Recombinant Chromobacterium violaceum ATP synthase epsilon chain (atpC)

What is the ATP synthase epsilon chain (atpC) in Chromobacterium violaceum?

The ATP synthase epsilon chain in C. violaceum is a regulatory subunit of the F-type ATP synthase, which functions as a molecular nanomotor converting the energy from proton translocation into ATP synthesis. As in other bacterial species, this subunit likely consists of approximately 130-140 amino acid residues with two distinct domains: an N-terminal domain that binds to the gamma subunit and a C-terminal domain (εCTD) that undergoes conformational changes in response to cellular energy conditions.

The epsilon chain serves as a critical regulator of ATP synthase activity, primarily preventing wasteful ATP hydrolysis under unfavorable energy conditions. This regulatory function is particularly important for bacteria that must adapt to changing environmental conditions, including pathogens like C. violaceum that transition between different host environments during infection .

How is the atpC gene structure in C. violaceum compared to other bacterial species?

While specific comparisons of C. violaceum atpC to other bacterial species are not directly addressed in the search results, broader patterns of conservation can be inferred. The "full-length" epsilon chains (~130-140 residues) are found in species across most bacterial phyla and exhibit inhibitory behavior in ATP synthase function .

Some bacterial lineages, including certain α-proteobacteria and members of Actinobacteria, Bacteroidetes, and Chlorobi phyla, completely lack the C-terminal domain of epsilon (εCTD). Other genera such as Thermotoga and Mycobacterium possess significantly shortened εCTD regions . As a member of the beta-proteobacteria, C. violaceum likely retains a full-length epsilon chain with an intact εCTD, since this structure appears to be conserved across diverse bacterial lineages.

The conservation of the εCTD in most bacterial species suggests its fundamental importance in ATP synthase regulation, which would apply to C. violaceum as well, despite variations in specific sequence and structure that may have evolved to suit particular ecological niches .

What are the structural features of bacterial ATP synthase epsilon chains relevant to C. violaceum research?

The bacterial ATP synthase epsilon chain typically exhibits two distinct conformational states that are critical to its regulatory function:

• The "down" or contracted state, where the C-terminal domain is positioned away from the catalytic core of the F₁ component, allowing unrestricted rotation of the gamma subunit.

• The "up" or extended state, where the C-terminal domain is inserted into the central cavity of the F₁ component, physically blocking rotation and inhibiting both ATP synthesis and hydrolysis .

The transition between these conformational states is influenced by:

• Nucleotide binding: The epsilon chain can respond to ATP/ADP ratio, with high ADP/Pi levels typically stabilizing the inhibitory "up" state.

• Proton motive force (pmf): Evidence suggests that pmf promotes release of the εCTD from its inserted, inhibitory state, thereby coupling energy status to ATP synthase regulation .

These structural features are likely conserved in C. violaceum and would be crucial considerations for researchers working with recombinant atpC, particularly when investigating its regulatory functions in isolation or in reconstituted systems.

What experimental methods are used for basic characterization of recombinant epsilon chains?

For basic characterization of recombinant C. violaceum ATP synthase epsilon chain, researchers typically employ the following methodological approaches:

• Expression optimization: Testing various expression systems (E. coli being the most common) with different induction conditions, temperature regimes, and fusion tags to maximize soluble protein yield.

• Purification strategies: Typically involving affinity chromatography (His-tag or other fusion tags), followed by size exclusion and/or ion-exchange chromatography to obtain pure protein.

• Conformational analysis: Circular dichroism spectroscopy to assess secondary structure content and thermal stability.

• Nucleotide binding assays: Fluorescence-based techniques or isothermal titration calorimetry to measure binding of ATP/ADP to the epsilon chain.

• Interaction studies: Pull-down assays or surface plasmon resonance to analyze binding to gamma subunit or other ATP synthase components.

The specific choice of methods would depend on the research questions being addressed. Based on studies of other bacterial epsilon chains, researchers should consider the protein's conformational dynamics and potential for aggregation when designing purification and analysis protocols .

How does the C-terminal domain of epsilon (εCTD) regulate ATP synthase activity?

