Recombinant Vibrio vulnificus ATP synthase subunit alpha (atpA), partial

What is the role of ATP synthase in Vibrio vulnificus?

ATP synthase in Vibrio vulnificus serves as the primary machinery for ATP production through oxidative phosphorylation. This multi-subunit enzyme complex utilizes the proton motive force (PMF) generated across the bacterial membrane to catalyze the synthesis of ATP from ADP and inorganic phosphate. In V. vulnificus, ATP synthase I has been identified as particularly important during infection, with the gene cluster (Vv1_1014 to Vv1_1022) showing significant increases in metabolic fluxes during in vivo analyses . The enzyme plays a crucial role in bacterial energy metabolism, which becomes especially important during the transition from estuarine environments to human hosts, where the bacterium must adapt to new energy requirements and environmental conditions.

How is ATP synthase activity regulated in response to environmental changes?

ATP synthase activity in V. vulnificus is regulated through multiple mechanisms that allow rapid adaptation to changing environmental conditions. During the transition from estuarine environments to host tissues, V. vulnificus undergoes extensive metabolic reprogramming involving over 150 genes identified as up-regulated in vivo . This reprogramming affects the regulation of ATP synthase both directly and indirectly.

The regulation appears to involve differential expression of electron transport chain components. Research has revealed that NADH dehydrogenase (complex I) shows significant up-regulation in vivo, while genes associated with complexes II through IV are down-regulated . This selective expression pattern suggests a specialized mechanism for generating the proton motive force required by ATP synthase under in vivo conditions.

Importantly, the ATP synthase I cluster itself shows increased metabolic fluxes during infection despite lacking corresponding transcriptional up-regulation . This indicates that post-transcriptional regulatory mechanisms, such as allosteric regulation, protein modifications, or alterations in enzyme kinetics, may play significant roles in modulating ATP synthase activity during infection.

What is the specific function of the alpha subunit (atpA) in the ATP synthase complex?

The alpha subunit (atpA) is a core component of the F₁ portion of ATP synthase, which forms the catalytic head of the enzyme complex. In the fully assembled ATP synthase, alpha subunits alternate with beta subunits to form a hexameric ring structure (α₃β₃) that contains the catalytic sites for ATP synthesis. While the beta subunits contain the primary catalytic sites, the alpha subunits play essential supporting roles in the catalytic mechanism.

The alpha subunit serves several critical functions within the ATP synthase complex:

• It contains nucleotide-binding sites that, while not directly catalytic, participate in the binding-change mechanism that drives ATP synthesis.

• It undergoes conformational changes during the catalytic cycle that are essential for enzyme function.

• It contributes to the stability and structural integrity of the F₁ complex.

• It participates in the transmission of conformational changes from the rotating central stalk to the catalytic sites.

In V. vulnificus, the alpha subunit is encoded within the ATP synthase I gene cluster (Vv1_1014 to Vv1_1022), which has been identified as crucial for energy production during infection . The proper function of atpA is essential for maintaining intracellular ATP levels, particularly under the oxygen-limited conditions encountered during infection.

How does ATP synthase contribute to V. vulnificus pathogenicity?

ATP synthase plays a crucial role in V. vulnificus pathogenicity by providing the energy necessary for various virulence-associated functions. Research has demonstrated that ATP synthase I is particularly important during V. vulnificus infection, with insertion mutants in the ATP synthase I gene cluster showing in vivo growth retardation and reduced intracellular ATP levels under oxygen-limited conditions .

The contribution of ATP synthase to pathogenicity includes:

• Supporting the energy requirements for the production and secretion of virulence factors, which are often energy-intensive processes.

• Enabling adaptation to the host environment through metabolic flexibility, allowing the bacterium to utilize available energy sources efficiently.

• Providing energy for stress response systems that protect the bacterium from host defense mechanisms.

• Maintaining cellular homeostasis and supporting bacterial replication within the host.

The metabolic reprogramming observed in V. vulnificus following infection includes modifications to its energy production pathways, with NADH dehydrogenase (complex I) emerging as a significant contributor to ATP synthase-mediated energy production in vivo . This specialized mechanism appears to be an adaptive strategy that enhances bacterial survival and virulence within the host environment.

