Recombinant Photorhabdus luminescens subsp. laumondii ATP synthase subunit c (atpE)

What is the biological significance of ATP synthase subunit c in Photorhabdus luminescens?

ATP synthase subunit c, encoded by the atpE gene in Photorhabdus luminescens, is a critical component of the bacterial ATP synthase complex. This protein plays an essential role in energy metabolism by forming the c-ring structure within the Fo domain of ATP synthase. The c-ring facilitates proton translocation across the membrane, which drives the rotational mechanism necessary for ATP synthesis . In P. luminescens, this energy generation system is particularly important as it supports the bacterium's complex lifecycle, including its symbiotic relationship with entomopathogenic nematodes and its pathogenicity toward insect hosts .

The ATP synthase complex in P. luminescens, like in other bacteria, consists of multiple subunits with a total molecular weight comparable to the human mitochondrial ATP synthase (~550 kDa) . The c subunit specifically forms a ring-like structure that rotates during ATP synthesis, with each 360° rotation resulting in the formation of three ATP molecules through conformational changes in the catalytic β subunits . This mechanism is critical for bacterial survival and virulence in both its symbiotic and pathogenic relationships.

How does the structure of ATP synthase subunit c relate to its function in P. luminescens?

The ATP synthase subunit c in P. luminescens, like in other organisms, adopts a hairpin-like structure with two transmembrane α-helices connected by a short loop. Multiple c subunits assemble to form the c-ring, which is embedded in the bacterial inner membrane. Each c subunit contains a conserved carboxylate residue (typically aspartate or glutamate) that is essential for proton binding and translocation .

The functional mechanism involves rotation of the c-ring driven by the proton motive force. As protons bind to the carboxylate residue on one side of the membrane and are released on the other side, they induce conformational changes that contribute to the rotation of the c-ring. This rotation is mechanically coupled to conformational changes in the F1 domain (containing α and β subunits), where ATP synthesis occurs .

The tight coupling between proton translocation through the c-ring and ATP synthesis in the catalytic sites demonstrates how structural features of the c subunit directly contribute to energy conversion efficiency. Additionally, the number of c subunits in the ring can vary between species, affecting the bioenergetic properties of the ATP synthase complex.

What techniques are commonly used to study recombinant P. luminescens ATP synthase subunit c?

Researchers studying recombinant P. luminescens ATP synthase subunit c typically employ a multi-technique approach:

• Recombinant Expression Systems: The atpE gene from P. luminescens is often cloned into expression vectors for production in E. coli systems. Codon optimization may be necessary to enhance expression in heterologous hosts.

• Protein Purification: Due to the hydrophobic nature of subunit c, specialized purification protocols using detergents or organic solvents are employed. Techniques include affinity chromatography with polyhistidine tags, size exclusion chromatography, and ion-exchange chromatography.

• Structural Analysis: Cryoelectron microscopy has proven valuable for studying membrane protein complexes like ATP synthase . This technique allows visualization of the protein in different conformational states, such as the prepore and pore states observed in TcdA1 from P. luminescens .

• Functional Assays: ATP synthesis or hydrolysis assays using reconstituted proteoliposomes are employed to assess the functional properties of the recombinant protein. These assays typically measure ATP production or phosphate release under various conditions.

• Site-Directed Mutagenesis: This technique is used to investigate the role of specific amino acid residues in the function of subunit c, particularly those involved in proton translocation and interaction with other subunits of the ATP synthase complex.

How does the recombinant expression of P. luminescens ATP synthase subunit c affect its structural integrity and functional properties?

Recombinant expression of P. luminescens ATP synthase subunit c presents several challenges that can impact its structural integrity and function. The highly hydrophobic nature of this protein often leads to expression difficulties including protein aggregation, improper folding, or toxicity to the host cells. These issues can be addressed through optimization strategies:

When expressed in E. coli systems, the use of specialized strains (e.g., C41(DE3) or C43(DE3)) designed for membrane protein expression can significantly improve yields while maintaining native structure. Additionally, fusion tags beyond standard hexahistidine tags, such as maltose-binding protein (MBP) or small ubiquitin-like modifier (SUMO), can enhance solubility without compromising functional properties.

Expression conditions critically affect protein quality. Lower induction temperatures (16-25°C) and reduced inducer concentrations often result in slower expression that favors proper membrane integration. The recombinant protein's structural integrity can be verified through circular dichroism spectroscopy to confirm the expected α-helical content characteristic of c subunits.