The C-terminal domain of the epsilon chain (εCTD) serves as a sophisticated regulatory switch that modulates ATP synthase activity in response to cellular energy status. This regulation involves several mechanisms:

• Physical inhibition: In its extended "up" conformation, the εCTD inserts into the central cavity of the F₁ component, physically blocking the rotation of the gamma subunit that is essential for catalysis. This inhibits both ATP synthesis and hydrolysis .

• Nucleotide sensing: The conformation of the εCTD responds to cellular nucleotide conditions. High ATP levels generally favor a non-inhibitory conformation, while elevated ADP/Pi levels stabilize the inhibitory state, allowing the bacterium to prevent wasteful ATP hydrolysis when energy is scarce .

• Response to proton motive force (pmf): Evidence indicates that pmf promotes release of the εCTD from its inhibitory position. This ensures that ATP synthesis proceeds when sufficient pmf is available to drive it, while hydrolysis is prevented when pmf is low .

• Coupling efficiency enhancement: Beyond simple inhibition, the εCTD contributes to the efficiency of coupling between F₁ and F₀ components, acting as a "bi-directional ratchet" that optimizes energy conversion in both directions .

• Reduction of futile cycling: The εCTD is particularly involved in preventing uncoupled ATPase activity, which would waste energy without contributing to pmf generation .

These regulatory mechanisms are likely conserved in C. violaceum, providing precise control over its energy metabolism in response to changing environmental conditions.

What are the most effective expression systems for producing functional recombinant C. violaceum atpC?

For researchers seeking to produce functional recombinant C. violaceum ATP synthase epsilon chain, several expression strategies warrant consideration:

• E. coli expression systems: These represent the most straightforward approach and have been successfully used for ATP synthase components from various bacterial species. Research indicates that thermophilic bacterial ATP synthase (PS3-F₀F₁) was effectively expressed in E. coli membranes . For the epsilon chain specifically:

  • BL21(DE3) derivatives with reduced protease activity are recommended

  • Codon optimization may improve expression levels

  • Growth at lower temperatures (16-25°C) after induction often enhances solubility

  • Fusion tags (SUMO, MBP) can improve solubility while maintaining function

• Cell-free expression systems: These can be advantageous for regulatory proteins that may be toxic when overexpressed in cells or that require specific folding conditions.

• Co-expression strategies: For functional studies, co-expressing the epsilon chain with its interaction partners (particularly the gamma subunit) can enhance stability and native folding. As noted in the search results, PS3-F₀F₁ components have been successfully co-expressed in E. coli membranes for functional studies .

Table 1: Comparison of Expression Systems for Recombinant ATP Synthase Components

The optimal choice depends on the intended application, with structural studies typically requiring higher purity and yield, while functional assays may prioritize native conformation and activity.

How can researchers effectively study the inhibitory mechanisms of C. violaceum epsilon chain?

Investigating the inhibitory mechanisms of C. violaceum ATP synthase epsilon chain requires sophisticated experimental approaches:

• Site-directed mutagenesis: Systematic mutation of residues in the C-terminal domain allows identification of specific amino acids critical for inhibitory function. Based on homology with other bacterial epsilon chains, researchers should target:

  • Conserved residues in the hinge region connecting N- and C-terminal domains

  • Residues predicted to interact with the gamma subunit

  • Potential nucleotide-binding residues

• Cross-linking experiments: This approach has proven valuable for studying epsilon chain function. As seen in research with PS3-F₀F₁, oxidation-induced disulfide formation between strategically placed cysteine residues in gamma and epsilon subunits can trap specific conformational states for functional analysis .

• Reconstitution into liposomes: Purified recombinant components can be reconstituted into liposomes to measure ATP synthesis and hydrolysis activities under controlled conditions. This approach allows manipulation of the pmf and nucleotide concentrations to assess their effects on epsilon-mediated regulation .

• Single-molecule techniques: These have been successfully applied to F₁-ATPase to measure rotation dynamics and the influence of regulatory factors. Single-molecule studies revealed that the efficiency of ATP synthesis was significantly reduced (30-50%) in constructs lacking the epsilon subunit .