What structural features of the ATP synthase alpha subunit are important for its function?

The ATP synthase alpha subunit contains several conserved structural features that are crucial for its function within the enzyme complex. These features have been elucidated through structural studies of ATP synthases from various organisms, providing insights applicable to V. vulnificus atpA.

Key structural features include:

• A nucleotide-binding domain with a classical Rossmann fold, characterized by alternating β-strands and α-helices that form a pocket for binding ATP or ADP.

• Highly conserved residues that interact with the phosphate groups of bound nucleotides, typically including a P-loop (phosphate-binding) motif.

• Interface regions that mediate interactions with neighboring beta subunits, forming the catalytic sites at the alpha-beta interfaces.

• Regions that interact with the central stalk (gamma subunit), allowing the transmission of rotational energy to conformational changes in the catalytic sites.

• Amino acid sequences that contribute to the stability of the hexameric ring structure.

The functional importance of these structural features is highlighted by their high conservation across bacterial species. In V. vulnificus, the ATP synthase I gene cluster that includes atpA has been identified as crucial for energy production during infection , indicating that the structural integrity and proper function of the alpha subunit are essential for bacterial pathogenicity.

How does NADH dehydrogenase contribute to ATP synthase-mediated energy production in V. vulnificus?

NADH dehydrogenase (complex I) plays a crucial role in energizing ATP synthase I for energy production in V. vulnificus, particularly during infection. Research has revealed a distinctive mechanism for ATP generation in vivo compared to in vitro conditions. While the classical model of cellular respiration involves five membrane-bound complexes working together, transcriptomic analyses of V. vulnificus during infection showed significant up-regulation of only complex I, while genes associated with complexes II through IV exhibited down-regulation .

These findings suggest that NADH dehydrogenase specifically contributes to proton motive force (PMF) generation and subsequent ATP synthesis during established V. vulnificus infections in vivo. This specialized role highlights an important adaptation in the bacterium's energy metabolism during infection, where complex I appears to become the primary driver of PMF generation to support ATP synthase I activity.

What are the differences in ATP synthase function between in vitro and in vivo conditions?

ATP synthase function in V. vulnificus exhibits remarkable differences between in vitro and in vivo conditions, reflecting the bacterium's metabolic adaptation to the host environment. Computational simulations using the genome-scale metabolic network model VvuMBEL943 have revealed that the ATP synthase I cluster exhibits significant increases in metabolic fluxes during in vivo analyses, despite not showing corresponding up-regulation at the transcriptional level .

Key differences include:

• Dependency on specific electron transport chain components: In vivo, ATP synthase appears highly dependent on NADH dehydrogenase (complex I) for PMF generation, while this dependency is less pronounced in vitro .

• Oxygen availability impact: Under the oxygen-limited conditions typically encountered during infection, ATP synthase I becomes particularly important for energy production. Insertion mutants in the ATP synthase I gene cluster have demonstrated in vivo growth retardation and reduced intracellular ATP levels specifically under oxygen-limited conditions .

• Energy output efficiency: The selective up-regulation of complex I combined with down-regulation of complexes II-IV in vivo suggests a potential shift in energy production efficiency, possibly representing an adaptation to the resources available within the host environment.

• Regulatory mechanisms: The discrepancy between transcriptional levels and metabolic flux suggests that post-transcriptional regulation may play a more significant role in controlling ATP synthase activity in vivo compared to in vitro.

These differences highlight the complex metabolic reprogramming that occurs in V. vulnificus during infection, with ATP synthase function being modulated to meet the specific energy requirements of pathogenesis.

How do mutations in ATP synthase genes affect bacterial virulence and survival in vivo?

Mutations in ATP synthase genes can significantly impact V. vulnificus virulence and survival within the host environment. Research has demonstrated that the ATP synthase I gene cluster (Vv1_1014 to Vv1_1022) is crucial for energy production during infection . Studies with insertion mutants in this cluster have shown in vivo growth retardation and reduced intracellular ATP levels under oxygen-limited conditions, highlighting the essential nature of ATP synthase I for bacterial survival during infection.