What are the comparative differences between ATP synthase subunit c from P. luminescens and other bacterial species?

Comparative analysis of ATP synthase subunit c across bacterial species reveals both conserved features and important differences that reflect evolutionary adaptations to different ecological niches:

Table 1: Comparative Analysis of ATP Synthase Subunit c Across Selected Bacterial Species

The variable stoichiometry of c-rings across species has significant bioenergetic consequences. With P. luminescens transitioning between the nematode gut and insect hemolymph, its ATP synthase likely shows adaptations to function across varying pH conditions, similar to the pH-responsive mechanism observed in the TcdA1 toxin pore formation . Notably, sequence analysis suggests P. luminescens may possess unique residues surrounding the conserved proton-binding site that modify pKa values, potentially optimizing function during the different stages of its lifecycle.

Comparative structural studies using cryo-EM or X-ray crystallography would be particularly valuable to elucidate how these differences contribute to the specific bioenergetic requirements of P. luminescens in its complex symbiotic and pathogenic relationships.

How can inhibitors of ATP synthase subunit c be used as research tools for studying P. luminescens pathogenicity?

ATP synthase inhibitors targeting subunit c provide powerful research tools for investigating P. luminescens pathogenicity mechanisms. By selectively disrupting energy metabolism, these compounds enable researchers to disentangle the complex relationship between bacterial energetics and virulence factor production.

The oligomycin class of inhibitors binds to the interface between subunit a and the c-ring, blocking proton translocation. Using these compounds at sub-lethal concentrations allows researchers to study how energy limitation affects toxin production in P. luminescens. This approach has revealed that certain virulence factors, including the tripartite ABC-type toxin complexes (Tcs), are differentially regulated under energy-limited conditions .

More selective tools include dicyclohexylcarbodiimide (DCCD), which covalently modifies the essential proton-binding carboxylate in subunit c. DCCD treatment studies have demonstrated that ATP synthesis inhibition significantly impacts the secretion efficiency of the TcdA1 toxin component, suggesting energy-dependent steps in the toxin export pathway .

When designing experiments with these inhibitors, careful titration is essential to distinguish specific effects on ATP synthase from non-specific membrane disruption. Combining inhibitor studies with genetic approaches, such as point mutations in the atpE gene that confer resistance to specific inhibitors, provides a powerful strategy to validate inhibitor specificity and study the consequences of ATP synthase disruption on P. luminescens pathogenicity mechanisms.

What are the challenges in studying the interaction between recombinant ATP synthase subunit c and other components of the ATP synthase complex?

Investigating interactions between recombinant ATP synthase subunit c and other components of the ATP synthase complex presents several methodological challenges:

The hydrophobic nature of subunit c complicates traditional protein-protein interaction assays. Co-immunoprecipitation experiments often yield false negatives due to detergent effects disrupting native interactions. Researchers have addressed this challenge by employing chemical crosslinking approaches prior to complex disruption, enabling the capture of transient or detergent-sensitive interactions.

Reconstitution systems that incorporate multiple ATP synthase components require precise lipid-to-protein ratios and membrane mimetics that support native-like interactions. Nanodiscs have emerged as a valuable technology in this context, providing a defined lipid environment while maintaining the solubility needed for biophysical studies.

Advanced spectroscopic techniques such as Förster resonance energy transfer (FRET) between labeled subunits can provide dynamic information about subunit interactions under various conditions. This approach has revealed how conformational changes in the c-ring are transmitted to other ATP synthase components during the catalytic cycle.

Among the most challenging aspects is studying the interface between subunit a and the c-ring, which forms the critical pathway for proton translocation. This interface undergoes subtle structural changes during rotation that are difficult to capture. Recent advances in high-speed atomic force microscopy offer promising capabilities for directly visualizing these dynamic interactions under near-physiological conditions.

What are the optimal conditions for expressing and purifying recombinant P. luminescens ATP synthase subunit c?

Optimizing the expression and purification of recombinant P. luminescens ATP synthase subunit c requires careful consideration of multiple parameters:

Expression System Selection:
The E. coli C43(DE3) strain has shown superior results for expressing membrane proteins like subunit c. This strain contains mutations that prevent the toxicity often associated with membrane protein overexpression. For the expression vector, pET systems with tightly controlled T7 promoters allow precise induction timing.

