Recombinant Chromobacterium violaceum ATP synthase gamma chain (atpG)

What is the structure and function of the ATP synthase gamma chain in C. violaceum?

The ATP synthase gamma chain (atpG) in Chromobacterium violaceum is a critical component of the F-type ATP synthase complex, which consists of two main functional units: F₁ and F₀. The gamma chain forms part of the central stalk of the F₁ domain and plays a crucial role in energy coupling between the proton-translocating F₀ portion and the catalytic F₁ portion.

In C. violaceum, as in other bacteria, the F₀F₁ ATP synthase functions as a multiprotein complex that harnesses the energy of proton gradients to synthesize ATP from ADP and inorganic phosphate. The gamma subunit specifically functions as the rotating central shaft that couples proton movement through F₀ to conformational changes in the catalytic sites of F₁ .

The C. violaceum ATP synthase is similar to the enzyme found in E. coli, belonging to the F₀F₁-type ATP synthase family that is crucial for bacterial energetic metabolism .

How conserved is the atpG gene across bacterial species compared to C. violaceum?

The atpG gene is highly conserved across bacterial species, with significant homology observed between C. violaceum and other bacteria. Sequence analysis shows approximately 48% homology between C. violaceum atpG and the corresponding gene in Desulfovibrio vulgaris . Conservation analysis of ATP synthase subunits reveals:

This high conservation reflects the essential nature of the ATP synthase complex in cellular energy metabolism across diverse bacterial species .

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

For recombinant expression of C. violaceum atpG, heterologous expression in E. coli is typically the most effective approach. Based on successful expression of other C. violaceum proteins, the following expression systems have proven effective:

• pET expression system: Using BL21(DE3) E. coli strains with the pET28a vector allows for high-level expression with an N-terminal or C-terminal His-tag for purification purposes .

• Induction conditions: Optimal expression is typically achieved by inducing cultures at OD₆₀₀ of 0.6-0.8 with 0.1-0.5 mM IPTG, followed by overnight incubation at 18°C to minimize inclusion body formation .

• Alternative vectors: For difficult-to-express proteins, fusion partners such as SUMO tag have been successfully used with C. violaceum proteins to enhance solubility .

For atpG specifically, ensuring proper folding often requires co-expression with other ATP synthase subunits or molecular chaperones to facilitate assembly of functional protein .

What purification strategies yield functional atpG protein?

Purification of functional atpG protein from C. violaceum requires strategies that maintain the protein's native conformation. Based on successful purification of ATP synthase components from other bacterial species, the following approach is recommended:

• Cell lysis: Gentle lysis methods using buffer containing 50 mM Tris-HCl (pH 7.5-8.0), 100-150 mM NaCl, 5-10% glycerol, and 1-5 mM MgCl₂ supplemented with protease inhibitors .

• Initial purification: For His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with imidazole gradient elution (20-250 mM) .

• Secondary purification: Size exclusion chromatography using Superdex 75 or 200 columns in buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 5% glycerol .

• Detergent considerations: If membrane association is a concern, mild detergents such as 0.03% (w/v) DDM (n-dodecyl β-D-maltoside) may be included in purification buffers .

For isolating intact and functional ATP synthase complexes containing the gamma chain, FLAG-tag affinity purification has been successfully applied to other bacterial species and could be adapted for C. violaceum .

How can the ATPase activity of recombinant C. violaceum atpG be assessed?

The ATPase activity of recombinant C. violaceum atpG can be assessed using several complementary approaches:

• Coupled enzyme assay: This spectrophotometric method measures ADP production by coupling ATP hydrolysis to NADH oxidation through pyruvate kinase and lactate dehydrogenase enzymes. The reaction is monitored by the decrease in absorbance at 340 nm .

  Reaction components:

  • 50 mM Tris-HCl (pH 8.0)

  • 5 mM MgCl₂

  • 2 mM ATP

  • 0.3 mM NADH

  • 2.5 mM phosphoenolpyruvate

  • 15-20 U/mL pyruvate kinase

  • 30 U/mL lactate dehydrogenase

  • Purified atpG protein/ATP synthase complex

• Malachite green phosphate assay: This colorimetric method directly measures inorganic phosphate released during ATP hydrolysis. The reaction with malachite green produces a colored complex that can be quantified at 620-640 nm .