The effects of ATP synthase mutations on virulence and survival are multi-faceted:

• Energy deficit: Mutations that compromise ATP synthase function reduce the bacterium's ability to generate energy, limiting resources available for virulence factor production, replication, and stress responses.

• Growth impairment: As demonstrated with both ATP synthase I mutants and the related NADH dehydrogenase mutant, defects in energy production pathways lead to significant growth retardation specifically under in vivo conditions .

• Adaptation capacity: ATP synthase mutations may limit the bacterium's ability to adapt to changing host environments, reducing its persistence during infection.

• Context-dependent effects: Importantly, the impact of ATP synthase mutations appears highly dependent on environmental conditions. The NADH dehydrogenase deletion mutant showed normal growth and ATP levels in vitro but exhibited pronounced defects in vivo , suggesting that ATP synthase deficiencies may have similarly context-dependent effects.

These findings highlight the potential of ATP synthase components as therapeutic targets, as their essential role in in vivo energy production makes them critical vulnerabilities in V. vulnificus pathogenesis.

What is the relationship between ATP synthase activity and metabolic reprogramming during infection?

The relationship between ATP synthase activity and metabolic reprogramming during V. vulnificus infection represents a sophisticated adaptation strategy that enhances bacterial survival within the host. Research has uncovered extensive metabolic reprogramming in V. vulnificus post-infection, with over 150 genes identified as up-regulated in vivo .

Within this reprogramming, ATP synthase occupies a central position:

• Flux-expression discrepancy: Computational simulations have revealed that the ATP synthase I cluster exhibits significant increases in metabolic fluxes during in vivo analyses, despite not showing corresponding up-regulation at the transcriptional level . This suggests that metabolic reprogramming affects ATP synthase activity through mechanisms beyond simple expression changes.

• Specialized PMF generation: The selective up-regulation of NADH dehydrogenase (complex I) alongside down-regulation of complexes II-IV represents a fundamental reconfiguration of the electron transport chain that directly impacts ATP synthase function .

• Adaptation to host resources: Metabolic reprogramming likely optimizes ATP synthase function for the specific nutrient and oxygen availability encountered during infection, maximizing energy production efficiency under challenging conditions.

• Integration with virulence mechanisms: The coordination between ATP synthase activity and virulence factor expression suggests that metabolic reprogramming synchronizes energy production with pathogenicity functions.

This intricate relationship highlights how V. vulnificus has evolved sophisticated regulatory mechanisms to ensure adequate energy production during infection, with ATP synthase activity being modulated as part of a broader metabolic adaptation strategy that enhances bacterial fitness within the host environment.

How can systems biology approaches enhance our understanding of ATP synthase function?

Systems biology approaches provide powerful frameworks for understanding ATP synthase function within the broader context of V. vulnificus metabolism and pathogenicity. The research on V. vulnificus energy metabolism demonstrates the value of these approaches in revealing non-intuitive relationships and context-specific functions .

Key applications and benefits include:

• Predictive modeling: Computational simulations using genome-scale metabolic network models, such as VvuMBEL943 for V. vulnificus, can predict metabolic fluxes through ATP synthase under different conditions. This approach revealed increased flux through ATP synthase I during infection despite lack of transcriptional up-regulation .

• Integration of multi-omics data: Combining transcriptomics, proteomics, and metabolomics provides a comprehensive view of ATP synthase regulation across different biological levels. This integration helped identify the discrepancy between gene expression and metabolic flux in V. vulnificus ATP synthase during infection .

• Network-level insights: Systems approaches can identify how ATP synthase interacts with other cellular processes, revealing unexpected relationships. This led to the discovery of the critical role of NADH dehydrogenase in energizing ATP synthase I specifically during infection .

• Context-specific function prediction: By analyzing network behavior under different conditions, systems biology can predict how ATP synthase function adapts to specific environments, explaining the observed differences between in vitro and in vivo phenotypes of energy metabolism mutants .