• Conformational analysis: Techniques such as hydrogen-deuterium exchange mass spectrometry (HDX-MS) can track conformational changes in the epsilon chain in response to nucleotides, pH changes, and interaction with other subunits.

These methodologies, used in complementary fashion, can provide a comprehensive understanding of how the C. violaceum epsilon chain regulates ATP synthase activity in response to changing cellular energy conditions.

What is the relationship between C. violaceum pathogenicity and ATP synthase regulation?

While the search results don't directly address connections between C. violaceum ATP synthase regulation and pathogenicity, several potential relationships can be inferred from our understanding of bacterial pathogenesis and ATP synthase function:

• Energy requirements during infection: C. violaceum causes severe infections in humans and animal models, including fulminant hepatitis in mice and bacteremia in humans . During infection, bacteria encounter diverse host microenvironments with varying nutrient availability, oxygen levels, and pH. Precise regulation of energy metabolism through the ATP synthase epsilon chain would be critical for survival under these changing conditions.

• Adaptation to host defense mechanisms: When C. violaceum encounters host immune cells, it must rapidly adapt its metabolism. The search results indicate that C. violaceum can escape from phagosomes in epithelial cells through mechanisms involving its type III secretion system (T3SS) . The energetics of T3SS assembly and function require ATP, potentially linking ATP synthase regulation to this virulence mechanism.

• Response to stress conditions: Host-induced stresses such as reactive oxygen species and nutrient limitation require metabolic adaptation. The epsilon chain's ability to prevent wasteful ATP hydrolysis under unfavorable conditions may contribute to stress tolerance during infection.

• Potential therapeutic target: Given its essential role in bacterial energy metabolism, the ATP synthase (including the epsilon chain) represents a potential target for antimicrobial development. Understanding C. violaceum-specific features of the epsilon chain could inform such efforts.

Future research should specifically investigate whether mutations affecting ATP synthase regulation influence C. violaceum virulence in animal models, and whether host conditions modulate epsilon chain conformation and function during infection.

How might the C. violaceum epsilon chain interact with other bacterial systems?

The potential interactions between the ATP synthase epsilon chain and other C. violaceum cellular systems represent an intriguing area for research:

• Quorum sensing integration: C. violaceum employs a sophisticated LuxIR-type quorum sensing system (CviI/CviR) that regulates virulence, pigment production, biofilm formation, and chitinase production . The energy-intensive processes regulated by quorum sensing likely require coordination with cellular energy status, potentially involving ATP synthase regulation. The search results indicate that quorum sensing controls virulence in C. violaceum , suggesting a possible functional connection with energy metabolism during infection.

• Type III secretion system (T3SS) energetics: C. violaceum possesses two T3SSs encoded in pathogenicity islands (Cpi-1/1a and Cpi-2), with Cpi-1/1a being critical for virulence . T3SS assembly and function require substantial energy input, suggesting potential coordination between T3SS activity and ATP synthase regulation, especially during host cell interaction.

• Metabolic checkpoint function: The epsilon chain's ability to respond to ATP/ADP ratios positions it as a potential metabolic checkpoint that could influence various cellular processes. In bacteria, energy status often serves as a master regulator of numerous pathways, including those involved in virulence.

• Response to environmental transitions: C. violaceum inhabits diverse environments, from aquatic ecosystems to mammalian hosts during infection . Transitions between these environments involve significant changes in metabolic demands, potentially requiring coordinated regulation of ATP synthase and other systems.

• Stress response integration: Under stress conditions (oxidative stress, nutrient limitation, antimicrobial exposure), bacteria often reallocate energy resources. The epsilon chain's regulatory function could participate in this reallocation by modulating ATP synthesis/hydrolysis in response to stress-induced metabolic changes.

Experimental approaches to investigate these potential interactions could include transcriptomic and proteomic analyses under various growth conditions, genetic manipulation of atpC combined with phenotypic characterization, and direct protein-protein interaction studies.

What methods can be used to investigate the conformational dynamics of C. violaceum epsilon chain?

Investigating the conformational dynamics of the C. violaceum ATP synthase epsilon chain requires specialized methodologies that can capture its transitions between regulatory states:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique can map regions of the protein that undergo conformational changes in response to nucleotides, pH, or interaction with other ATP synthase components. By measuring the rate of hydrogen exchange in different parts of the protein under various conditions, researchers can identify which regions become more exposed or protected.