• Luciferin/luciferase-based ATP detection: For measuring ATP synthesis activity (rather than hydrolysis), this highly sensitive method quantifies ATP produced by the enzyme when provided with ADP and inorganic phosphate in the presence of an artificial proton gradient .

It's important to note that isolated atpG may show limited activity compared to the intact ATP synthase complex, as the gamma subunit functions within the context of the complete F₁F₀ structure .

What factors influence nucleotide binding properties of C. violaceum atpG?

Several factors influence the nucleotide binding properties of C. violaceum atpG:

• Divalent cations: Mg²⁺ is essential for nucleotide binding, typically at a concentration of 2-5 mM. Ca²⁺ can also support binding but generally results in decreased catalytic activity .

• pH: Optimal nucleotide binding typically occurs at pH 7.5-8.0, with significant decreases in binding affinity observed outside this range .

• Salt concentration: Ionic strength affects binding, with optimal conditions typically in the range of 50-150 mM NaCl or KCl .

• Temperature: While C. violaceum grows optimally at 30°C, nucleotide binding studies are often performed at 25°C to balance binding affinity with protein stability .

• Conformational state: The nucleotide binding properties are influenced by interactions with other ATP synthase subunits, particularly the alpha and beta subunits, which form the catalytic interface .

Research has shown that "nucleotide binding is crucial for in vitro F₁ assembly, whereas ATP hydrolysis appears to be less critical" for the formation of functional complexes .

How does atpG interact with other subunits of the C. violaceum ATP synthase complex?

The atpG (gamma chain) of C. violaceum ATP synthase interacts with multiple subunits to form a functional enzyme complex:

• Interactions with alpha/beta subunits: The gamma chain rotates within the hexameric ring of alternating alpha and beta subunits, making asymmetric contacts that drive conformational changes during catalysis. These interactions are primarily hydrophobic with specific electrostatic contacts at the C-terminal domain of gamma .

• Interactions with epsilon and c-ring: The gamma subunit connects to the membrane-embedded c-ring through interactions with the epsilon subunit, forming the complete rotor assembly. This connection is crucial for coupling proton translocation to ATP synthesis .

• Interactions with delta and b subunits: The gamma subunit also interacts with the peripheral stalk composed of delta and b subunits, which serves as a stator against which the central rotor turns.

A structural model based on homology with other bacterial F-type ATP synthases suggests that these interactions in C. violaceum follow the general architectural principles seen in other bacteria, with specific sequence variations that may fine-tune the enzyme's function to the organism's ecological niche and metabolic requirements .

What techniques are effective for studying protein-protein interactions involving C. violaceum atpG?

Several techniques have proven effective for studying protein-protein interactions involving bacterial ATP synthase components that can be applied to C. violaceum atpG:

• Co-immunoprecipitation (Co-IP): Using antibodies against atpG or epitope tags (FLAG, His) to pull down interaction partners. This approach has been successfully used with other bacterial ATP synthases .

• Surface plasmon resonance (SPR): This label-free technique allows real-time monitoring of binding kinetics between immobilized atpG and other subunits flowing in solution.

• Crosslinking coupled with mass spectrometry: Chemical crosslinking followed by proteolytic digestion and mass spectrometric analysis can identify interaction interfaces at amino acid resolution .

• Förster resonance energy transfer (FRET): By labeling atpG and potential interaction partners with appropriate fluorophores, interactions can be monitored in real-time, potentially even in living cells.

• Bacterial two-hybrid systems: These genetic approaches can be used to screen for interactions in a cellular context, though results should be validated with biochemical methods.

When applying these techniques to C. violaceum atpG, consideration should be given to the membrane association of the complete complex and the potential requirement for detergents or membrane mimetics to maintain native conformations during analysis .

How is atpG expression regulated in C. violaceum under different growth conditions?

The expression of atpG in C. violaceum is regulated in response to changing environmental and metabolic conditions:

• Energy status: ATP synthase expression, including atpG, is upregulated during energy-limited conditions and downregulated when cellular ATP levels are high, as part of a feedback mechanism.

• Growth phase: Expression is typically higher during exponential growth phase than in stationary phase, correlating with higher energy demands during rapid growth .

