Recombinant Photorhabdus luminescens subsp. laumondii ATP synthase subunit delta (atpH)

What is Photorhabdus luminescens and why is it significant for research?

Photorhabdus luminescens is a gram-negative luminescent gammaproteobacterium with a fascinating dual lifestyle, functioning both as a symbiont and a pathogen . It maintains a mutualistic relationship with nematodes from the Heterorhabditis genus, colonizing the intestinal tract of the nematode during its symbiotic stage . During its pathogenic stage, the bacterium-nematode complex invades and kills various insect larvae . The genome sequence of P. luminescens subsp. laumondii TT01 has been fully deciphered, making it an excellent model organism for studying host-microbe interactions, bioluminescence mechanisms, and bacterial adaptation strategies . Its unique properties have made it valuable for research in microbiology, entomology, and biotechnology applications, including as an alternative organism for recombinant luminescent bioreporters due to its stable bioluminescence expression .

What is the ATP synthase delta subunit (atpH) and what is its role in P. luminescens?

The ATP synthase subunit delta (atpH) is a component of the F-type ATP synthase complex, specifically part of the F1 sector . In P. luminescens, as in other bacteria, this protein plays a crucial role in energy metabolism by participating in the synthesis of ATP through oxidative phosphorylation. The delta subunit functions as part of the peripheral stalk of ATP synthase, connecting the F1 and F0 portions of the enzyme complex and contributing to its stability during rotational catalysis. The recombinant protein consists of 177 amino acids with the sequence starting with MSEFATVARP and ending with RLDRLTDVLQS . This protein is part of the bacterial energy production machinery that enables P. luminescens to maintain its metabolic activities during both symbiotic and pathogenic phases of its lifecycle.

How does the structure of P. luminescens atpH compare to ATP synthase subunits in other bacterial species?

P. luminescens atpH shares structural similarities with ATP synthase delta subunits from other bacterial species, particularly within the Enterobacteriaceae family. The protein has a Uniprot accession number of Q7NA91 and maintains the characteristic domains found in bacterial F-type ATPase delta subunits . Structurally, the P. luminescens atpH contains regions that interact with both the alpha/beta subunits of the F1 sector and components of the peripheral stalk, maintaining the structural integrity of the ATP synthase complex.

Comparative analysis with other bacterial delta subunits shows conservation in functional domains while maintaining species-specific variations that may relate to the unique environmental adaptations of P. luminescens. Unlike many other bacterial species, P. luminescens must function effectively within both insect hemolymph during pathogenesis and nematode gut environments during symbiosis, which may influence structural features of its energy-generating proteins including atpH .

How does quorum sensing via AI-2 affect ATP synthase expression and function in P. luminescens?

Research indicates that AI-2 quorum sensing in P. luminescens regulates over 300 targets involved in various cellular compartments and metabolic pathways . While direct regulation of atpH has not been specifically documented in the provided literature, the global regulatory effects of AI-2 likely influence energy metabolism including ATP synthase expression. P. luminescens contains the luxS gene responsible for AI-2 synthesis, and studies with luxS-deficient mutants show significant transcriptomic and proteomic changes .

AI-2 appears high in the regulatory hierarchy, controlling multiple transcriptional regulators and exhibiting dose-dependent effects . For researchers investigating ATP synthase regulation, it's important to consider that the quorum sensing system may modulate atpH expression in response to cell density, potentially optimizing energy production during different stages of the bacterial lifecycle. Experiments comparing wild-type and luxS-deficient strains could reveal whether ATP synthase components are directly or indirectly regulated by this quorum sensing system, particularly during the transition from symbiotic to pathogenic states.

What are the challenges in expressing and purifying functional recombinant P. luminescens atpH for structural studies?

Expression and purification of functional recombinant P. luminescens atpH presents several technical challenges that researchers should consider:

• Expression system selection: While E. coli is commonly used and confirmed as the expression system for commercial recombinant atpH , optimization of expression conditions is crucial for obtaining properly folded protein. For ATP synthase components, which normally exist in multi-subunit complexes, expressing individual subunits may lead to folding issues or aggregation.

• Protein solubility: The delta subunit interfaces with multiple other ATP synthase components in its native state. When expressed alone, it may exhibit reduced solubility or form inclusion bodies, requiring optimization of solubilization and refolding protocols.