• Therapeutic target identification: Systems-level analysis can identify vulnerabilities in bacterial energy metabolism networks that could be exploited for antimicrobial development, with ATP synthase and its regulatory network representing potential targets.

The discovery of metabolic reprogramming in V. vulnificus post-infection exemplifies how systems approaches can reveal complex adaptive strategies that would be difficult to discern through reductionist methods alone .

What are the best methods for expressing and purifying recombinant V. vulnificus atpA?

Successful expression and purification of recombinant V. vulnificus ATP synthase subunit alpha (atpA) requires careful consideration of expression systems, vectors, and purification strategies. Based on approaches used for related proteins, the following methodologies are recommended:

For expression:

• Expression host selection: Escherichia coli BL21(DE3) or Rosetta strains are generally preferred for expressing V. vulnificus proteins due to their high expression levels and reduced protease activity. The Rosetta strain may be particularly advantageous if atpA contains rare codons.

• Vector design: pET-based expression vectors with T7 promoters offer strong, inducible expression. Adding an N-terminal or C-terminal His₆-tag facilitates purification while minimizing interference with protein function.

• Optimized conditions: Lowering the post-induction temperature to 16-20°C and using moderate IPTG concentrations (0.1-0.5 mM) often improves soluble protein yield by reducing inclusion body formation.

For purification:

• Affinity chromatography: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is effective for His-tagged atpA purification.

• Buffer optimization: Including ATP or ADP (1-5 mM) and Mg²⁺ (5-10 mM) in purification buffers can stabilize the protein structure.

• Additional purification steps: Ion exchange chromatography followed by size exclusion chromatography can achieve high purity necessary for structural and functional studies.

Similar approaches have been successfully employed for cloning and expressing V. vulnificus genes in previous studies . When expressing atpA for functional studies, co-expression with other ATP synthase subunits may be necessary, as the alpha subunit alone might not exhibit full functionality.

How can one effectively measure ATP synthase activity in V. vulnificus?

Measuring ATP synthase activity in V. vulnificus can be accomplished through several complementary approaches, each with specific advantages for different experimental questions:

• Intracellular ATP quantification: The luciferase-based ATP assay provides a sensitive measure of total cellular ATP levels, which reflect ATP synthase activity in vivo. This approach was successfully used in the study of NADH dehydrogenase mutants and involves:

  • Harvesting cells from various growth conditions

  • Cell lysis in appropriate buffer

  • ATP measurement using recombinant firefly luciferase and D-luciferin substrate

  • Normalization by cell number or protein content

  • Comparison to standard curve generated using control ATP samples

• Membrane vesicle assays: For more direct measurement of ATP synthase activity:

  • Preparation of inverted membrane vesicles from V. vulnificus

  • Measurement of ATP synthesis upon addition of NADH or succinate (to generate PMF)

  • Quantification of synthesized ATP using the luciferase assay

  • Inclusion of controls with specific inhibitors (e.g., DCCD for ATP synthase)

• Proton translocation measurement: Using pH-sensitive fluorescent probes like ACMA (9-amino-6-chloro-2-methoxyacridine) to monitor proton translocation associated with ATP synthase activity.

• Oxygen consumption assays: Measuring respiratory activity coupled to ATP synthesis using an oxygen electrode system.

The choice of method depends on the specific aspect of ATP synthase function being investigated. For assessing the role of ATP synthase in vivo, the intracellular ATP measurement approach has proven particularly valuable, as it directly demonstrates the impact of mutations on cellular energy levels under physiologically relevant conditions .

What animal models are most effective for studying V. vulnificus ATP synthase function in vivo?

Several animal models have been established for studying V. vulnificus pathogenesis, with the rat peritoneal infection model demonstrating particular utility for investigating ATP synthase function in vivo. This model was successfully employed in the study of NADH dehydrogenase's contribution to ATP synthase-mediated energy production .