• FRET (Förster Resonance Energy Transfer): By introducing fluorescent probes at strategic positions in the epsilon chain, researchers can monitor conformational changes in real-time. This approach has been particularly valuable for studying the transition between "up" and "down" states of the epsilon C-terminal domain.

• Cross-linking coupled with mass spectrometry: As mentioned in search result 2, cross-linking has been used to study ATP synthase regulation . This approach can trap specific conformational states and identify interaction interfaces between the epsilon chain and other subunits, particularly gamma.

• Single-molecule techniques: These approaches can directly observe conformational dynamics of individual molecules, avoiding ensemble averaging. Techniques such as single-molecule FRET or high-speed atomic force microscopy have been applied to ATP synthase components to visualize conformational changes during function.

• Molecular dynamics simulations: Computational approaches can model the conformational dynamics of the epsilon chain based on structural data from homologous proteins. These simulations can predict the energetics of conformational transitions and the effects of mutations or ligand binding.

• EPR spectroscopy: By introducing spin labels at specific positions, electron paramagnetic resonance spectroscopy can measure distances between labeled sites and track conformational changes under different conditions.

Table 2: Comparison of Methods for Studying Epsilon Chain Conformational Dynamics

These complementary approaches can provide a comprehensive understanding of how the C. violaceum epsilon chain transitions between regulatory states in response to cellular energy conditions.

What data analysis approaches are optimal for interpreting epsilon chain functional studies?

When analyzing data from functional studies of C. violaceum ATP synthase epsilon chain, researchers should consider the following analytical approaches:

• Kinetic modeling: ATP synthase regulation involves complex kinetics with multiple states. Fitting experimental data to mathematical models can provide insights into:

  • Rate constants for transitions between regulatory states

  • Effects of mutations on these transitions

  • Influence of nucleotides and pmf on regulatory dynamics

• Comparative analysis: Comparing the C. violaceum epsilon chain with homologs from other bacterial species can identify conserved and unique features. The search results indicate significant variation in epsilon chain structure and function across bacterial lineages . Systematic analysis of these differences in relation to ecological niches can reveal adaptations specific to C. violaceum.

• Structure-function correlation: Integrating structural data with functional measurements allows mapping of critical residues and regions. This approach can identify:

  • Hinge regions important for conformational changes

  • Interaction interfaces with other ATP synthase subunits

  • Potential binding sites for regulatory molecules

• Systems biology integration: Placing epsilon chain function in the broader context of C. violaceum metabolism requires integration with other datasets:

  • Transcriptomic data under various growth conditions

  • Metabolomic profiles showing changes in ATP/ADP ratio

  • Proteomic analysis of ATP synthase subunit expression

• Statistical approaches for single-molecule data: When analyzing single-molecule measurements (as used in several ATP synthase studies ), specialized statistical methods are required to account for molecular heterogeneity and rare events.

These analytical approaches should be applied with consideration of the specific research questions being addressed and the nature of the experimental data collected.

How can researchers design experiments to investigate the role of C. violaceum ATP synthase in pathogenicity?

Designing robust experiments to explore connections between ATP synthase regulation and C. violaceum pathogenicity requires multifaceted approaches:

• Genetic manipulation strategies:

  • Create atpC deletion mutants and complemented strains

  • Engineer point mutations targeting specific regulatory features (εCTD hinge region, interface with gamma subunit)

  • Develop conditional expression systems to modulate atpC expression during infection

• In vitro infection models:

  • Compare wild-type and atpC mutant strains in macrophage infection assays

  • Assess ATP synthase activity during different phases of host cell interaction

  • Measure the effects of host-relevant conditions (pH, oxygen limitation) on epsilon chain conformation

• Animal infection studies:

  • The search results indicate that C. violaceum causes fulminant hepatitis in mice , providing a relevant model

  • Compare tissue colonization and survival of wild-type and atpC mutant strains

  • Assess bacterial energy metabolism in vivo using metabolomic approaches

• Integration with virulence mechanisms:

  • Investigate potential coordination between ATP synthase regulation and T3SS expression/activity

  • The search results highlight the importance of T3SS for C. violaceum virulence

  • Examine whether atpC mutations affect T3SS-dependent phenotypes

• Host response considerations:

  • Assess whether ATP synthase components are recognized by host immune systems

  • Investigate whether epsilon chain regulation influences bacterial survival in the face of host defenses

  • The search results indicate that C. violaceum infection is controlled in healthy mice by the NLRC4 inflammasome

• Translational potential:

  • Screen for small molecules that specifically target C. violaceum epsilon chain

  • Assess whether such compounds affect bacterial virulence

These experimental approaches should incorporate appropriate controls and employ complementary methodologies to establish robust connections between ATP synthase regulation and pathogenicity.

What are the future research directions for C. violaceum ATP synthase epsilon chain studies?

Future research on the C. violaceum ATP synthase epsilon chain should address several key knowledge gaps and opportunities:

• Structural characterization: Determining the high-resolution structure of C. violaceum epsilon chain in different conformational states would provide a foundation for understanding its regulatory mechanism. This should include both X-ray crystallography and cryo-EM approaches to capture dynamic conformational states.

• Species-specific features: Comparative studies with epsilon chains from other bacterial species could reveal adaptations specific to C. violaceum's ecological niche and pathogenic lifestyle. The search results indicate significant variation in epsilon chain structure and function across bacterial lineages , suggesting potential specialization.

• Integration with virulence mechanisms: Exploring connections between ATP synthase regulation and C. violaceum's virulence factors, particularly the T3SS identified as critical for pathogenicity , could reveal novel aspects of bacterial energy management during infection.

• Regulatory networks: Investigating potential coordination between ATP synthase regulation and other cellular systems, including the quorum sensing system described in the search results , would provide insights into how C. violaceum integrates energy metabolism with other aspects of its biology.

• Therapeutic targeting: Given the importance of ATP synthase for bacterial survival, exploring the epsilon chain as a potential target for antimicrobial development could lead to novel therapeutic approaches for C. violaceum infections, which are often resistant to multiple antibiotics.

• Technological innovations: Developing new methodologies to study epsilon chain dynamics in living bacteria, perhaps through fluorescent biosensors or in vivo crosslinking approaches, could bridge the gap between in vitro mechanistic studies and physiological relevance.

These research directions would collectively advance our understanding of how C. violaceum regulates its energy metabolism in different environments, including during host infection, and could reveal novel principles of bacterial bioenergetics with broader implications.

What are the technical challenges that researchers should anticipate when working with recombinant C. violaceum atpC?

Researchers working with recombinant C. violaceum ATP synthase epsilon chain should be prepared to address several technical challenges:

• Protein stability issues: The epsilon chain undergoes significant conformational changes as part of its regulatory function . These dynamic properties can make the isolated protein unstable or prone to aggregation. Strategies to address this include:

  • Optimizing buffer conditions (ionic strength, pH, additives like glycerol)

  • Co-expression with stabilizing partners, particularly the gamma subunit

  • Engineering constructs that stabilize specific conformational states

• Functional assessment complexity: Unlike enzymes with easily measurable catalytic activities, assessing the regulatory function of the epsilon chain requires sophisticated approaches:

  • Reconstitution with other ATP synthase components

  • Development of conformation-specific antibodies or probes

  • Establishing reliable assays for conformational transitions

• Expression optimization: Achieving sufficient yields of properly folded protein may require extensive optimization:

  • Testing multiple expression systems and conditions

  • Exploring various fusion partners and solubility tags

  • Developing specialized refolding protocols if inclusion bodies form

• Conformational heterogeneity: The epsilon chain likely exists in an equilibrium between different conformational states , complicating structural and functional studies:

  • Methods may be needed to enrich for specific conformations

  • Data analysis must account for mixed populations

  • Conditions that shift the equilibrium should be identified

• Species-specific features: While lessons can be drawn from studies of other bacterial epsilon chains, C. violaceum-specific features may require tailored approaches:

  • Codon optimization for expression hosts

  • Identification of C. violaceum-specific interaction partners

  • Consideration of unique post-translational modifications