• Functional assessment: Unlike enzymatic subunits, the delta subunit's structural role makes functional assessment challenging. Researchers might need to evaluate proper folding through circular dichroism spectroscopy or binding assays with partner subunits.

• Stability considerations: Purified atpH has specified storage recommendations, with liquid forms having a shelf life of 6 months at -20°C/-80°C and lyophilized forms extending to 12 months . Addition of glycerol (5-50%) is recommended for long-term storage.

For successful structural studies, researchers should consider co-expression with interacting partners or stabilizing mutations that might enhance structural integrity while maintaining native conformation.

How does the function of ATP synthase in P. luminescens relate to its ability to overcome oxidative stress during insect infection?

P. luminescens faces significant oxidative stress during insect infection, encountering reactive oxygen species (ROS) including superoxide ions, hydrogen peroxide, and peroxynitrite as part of the insect's immune response . ATP synthase function may be integrally connected to the bacterium's oxidative stress resistance through several mechanisms:

• Energy provision for defense systems: ATP synthase generates the energy currency (ATP) required for powering oxidative stress defense enzymes such as catalases, peroxidases, and superoxide dismutases.

• Maintenance of proton motive force: The F0F1 ATP synthase complex, which includes atpH, helps maintain the proton motive force across the bacterial membrane. This electrochemical gradient is critical for numerous cellular processes including those involved in stress response.

• Potential regulatory connections: Studies of luxS-deficient strains show increased oxidative stress resistance is regulated by AI-2 quorum sensing . There may be regulatory networks connecting quorum sensing, energy metabolism through ATP synthase, and oxidative stress resistance.

Researchers investigating these connections could design experiments comparing wild-type and ATP synthase-modified strains for survival in the presence of H₂O₂ or other oxidative agents, similar to experiments conducted with luxS mutants . The bacterial growth response to H₂O₂ exposure (0.5-1 mM) could serve as a measurable indicator of oxidative stress resistance mechanisms potentially connected to ATP synthase function.

What are the optimal protocols for recombinant expression and purification of P. luminescens atpH?

Based on published methodologies for P. luminescens proteins, the following protocol framework is recommended for atpH expression and purification:

Expression System and Conditions:

• Clone the P. luminescens atpH gene into an expression vector such as pET-22b using NdeI and XhoI restriction sites

• Transform into E. coli BL21(DE3) containing a plasmid expressing the lacI repressor (e.g., pDIA17)

• Culture in rich media such as Hyper Broth to achieve high cell density

• Induce expression at OD₆₀₀ of approximately 3.0 with 3 mM IPTG for 2 hours

Purification Strategy:

• Harvest cells by centrifugation and disrupt using mechanical methods (e.g., FastPrep apparatus)

• Prepare cell lysate in buffer containing 20 mM sodium phosphate (pH 7.2) and 200 mM NaCl

• Remove cell debris by centrifugation at 7,500 × g for 20 minutes

• Purify using affinity chromatography if a tag was incorporated, or ion exchange chromatography

• Assess purity by SDS-PAGE (target >85% purity)

Storage and Handling:

• For liquid preparations, store at -20°C/-80°C with expected shelf life of 6 months

• For lyophilized preparations, store at -20°C/-80°C with expected shelf life of 12 months

• For working solutions, reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol to 5-50% final concentration for long-term storage

• Avoid repeated freeze-thaw cycles

This methodology can be adjusted based on specific research requirements and the intended applications of the purified protein.

How can researchers effectively evaluate the functional integrity of recombinant atpH?

Assessing the functional integrity of recombinant atpH presents unique challenges because the delta subunit serves primarily structural roles in the ATP synthase complex rather than exhibiting enzymatic activity. Researchers can employ the following complementary approaches:

Structural Assessment Methods:

• Circular Dichroism (CD) Spectroscopy: To analyze secondary structure content and compare with predicted structural elements

• Size Exclusion Chromatography: To evaluate oligomeric state and detect potential aggregation

• Thermal Shift Assays: To assess protein stability and proper folding

• Limited Proteolysis: To examine domain organization and accessibility

Functional Interaction Assays:

• Binding Assays with Partner Subunits: Using surface plasmon resonance or isothermal titration calorimetry to measure interactions with other ATP synthase components

• Reconstitution Studies: Combining recombinant atpH with other purified ATP synthase subunits to assess complex formation

• Complementation Assays: Testing whether the recombinant protein can restore function in atpH-deficient bacterial strains

Data Analysis Approach:

By combining multiple assessment methods, researchers can build a comprehensive profile of the recombinant atpH protein's structural integrity and functional capacity.