• Oxygen availability: C. violaceum can grow under both aerobic and anaerobic conditions, and ATP synthase expression is differentially regulated to adapt to energy generation through either oxidative phosphorylation or anaerobic respiration using nitrate or fumarate as alternative electron acceptors .

• pH stress: During alkaline stress, ATP synthase components including gamma chain may be upregulated, similar to patterns observed in related bacteria like Desulfovibrio vulgaris .

• Quorum sensing influence: While direct regulation of atpG by quorum sensing has not been specifically demonstrated, the CviI/CviR quorum sensing system in C. violaceum regulates numerous metabolic functions and may influence energy metabolism indirectly .

The gene is likely organized in an operon with other ATP synthase components, typical of bacterial ATP synthase genes, allowing coordinated expression of the entire complex .

What role does atpG play in C. violaceum's adaptability to diverse ecosystems?

The atpG subunit, as part of the ATP synthase complex, contributes significantly to C. violaceum's remarkable adaptability to diverse ecosystems:

• Metabolic flexibility: The ATP synthase complex enables C. violaceum to efficiently generate energy under various growth conditions, including aerobic respiration, anaerobic respiration using alternative electron acceptors (nitrate, fumarate), and possibly during fermentative metabolism .

• pH adaptation: The ATP synthase helps maintain intracellular pH homeostasis, particularly important for C. violaceum which can be found in environments with varying pH levels .

• Temperature adaptation: As a bacterium found in tropical and subtropical regions, C. violaceum requires robust energy generation systems that function effectively across temperature variations .

• Stress response: During environmental stress conditions, the ATP synthase complex helps maintain energy homeostasis, crucial for expressing stress-response proteins and maintaining cellular functions .

• Virulence and pathogenesis: Although primarily an environmental bacterium, C. violaceum can cause opportunistic infections in humans. Energy metabolism, including ATP synthesis, supports various virulence mechanisms including toxin production, motility, and type III/VI secretion systems .

The efficient operation of ATP synthase, with atpG as a key component in energy coupling, likely underpins C. violaceum's ability to thrive in diverse ecological niches from soil and water environments to occasional host infection scenarios .

How can recombinant C. violaceum atpG be used to study bacterial energy metabolism regulation?

Recombinant C. violaceum atpG can serve as a powerful tool for investigating bacterial energy metabolism regulation through several advanced approaches:

• Site-directed mutagenesis studies: By creating specific mutations in conserved regions of atpG, researchers can probe structure-function relationships and regulatory mechanisms. Key targets include:

  • The DELSEED motif involved in subunit interactions

  • Residues at the interface with alpha/beta subunits

  • The coiled-coil regions critical for torque generation

• Inhibitor binding studies: Using recombinant atpG to screen and characterize small molecule modulators of ATP synthase activity can reveal regulatory mechanisms and potential antimicrobial targets.

• Interspecies chimeric constructs: Creating chimeric proteins containing regions from different bacterial species can help identify species-specific regulatory elements and adaptation mechanisms.

• Systems biology integration: Combining atpG functional studies with transcriptomics, proteomics, and metabolomics data from C. violaceum under different conditions can reveal regulatory networks controlling energy metabolism .

• In vitro reconstitution experiments: Assembling functional ATP synthase complexes with defined subunit compositions to study the influence of different gamma chain variants on the enzyme's regulatory properties .

These approaches can particularly illuminate how C. violaceum adapts its energy metabolism when transitioning between aerobic and anaerobic respiration or when expressing virulence factors under different environmental conditions .

What insights can studies of C. violaceum atpG provide about bacterial adaptation to environmental stresses?

Studies of C. violaceum atpG can provide unique insights into bacterial adaptation mechanisms:

• pH adaptation mechanisms: By examining atpG function and expression under different pH conditions, researchers can understand how C. violaceum maintains energy homeostasis during pH stress. This is particularly relevant as ATP synthase must operate effectively across diverse pH environments that C. violaceum encounters .

• Temperature-dependent regulation: Investigating how atpG function changes across temperature ranges can reveal adaptations that allow C. violaceum to thrive in tropical and subtropical regions with temperature fluctuations .

• Nutrient limitation responses: Studying atpG regulation during nutrient limitation can reveal how bacteria prioritize energy allocation during stress conditions, particularly the balance between ATP conservation and production .