Expression Protocol:

• Culture growth: LB medium supplemented with 0.4% glucose at 37°C until OD600 reaches 0.6-0.8

• Temperature shift: Reduce to 18-20°C prior to induction

• Induction: 0.1-0.3 mM IPTG

• Post-induction: Continue expression for 16-18 hours at the reduced temperature

Purification Strategy:
The purification of ATP synthase subunit c is challenging due to its hydrophobic nature and tendency to form oligomeric structures. A sequential approach yields the best results:

• Membrane isolation through differential centrifugation

• Solubilization using a mild detergent mixture (typically 2% n-dodecyl β-D-maltoside with 0.5% digitonin)

• Immobilized metal affinity chromatography (IMAC) using a C-terminal polyhistidine tag

• Size exclusion chromatography to separate monomeric from oligomeric forms

Critical Parameters for Success:

• Buffer composition: 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol, and 0.05% detergent for stabilization

• Addition of 5 mM DTT to prevent oxidation of cysteine residues

• Inclusion of 0.1 mg/mL cardiolipin during purification to maintain native-like lipid interactions

• Storage at -80°C in small aliquots to prevent freeze-thaw damage

This optimized protocol typically yields 1-2 mg of purified protein per liter of bacterial culture with >90% purity as assessed by SDS-PAGE analysis.

How can researchers effectively assess the functional integrity of recombinant ATP synthase subunit c?

Assessing the functional integrity of recombinant ATP synthase subunit c requires evaluating both its structural properties and ability to participate in ATP synthesis when incorporated into appropriate complexes:

Structural Integrity Assessment:

• Circular Dichroism (CD) Spectroscopy: The ATP synthase subunit c typically shows characteristic α-helical signatures with negative peaks at 208 and 222 nm. The ratio between these peaks provides information about the protein's folding state.

• Thermal Stability Analysis: Using differential scanning calorimetry or thermal shift assays to determine the melting temperature (Tm) of the purified protein. Properly folded subunit c should display a cooperative unfolding transition.

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS): This technique assesses whether the purified protein forms the expected oligomeric assemblies (c-rings) or exists as monomers in detergent solutions.

Functional Assessment Methods:

• Reconstitution Assays:

  • Incorporate purified subunit c into liposomes with defined lipid composition

  • Add additional ATP synthase components (either purified or as subcomplexes)

  • Measure proton translocation using pH-sensitive fluorescent dyes such as ACMA (9-amino-6-chloro-2-methoxyacridine)

• ATP Synthesis Activity:

  • Generate a proton gradient across the proteoliposome membrane using pH jump or valinomycin/K+ methods

  • Measure ATP production using luciferase-based luminescence assays

  • Compare activity rates to those obtained with native ATP synthase complexes

• Inhibitor Binding Studies:

  • Measure binding of specific inhibitors (e.g., DCCD) that target subunit c

  • Determine binding constants and compare with those of native protein

  • Assess whether expected functional consequences (inhibition of proton translocation) occur

A comprehensive assessment would include all these approaches, as structural integrity does not guarantee functional competence. The gold standard for confirming full functionality is demonstrating ATP synthesis activity when the recombinant subunit c is incorporated into a complete ATP synthase complex.

What techniques can be used to study the structural dynamics of ATP synthase subunit c during proton translocation?

Investigating the structural dynamics of ATP synthase subunit c during proton translocation requires specialized techniques that can capture transient states and conformational changes:

Site-Directed Spin Labeling (SDSL) with Electron Paramagnetic Resonance (EPR) Spectroscopy:
This approach involves introducing cysteine residues at strategic positions within the subunit c sequence for subsequent labeling with nitroxide spin probes. EPR measurements can then detect changes in local mobility and distances between labeled sites during proton translocation events. Continuous wave EPR provides information about the mobility of the spin label, while pulsed EPR techniques such as DEER (Double Electron-Electron Resonance) measure distances between labels in the range of 1.5-8 nm.

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):
HDX-MS measures the rate at which backbone amide hydrogens exchange with deuterium in the solvent. This exchange rate is dependent on hydrogen bonding and solvent accessibility, providing insights into conformational changes and protein dynamics. By comparing exchange rates under different conditions (e.g., in the presence or absence of a proton gradient), researchers can identify regions of subunit c that undergo structural changes during proton translocation.

Single-Molecule FRET (smFRET):
This technique monitors distance changes between donor and acceptor fluorophores attached to specific residues in subunit c. By observing individual molecules, researchers can detect transient states and conformational heterogeneity that would be averaged out in bulk measurements. Recent advances in total internal reflection fluorescence (TIRF) microscopy allow for real-time observation of c-ring rotation during ATP synthesis.