What experimental approaches can be used to investigate the role of atpH in P. luminescens bioluminescence and pathogenicity?

Investigating the connection between ATP synthase function, specifically the delta subunit (atpH), and P. luminescens bioluminescence and pathogenicity requires targeted experimental approaches:

Genetic Manipulation Strategies:

• Construction of atpH mutants: Generate knockout, knockdown, or site-directed mutants of atpH using CRISPR-Cas9 or allelic exchange methods

• Complementation studies: Restore atpH function through plasmid-based expression to confirm phenotypes are specifically related to atpH modification

• Conditional expression systems: Develop strains with inducible atpH expression to study dose-dependent effects

Phenotypic Assays:

• Bioluminescence measurements: Quantify light production using a luminometer (e.g., Lumat LB9507) with 2-second integration times of 10 μl culture samples

• Insect virulence assays: Assess pathogenicity using established lepidopteran models such as Spodoptera littoralis

• Oxidative stress resistance: Challenge bacteria with hydrogen peroxide (0.5-1 mM) and monitor growth to assess connections between energy metabolism and stress resistance

• Energy status assessment: Measure ATP/ADP ratios and adenylate energy charge (AEC)

Molecular Analysis:

• Transcriptome profiling: Compare gene expression patterns between wild-type and atpH-modified strains, focusing on bioluminescence (lux) genes and virulence factors

• Proteomics: Identify changes in protein expression patterns, particularly in energy metabolism and bioluminescence pathways

• Metabolic flux analysis: Trace carbon flow through central metabolism to understand energetic consequences of atpH modification

By coordinating these experimental approaches, researchers can establish potential causal relationships between ATP synthase function and the distinctive phenotypic characteristics of P. luminescens. The resulting data could be particularly valuable in understanding how energy metabolism interfaces with specialized functions like bioluminescence and virulence mechanisms.

How does ATP synthase function integrate with quorum sensing networks in regulating P. luminescens virulence?

The integration of ATP synthase function with quorum sensing networks in P. luminescens represents a complex regulatory system that likely coordinates energy production with population density-dependent virulence:

Evidence for Regulatory Integration:
Research indicates that AI-2, produced by the LuxS enzyme, regulates over 300 targets in P. luminescens, affecting various metabolic pathways . This quorum sensing system appears to function high in the regulatory hierarchy, controlling multiple transcriptional regulators in a dose-dependent manner . While direct regulation of ATP synthase components by AI-2 is not explicitly documented in the available literature, the extensive reach of AI-2 regulation suggests potential coordination between energy metabolism and virulence mechanisms.

Key Regulatory Connections:

• Oxidative stress resistance: AI-2 increases oxidative stress resistance, which is crucial for overcoming the insect host's immune response involving reactive oxygen species . ATP synthase would need to maintain energy production under these stressful conditions, suggesting coordinated regulation.

• Bioluminescence modulation: AI-2 regulates bioluminescence by controlling spermidine synthesis . Since bioluminescence requires significant energy investment, coordination with ATP production via ATP synthase would be metabolically advantageous.

• Virulence attenuation: luxS-deficient strains exhibit attenuated virulence against lepidopteran hosts . This suggests that quorum sensing affects pathogenicity mechanisms that may have associated energetic requirements supplied by ATP synthase.

Proposed Regulatory Model:

Researchers investigating these connections should consider experimental designs comparing wild-type, luxS-deficient, and ATP synthase-modified strains across metrics of energy production, stress resistance, bioluminescence, and virulence to further elucidate these integrated regulatory networks.

What comparative insights can be gained by studying ATP synthase delta subunits across different bacterial symbionts and pathogens?