• Oxidative stress responses: As C. violaceum transitions between oxygen-rich and oxygen-limited environments, changes in atpG function and ATP synthase activity can illuminate adaptations to varying redox conditions and oxidative stress .

• Host interaction adaptations: For an opportunistic pathogen like C. violaceum, comparing atpG function during environmental versus host-associated growth can reveal adaptations to the unique stresses encountered during infection .

This research is especially valuable because C. violaceum occupies an ecological niche between free-living environmental bacteria and opportunistic pathogens, making it an excellent model for studying metabolic adaptations across diverse conditions .

How does C. violaceum atpG compare structurally and functionally with homologs from other bacteria?

Comparative analysis of C. violaceum atpG with homologs from other bacteria reveals important structural and functional insights:

This comparative approach provides insights into both the universal aspects of ATP synthase function and the specific adaptations that optimize the enzyme for C. violaceum's ecological niche .

What evolutionary insights can be gained from studying C. violaceum atpG?

Studying C. violaceum atpG from an evolutionary perspective offers several important insights:

• Ancient conserved machinery: The ATP synthase complex represents one of the most ancient and conserved energy-converting machineries. Analysis of C. violaceum atpG in this context helps understand the minimal conserved elements required for function versus adaptable regions.

• Horizontal gene transfer assessment: Comparing atpG sequences can help identify potential horizontal gene transfer events in the evolution of energy metabolism among proteobacteria.

• Adaptive evolution signatures:

  • Regions under positive selection may indicate adaptations to specific environmental pressures

  • The ratio of synonymous to non-synonymous substitutions across different domains of atpG can reveal functional constraints versus adaptive regions

• Co-evolution with other ATP synthase subunits: Analysis of evolutionary rates across different ATP synthase components can reveal co-evolutionary relationships that maintain functional interactions between subunits.

• Paralogs and gene duplication events: Some bacteria contain multiple ATP synthase operons adapted for different conditions; evolutionary analysis can clarify the relationship of C. violaceum atpG to such paralogs.

These evolutionary insights provide context for understanding why certain residues and regions are invariant across all bacteria, while others show species-specific variations that likely represent adaptations to particular ecological niches .

What are common challenges in expressing and purifying functional C. violaceum atpG, and how can they be addressed?

Researchers frequently encounter several challenges when working with C. violaceum atpG:

• Protein solubility issues:

  • Challenge: atpG may form inclusion bodies when overexpressed.

  • Solution: Lower induction temperature (16-18°C), reduce IPTG concentration (0.1-0.2 mM), or use solubility-enhancing fusion tags like SUMO or MBP .

• Improper folding:

  • Challenge: Isolated atpG may not adopt native conformation without other ATP synthase subunits.

  • Solution: Co-express with other components of the F1 domain or purify under native conditions that preserve existing complexes .

• Loss of activity during purification:

  • Challenge: Activity diminishes with each purification step.

  • Solution: Include nucleotides (ATP/ADP) and Mg²⁺ in purification buffers, minimize purification steps, and avoid freeze-thaw cycles .

• Protein instability:

  • Challenge: Purified protein shows degradation during storage.

  • Solution: Store with 10-20% glycerol, avoid multiple freeze-thaw cycles, and consider storage at -80°C in small single-use aliquots .

• Contamination with endogenous E. coli ATP synthase components:

  • Challenge: Co-purification of host proteins.

  • Solution: Use affinity tags combined with ion exchange chromatography, or consider expression in ATP synthase-deficient E. coli strains .

• Difficulties assessing activity:

  • Challenge: Isolated atpG shows limited activity outside the complete complex.

  • Solution: Develop assays that measure specific aspects like nucleotide binding rather than catalytic activity, or reconstitute with minimal complex components .

Researchers have found that "nucleotide binding is crucial for in vitro F₁ assembly, whereas ATP hydrolysis appears to be less critical," suggesting the inclusion of nucleotides during purification is particularly important .

What analytical methods are most effective for characterizing the structural integrity of recombinant C. violaceum atpG?