Molecular Dynamics (MD) Simulations:
Computational approaches complement experimental techniques by providing atomic-level insights into proton translocation mechanisms. All-atom MD simulations can model the protonation/deprotonation events at the conserved carboxylate residue and the resulting conformational changes. Coarse-grained simulations enable modeling of the entire c-ring dynamics over longer timescales relevant to the complete rotational cycle.

The most comprehensive understanding comes from integrating multiple techniques. For example, distance constraints derived from EPR or smFRET experiments can be used to refine MD simulations, while simulation predictions can guide the selection of residues for experimental labeling.

How should researchers interpret contradictory results between in vitro and in vivo studies of P. luminescens ATP synthase function?

When faced with contradictory results between in vitro and in vivo studies of P. luminescens ATP synthase function, researchers should systematically analyze several factors that could explain these discrepancies:

Contextual Differences Analysis:
The cellular environment in vivo contains numerous factors absent from simplified in vitro systems. The ATP synthase interacts with other cellular components and operates within the context of membrane heterogeneity, crowding effects, and metabolic networks. Evidence from studies on bacterial pathogens indicates that ATP synthase function can be modulated by membrane lipid composition, which differs between artificial membranes used in vitro and the native bacterial membrane .

Regulatory Mechanisms:
In vivo, ATP synthase activity is subject to regulation through various mechanisms, including post-translational modifications, protein-protein interactions, and allosteric regulation by metabolites. For example, the dual functionality of ATP synthase in both ATP synthesis and cell death pathways (as observed in mitochondrial systems) suggests regulatory complexity that may not be replicated in reconstituted systems .

Experimental Conditions Gap:
The specific conditions used in vitro may not accurately reflect the physiological environment encountered by P. luminescens during its complex lifecycle. This bacterium transitions between symbiosis with nematodes and pathogenicity in insects, experiencing different pH environments. Research on the TcdA1 toxin shows that P. luminescens proteins can be sensitive to pH changes, with membrane insertion triggered at both low and high pH . Similar pH-dependent effects might influence ATP synthase function differently between controlled in vitro conditions and the variable in vivo environment.

Methodological Approach to Resolve Contradictions:

This systematic analysis approach helps distinguish between genuine biological differences and artifacts of experimental methodology, leading to a more accurate understanding of ATP synthase function in P. luminescens.

What are the key considerations when comparing P. luminescens ATP synthase subunit c with homologous proteins from other bacterial species?

When conducting comparative analyses of P. luminescens ATP synthase subunit c with homologous proteins from other bacterial species, researchers should consider several critical factors to ensure meaningful interpretations:

Sequence Alignment Quality:
The high hydrophobicity and relatively small size of subunit c can make sequence alignments challenging. Researchers should use specialized algorithms designed for membrane proteins that account for the physicochemical properties of transmembrane segments. Multiple alignment approaches should be employed to identify truly conserved residues versus alignment artifacts. Special attention should be paid to the proton-binding site, typically containing a conserved carboxylate residue (Asp or Glu) that is essential for function.

Structural Context Integration:
Beyond primary sequence, the structural context of amino acid differences must be considered. Substitutions in transmembrane regions may alter helix-helix packing in the c-ring, while changes in loop regions might affect interactions with other ATP synthase subunits. Homology modeling based on available high-resolution structures (such as those from E. coli or mycobacterial species) can provide valuable insights into the structural consequences of sequence variations.

Evolutionary and Ecological Context:
Interpretations should consider the unique ecological niche of P. luminescens. As an insect pathogen with a complex lifecycle involving symbiosis with nematodes, P. luminescens experiences diverse environmental conditions that may have driven specific adaptations in ATP synthase function. For instance, the ability of P. luminescens to function at both acidic and alkaline pH conditions during different lifecycle stages might be reflected in properties of its ATP synthase components .

Functional Convergence versus Divergence:
When identifying differences, it's crucial to distinguish between:

• Functionally neutral variations that don't significantly impact ATP synthase operation

• Adaptive variations that optimize function for specific environmental conditions

• Compensatory variations that maintain function despite changes elsewhere in the complex

This assessment requires integration of structural data with functional assays across different species. For example, while the essential proton-binding mechanism is conserved across bacteria, the c-ring stoichiometry (number of c subunits per ring) varies between species, affecting the bioenergetic efficiency of ATP synthesis.

By carefully considering these factors, researchers can avoid misinterpretations and extract valuable insights about the evolutionary adaptations of ATP synthase across bacterial species with different lifestyles and ecological niches.