Comparative analysis of ATP synthase delta subunits across bacterial symbionts and pathogens provides valuable insights into evolutionary adaptations related to different ecological niches and lifestyles:

Structural and Functional Conservation:
The ATP synthase delta subunit serves as a critical component of the peripheral stalk in F-type ATP synthases across bacterial species. Comparative studies can reveal:

• Core conserved domains: Identifying invariant regions that maintain essential structural and functional properties

• Variable regions: Pinpointing adaptations that may relate to specific environmental conditions

• Phylogenetic relationships: Tracing evolutionary lineages based on sequence conservation patterns

Adaptive Specializations in Dual-Lifestyle Organisms:
P. luminescens represents an interesting case as both a symbiont and pathogen. Comparative analysis with other dual-lifestyle organisms might reveal:

• Energy efficiency adaptations: Modifications that optimize ATP production under different host conditions

• Regulatory interfaces: Variations in regions that might interact with lifestyle-specific regulatory systems

• Stress resistance features: Structural adaptations that maintain functionality under host-imposed stresses

Methodological Framework for Comparative Analysis:

• Sequence alignment: Compare atpH sequences across selected symbionts, pathogens, and free-living relatives

• Structural modeling: Generate and compare predicted structures to identify functionally significant variations

• Expression pattern analysis: Examine differential expression of atpH under symbiotic versus pathogenic conditions

• Functional complementation: Test cross-species functionality by expressing atpH variants in heterologous hosts

Sample Comparative Data:

This comparative approach would provide insights into how energy-generating machinery has evolved to support different bacterial lifestyles, with potential applications in understanding host-microbe interactions and developing targeted antimicrobials.

What emerging technologies could advance our understanding of ATP synthase function in P. luminescens?

Several cutting-edge technologies and approaches show promise for deepening our understanding of ATP synthase function in P. luminescens:

Advanced Structural Biology Techniques:

• Cryo-electron microscopy (Cryo-EM): Enables visualization of the entire ATP synthase complex at near-atomic resolution without crystallization, potentially revealing P. luminescens-specific structural features

• Single-particle analysis: Allows examination of conformational heterogeneity and dynamic states of the ATP synthase complex

• Integrative structural biology: Combines multiple techniques (X-ray crystallography, NMR, SAXS) to build comprehensive structural models of the ATP synthase complex

Real-time Monitoring Systems:

• FRET-based sensors: Development of fluorescence resonance energy transfer sensors to monitor ATP synthase conformational changes and activity in living cells

• Bioluminescence resonance energy transfer (BRET): Leverage P. luminescens' natural luminescence for studying protein-protein interactions involving ATP synthase components

• Single-molecule techniques: Apply methods like magnetic tweezers or optical traps to study the rotational mechanics of individual ATP synthase complexes

Genome Editing and Systems Biology:

• CRISPR-Cas9 precise engineering: Create subtle mutations in atpH to investigate structure-function relationships without disrupting the entire complex

• Multi-omics integration: Combine transcriptomics, proteomics, and metabolomics to understand how ATP synthase function is integrated with global cellular processes

• In silico modeling: Develop computational models of P. luminescens energy metabolism incorporating ATP synthase function under different environmental conditions

Proposed Validation Experiments:

These emerging technologies could provide unprecedented insights into how ATP synthase function is adapted to support P. luminescens' unique dual lifestyle as both an insect pathogen and nematode symbiont.

How might understanding ATP synthase in P. luminescens contribute to the development of novel bioluminescent research tools?

P. luminescens has already shown promise as an alternative organism for recombinant luminescent bioreporters, with bioluminescence expression that remains stable under various conditions . Further understanding of ATP synthase's role in energy provision for bioluminescence could enable the development of advanced research tools:

Enhanced Bioluminescent Reporters:

• Energy-optimized reporters: By understanding the relationship between ATP synthase function and bioluminescence, researchers could design reporters with improved signal strength and duration

• Metabolic state indicators: Develop systems where bioluminescence intensity correlates with specific aspects of cellular energy status

• ATP-dependent luminescence systems: Create synthetic biology tools that couple ATP synthase activity directly to light production for real-time monitoring

Experimental Applications:

• Preservative efficacy testing: Building on existing biosensor applications , develop ATP synthase-modulated reporters for antimicrobial screening

• Environmental toxicity sensors: Create systems where alterations in ATP synthase function due to environmental toxins produce measurable changes in bioluminescence

• Host-pathogen interaction models: Utilize bioluminescent P. luminescens with modified ATP synthase components to track energy utilization during infection processes

Technical Innovation Potential:

By harnessing the natural connection between energy metabolism through ATP synthase and bioluminescence in P. luminescens, researchers could develop a new generation of research tools with applications spanning from basic science to applied fields like environmental monitoring and drug discovery.