Multiple analytical methods can effectively assess the structural integrity of recombinant C. violaceum atpG:

• Circular dichroism (CD) spectroscopy:

  • Provides information on secondary structure content

  • Useful for comparing wild-type and mutant proteins

  • Can monitor thermal denaturation to assess stability

  • Typical properly folded atpG shows characteristic alpha-helical patterns with negative peaks at 208 and 222 nm

• Intrinsic fluorescence spectroscopy:

  • Monitors tertiary structure through tryptophan and tyrosine fluorescence

  • Properly folded atpG typically shows distinctive emission maxima

  • Changes in spectra can indicate unfolding or altered conformation

• Limited proteolysis:

  • Properly folded protein shows distinctive digestion patterns

  • Can identify stable domains and flexible regions

  • Useful for comparing wild-type and mutant variants

• Differential scanning calorimetry (DSC):

  • Measures thermal transitions directly related to protein unfolding

  • Provides quantitative stability parameters (Tm, ΔH)

  • Can detect presence of multiple domains with distinct stabilities

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS):

  • Determines absolute molecular weight and oligomeric state

  • Detects aggregation or incorrect assembly

  • Can compare experimental values with theoretical predictions

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Maps solvent-accessible regions of the protein

  • Identifies stable core domains versus flexible regions

  • Useful for comparing variants or conditions affecting structure

For atpG specifically, comparing nucleotide binding properties (using fluorescent ATP analogues) between purified protein and reference standards can also serve as a functional indicator of structural integrity .

What emerging research areas might benefit from studies of C. violaceum atpG?

Several emerging research areas could be significantly advanced through studies of C. violaceum atpG:

• Synthetic biology applications:

  • Engineering bacterial ATP synthases with altered efficiency or regulatory properties

  • Developing cellular energy production modules for synthetic organisms

  • Creating biosensors based on conformational changes in atpG

• Antimicrobial development:

  • Identifying specific inhibitors targeting bacterial ATP synthases

  • Understanding species-specific differences that could enable selective targeting

  • Developing combination therapies targeting energy metabolism during infection

• Biotechnological applications:

  • Engineering bacterial strains with enhanced ATP production for biotechnology processes

  • Development of nano-rotary motors based on the F₁ complex

  • Creating artificial photosynthetic systems incorporating ATP synthase components

• Environmental adaptation studies:

  • Understanding bacterial adaptation to changing environments

  • Exploring energy metabolism in extremophiles by comparative studies

  • Investigating microbial community interactions mediated by energy status

• Systems biology integration:

  • Modeling bacterial metabolism with accurate parameters from purified components

  • Understanding global regulatory networks integrating energy status with other cellular processes

  • Quantitative analysis of bacterial fitness landscapes under energy constraints

These research directions are particularly relevant given C. violaceum's position as both an environmental organism and opportunistic pathogen, making its energy metabolism adaptations broadly applicable to understanding bacterial adaptability .

What novel experimental approaches could advance our understanding of C. violaceum atpG function?

Several cutting-edge experimental approaches could significantly advance our understanding of C. violaceum atpG function:

• Single-molecule techniques:

  • Single-molecule FRET to monitor conformational changes during catalysis

  • Optical or magnetic tweezers to measure force generation by the gamma subunit

  • High-speed AFM to visualize rotational dynamics in real-time

• Cryo-electron microscopy:

  • High-resolution structures of C. violaceum ATP synthase in different catalytic states

  • Visualization of specific conformational changes induced by nucleotide binding

  • Comparative structural analysis with other bacterial ATP synthases

• In situ approaches:

  • Cryo-electron tomography to visualize ATP synthase in its native membrane environment

  • Super-resolution microscopy to track ATP synthase distribution and dynamics in living cells

  • Correlative light and electron microscopy to link function with ultrastructure

• Genetic approaches:

  • CRISPR-Cas9 genome editing of C. violaceum to create precise atpG variants

  • Global genetic interaction screens to identify functional relationships

  • In vivo protein-protein interaction mapping using proximity labeling (BioID, APEX)

• Computational approaches:

  • Molecular dynamics simulations of the complete ATP synthase complex

  • Machine learning analysis of sequence-function relationships across bacterial species

  • Multiscale modeling linking molecular function to cellular physiology

• Time-resolved structural methods:

  • Time-resolved X-ray/neutron scattering to capture intermediate states

  • Temperature-jump coupled spectroscopic methods to follow conformational changes