What are the emerging techniques that could advance our understanding of P. luminescens ATP synthase function?

Several cutting-edge techniques are poised to revolutionize our understanding of P. luminescens ATP synthase function and dynamics:

Cryo-Electron Tomography (cryo-ET):
This technique allows visualization of macromolecular complexes like ATP synthase in their native cellular environment without extraction or purification. Recent advances in sample preparation, direct electron detectors, and phase plates have improved resolution to near-atomic levels. Applied to P. luminescens, cryo-ET could reveal not only the structure of ATP synthase but also its distribution and organization within the bacterial membrane during different lifecycle stages, providing insights into its role in energy metabolism during both symbiotic and pathogenic phases.

Time-Resolved Serial Femtosecond Crystallography:
Using X-ray free-electron lasers (XFELs), this method captures structural snapshots of proteins at femtosecond timescales. For P. luminescens ATP synthase components, this could provide unprecedented views of conformational changes during the catalytic cycle, particularly the transient states during proton translocation through subunit c. These datasets would significantly enhance our understanding of the mechanistic details underlying energy conversion in this bacterial species.

AlphaFold2 and Integrative Structural Biology:
Deep learning approaches like AlphaFold2 have dramatically improved protein structure prediction capabilities. For P. luminescens ATP synthase, combining AlphaFold2 predictions with sparse experimental data (from crosslinking, mass spectrometry, or low-resolution EM) could produce highly accurate structural models of the complete complex in different functional states. This integrative approach is particularly valuable for membrane protein complexes that remain challenging for traditional structural biology methods.

In-cell NMR Spectroscopy:
Recent developments have made it possible to perform NMR studies in living cells. Applied to isotopically labeled P. luminescens, this technique could monitor structural and dynamic changes in ATP synthase components under physiological conditions and in response to environmental changes that mimic those encountered during the bacterium's lifecycle. This approach would bridge the gap between in vitro studies and the complex cellular environment.

Combining these emerging techniques with established methods will provide a more comprehensive understanding of how P. luminescens ATP synthase functions within the broader context of bacterial physiology and host-pathogen interactions.

How might research on P. luminescens ATP synthase contribute to understanding bacterial pathogenicity mechanisms?

Research on P. luminescens ATP synthase has significant potential to enhance our understanding of bacterial pathogenicity mechanisms through several interconnected avenues:

Energy-Pathogenicity Coupling:
ATP synthase functions as the primary energy production machinery in bacteria, directly influencing the energy available for virulence factor production and secretion. Studies of P. luminescens ATP synthase could reveal how energy metabolism is reconfigured during the transition from the symbiotic to the pathogenic lifestyle. This bacterium produces numerous virulence factors, including the tripartite ABC-type toxin complexes (Tcs), which require substantial energy for synthesis and secretion . Understanding how ATP synthase activity is regulated during infection could provide insights into the energetic requirements of pathogenicity.

Membrane Potential and Virulence Factor Secretion:
The proton gradient maintained by ATP synthase contributes to bacterial membrane potential, which is critical for various secretion systems that deliver virulence factors to host cells. In P. luminescens, the syringe-like mechanism observed in the TcdA1 toxin complex depends on membrane insertion triggered by pH changes . Research could explore how ATP synthase activity influences membrane potential and subsequently affects the efficiency of toxin delivery systems during insect infection.

Adaptation to Host Environments:
During its lifecycle, P. luminescens must adapt to varying environments within the nematode vector and insect host. These different niches present challenges including pH fluctuations, nutrient availability changes, and host defense mechanisms. ATP synthase function may be modulated to optimize energy production under these varying conditions. Comparative studies of ATP synthase activity in different lifecycle stages could identify adaptive mechanisms that enable successful host colonization.

Host Cell Apoptosis Induction:
Research on P. asymbiotica, a related species that is pathogenic to both insects and humans, has shown that this bacterium can induce apoptosis in host cells, particularly macrophages . ATP synthase has been implicated in apoptotic pathways through the mitochondrial permeability transition pore . Investigating whether P. luminescens ATP synthase components interact with host cellular machinery could reveal novel mechanisms by which bacterial pathogens manipulate host cell death pathways.

This research direction is particularly valuable because the principles elucidated in P. luminescens may apply to other bacterial pathogens, including those that affect humans. The unique lifecycle of P. luminescens provides a model system for studying the metabolic adaptations that underlie transitions between different host environments, a challenge faced by many pathogens.