoichiometry of ATP synthase components. Similar quality control mechanisms likely operate in M. succiniciproducens, though with species-specific adaptations reflecting its unique physiology and environmental niche.
What emerging technologies will advance our understanding of M. succiniciproducens ATP synthase?
Several cutting-edge technologies are poised to revolutionize our understanding of ATP synthase structure, function, and regulation in M. succiniciproducens:
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Cryo-Electron Microscopy:
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Enables visualization of ATP synthase structure at near-atomic resolution
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Can capture different conformational states during the catalytic cycle
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Provides insights into how peripheral stalk subunits like atpF stabilize the complex
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Single-Molecule Techniques:
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Direct observation of rotational catalysis in individual ATP synthase complexes
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Measurement of torque generation and energy transduction efficiency
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Correlation of structural dynamics with functional outputs
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Advanced Metabolic Flux Analysis:
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Integration of ATP synthase function with whole-cell metabolism
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Real-time monitoring of metabolic responses to ATP synthase perturbation
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Constraint-based modeling to predict system-level effects of atpF modifications
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Synthetic Biology Approaches:
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Design of minimal ATP synthase complexes with defined components
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Creation of hybrid systems incorporating components from different species
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Engineering of novel regulatory mechanisms for controlled expression
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Systems Biology Integration:
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Multi-omics data integration for comprehensive understanding
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Machine learning approaches to identify non-obvious patterns and relationships
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Predictive modeling of how ATP synthase modifications affect cell physiology
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These technologies will build upon the foundation established by genome-scale metabolic-flux analysis of M. succiniciproducens , enabling researchers to develop increasingly sophisticated models of how ATP synthase function integrates with the specialized metabolism of this organism.
How might understanding atpF function contribute to broader research on bacterial energy metabolism?
Research on M. succiniciproducens atpF has implications that extend far beyond this specific organism:
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Fundamental Bioenergetic Principles:
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Insights into the molecular mechanisms of chemiosmotic energy conservation
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Understanding of structure-function relationships in rotary molecular machines
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Elucidation of protein-protein interactions in multisubunit complexes
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Metabolic Engineering Applications:
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Strategies for optimizing cellular energetics in biotechnological applications
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Approaches for balancing ATP supply and demand in engineered pathways
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Methods for redirecting metabolic flux toward desired products
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Evolutionary Biology:
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Understanding of how ATP synthase co-evolved with cellular metabolism
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Insights into adaptation to specialized ecological niches
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Identification of conserved bioenergetic principles across diverse life forms
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Comparative Biochemistry:
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Frameworks for comparing energy conservation strategies across species
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Recognition of common principles and species-specific adaptations
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Identification of convergent solutions to bioenergetic challenges
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Research on M. succiniciproducens has already contributed significantly to our understanding of bacterial metabolism, particularly through the development of metabolically engineered strains capable of producing succinic acid without by-product formation . Further studies focusing on ATP synthase function will continue to provide insights applicable to diverse fields including biochemistry, synthetic biology, and bioengineering.