The rat peritoneal infection model involves:

• Preparation of bacterial suspensions (typically 2 ml of 5 × 10⁵ CFU/mL) in dialysis tubes

• Surgical implantation of these tubes into rat peritoneal cavities

• Incubation for a defined period (e.g., 4 hours)

• Retrieval of bacteria and analysis of growth and ATP levels

This model effectively simulates in vivo conditions encountered during infection, including oxygen limitation and host defense factors. It successfully demonstrated that the NADH dehydrogenase deletion mutant exhibited pronounced growth retardation and reduced intracellular ATP levels specifically under in vivo conditions .

Alternative animal models include:

• Mouse infection models: Often requiring iron overloading to increase susceptibility to V. vulnificus

• Zebrafish models: Offering advantages for real-time visualization of infection processes

• Invertebrate models: Providing ethical and cost advantages for initial screening studies

The choice of model should be guided by the specific aspects of ATP synthase function being investigated. The rat peritoneal infection model has proven particularly valuable for energy metabolism studies as it allows direct recovery of bacteria for subsequent biochemical analyses, facilitating direct measurement of intracellular ATP levels under in vivo conditions .

What molecular genetic techniques are most useful for studying ATP synthase genes?

Several molecular genetic techniques have proven particularly valuable for studying ATP synthase genes in V. vulnificus:

• Allelic exchange mutagenesis: This method allows precise deletion or modification of ATP synthase genes. The approach used for generating the NADH dehydrogenase deletion mutant can be adapted for atpA studies:

  • Amplification of upstream and downstream fragments flanking the target gene

  • Fusion of these fragments through crossover PCR

  • Cloning into appropriate vectors (e.g., TOPO® TA vector)

  • Selection of recombinants through marker-based strategies

• Complementation analysis: Essential for confirming that observed phenotypes are specifically due to the targeted mutation:

  • Cloning the wild-type gene into an expression vector

  • Introduction into the mutant strain

  • Verification of phenotype restoration, as demonstrated with the NADH dehydrogenase revertant strain

• Reporter gene fusions: For studying gene expression patterns:

  • Fusion of ATP synthase gene promoters with reporter genes (e.g., lacZ, gfp)

  • Monitoring expression under different conditions to understand regulation

• Site-directed mutagenesis: For investigating specific functional residues:

  • Introduction of point mutations in catalytic sites or regulatory regions

  • Analysis of consequences on ATP synthase function and bacterial physiology

• RT-qPCR: For quantifying gene expression levels under different conditions, as used to assess transcriptional expression of electron transport chain components in the referenced study .

These techniques, combined with biochemical and physiological assays, provide a comprehensive toolkit for investigating ATP synthase function and regulation in V. vulnificus, enabling researchers to connect genetic modifications with functional outcomes at both molecular and organismal levels.

How can structural biology approaches enhance our understanding of V. vulnificus ATP synthase?

Structural biology approaches offer powerful tools for understanding the molecular details of V. vulnificus ATP synthase function and regulation. While the provided materials don't directly discuss structural studies of V. vulnificus ATP synthase, several approaches can be applied based on methods used for related systems:

• X-ray crystallography: Determining high-resolution structures of isolated ATP synthase components, particularly the F₁ portion containing the alpha subunit. This can reveal:

  • Precise arrangement of catalytic and regulatory sites

  • Conformational states associated with different steps of the catalytic cycle

  • Binding interfaces between subunits

  • Structural basis for nucleotide binding and hydrolysis

• Cryo-electron microscopy (cryo-EM): Particularly valuable for studying the intact ATP synthase complex:

  • Visualization of the complete enzyme architecture

  • Identification of conformational changes during catalysis

  • Characterization of membrane integration of the F₀ portion

  • Structural differences between active and inactive states

• Nuclear magnetic resonance (NMR) spectroscopy: Useful for studying dynamic aspects of smaller ATP synthase components:

  • Conformational dynamics in solution

  • Protein-ligand interactions

  • Local structural changes upon nucleotide binding

• Molecular dynamics simulations: Complementing experimental approaches to understand:

  • Conformational flexibility not captured in static structures

  • Energy transduction mechanisms

  • Water and ion movements through the complex

• Hydrogen/deuterium exchange mass spectrometry (HDX-MS): For probing conformational changes and protein dynamics:

  • Identification of regions with altered solvent accessibility during catalysis

  • Detection of allosteric networks within the complex

Structural insights would be particularly valuable for understanding the unique features of V. vulnificus ATP synthase that contribute to its specialized function during infection, potentially revealing adaptation mechanisms that could be targeted for antimicrobial development.

How should researchers interpret ATP levels when comparing wild-type and mutant V. vulnificus strains?

Key considerations include:

What are the key considerations when designing experiments to study ATP synthase function?

Designing robust experiments to study ATP synthase function in V. vulnificus requires careful consideration of several key factors:

The study of NADH dehydrogenase's role in ATP synthase-mediated energy production demonstrates the value of this comprehensive approach, revealing function-specific roles that would not have been detected with more limited experimental designs .

How can researchers effectively compare results from in vitro and in vivo ATP synthase studies?

Effectively comparing results from in vitro and in vivo ATP synthase studies requires careful consideration of the fundamental differences between these experimental contexts. The research on V. vulnificus energy metabolism provides several valuable strategies for such comparisons :

• Parallel experimental design: Conduct as many measurements as possible using identical methods across both conditions. The referenced study measured intracellular ATP levels and growth parameters using the same methodologies in both in vitro cultures and samples from the rat peritoneal infection model , enabling direct comparison.

• Normalization strategies: Develop consistent normalization approaches that work across different experimental contexts. In the NADH dehydrogenase study, ATP measurements were normalized to viable cell counts in both in vitro and in vivo samples , ensuring comparability despite different growth environments.

• Bridging experiments: When direct comparisons are challenging, design intermediate conditions that progressively introduce in vivo-like features to in vitro experiments. For V. vulnificus, this might include oxygen-limited in vitro cultures that partially mimic the in vivo environment.

• Systems biology integration: Use computational modeling to predict how network-level properties might create condition-specific dependencies. The genome-scale metabolic network model VvuMBEL943 helped identify increased metabolic flux through ATP synthase I during infection despite lack of transcriptional up-regulation .

• Multi-omics correlation: When possible, correlate functional measurements with transcriptomic, proteomic, or metabolomic data to understand the underlying mechanisms of observed differences. The identification of up-regulated NADH dehydrogenase expression in vivo provided mechanistic insight into the observed functional differences.

How can researchers integrate ATP synthase findings into broader understanding of bacterial metabolism?

Integrating ATP synthase findings into a broader understanding of bacterial metabolism requires connecting enzyme-level observations with system-wide metabolic networks and physiological outcomes. The research on V. vulnificus energy metabolism demonstrates several effective strategies for such integration :

• Systems biology frameworks: Using genome-scale metabolic models like VvuMBEL943 to place ATP synthase function within the context of entire metabolic networks. This approach revealed increased metabolic flux through ATP synthase I during infection despite lack of transcriptional up-regulation , highlighting its central role in metabolic adaptation.

• Multi-omics integration: Combining ATP synthase functional data with transcriptomic, proteomic, and metabolomic analyses to understand regulatory mechanisms. The discovery of up-regulated NADH dehydrogenase expression in vivo alongside down-regulation of other respiratory complexes provided critical context for understanding ATP synthase function during infection.

• Phenotypic correlation: Connecting biochemical measurements with physiological outcomes. The correlation between reduced intracellular ATP levels and growth retardation in the NADH dehydrogenase mutant specifically under in vivo conditions established the functional significance of the observed metabolic changes.

• Comparative analysis across conditions: Examining how ATP synthase function varies between different growth conditions reveals adaptation strategies. The condition-specific dependency on NADH dehydrogenase for energy production highlighted metabolic flexibility as a key feature of V. vulnificus pathogenicity.

• Network-based interpretation: Analyzing how perturbations to ATP synthase affect other metabolic pathways. The metabolic reprogramming observed in V. vulnificus following infection, involving over 150 up-regulated genes , represents a coordinated response that integrates energy production with various aspects of bacterial physiology.

By applying these integrative approaches, researchers can move beyond isolated observations of ATP synthase function to understand its role within the complex, adaptive metabolic networks that underlie V. vulnificus pathogenicity.