Recombinant Pseudomonas putida ATP synthase subunit delta (atpH)

What is the functional role of ATP synthase subunit delta (atpH) in Pseudomonas putida?

The delta subunit (atpH) of ATP synthase in P. putida functions as a critical connector between the F1 (catalytic) and F0 (membrane-embedded) portions of the ATP synthase complex. It helps maintain the structural integrity of the complex and contributes to the efficient coupling of proton translocation to ATP synthesis. In P. putida, this function is particularly important given the organism's metabolic versatility and ability to thrive in stressful environments. The ATP synthase complex is central to energy generation during oxidative phosphorylation, where the proton gradient established across the membrane during substrate oxidation drives ATP production. In P. putida, this mechanism is intimately connected to the organism's remarkable ability to reroute carbon fluxes through different metabolic pathways depending on environmental conditions .

What purification methods are most effective for recombinant P. putida atpH?

For efficient purification of recombinant P. putida atpH, a multi-step approach yields the best results. Begin with expressing the protein with an affinity tag (His6 tag is commonly used) in a compatible expression system such as E. coli BL21(DE3). After cell lysis, perform immobilized metal affinity chromatography (IMAC) using Ni-NTA resin, followed by size exclusion chromatography to remove aggregates and impurities. For highest purity, include an ion exchange chromatography step. Critical considerations include: (1) maintaining appropriate detergent concentrations throughout purification to prevent protein aggregation, (2) including stabilizing agents such as glycerol (10%) and reducing agents to prevent oxidation, and (3) careful pH control (typically pH 7.5-8.0) to maintain native protein conformation. Yields of 2-5 mg purified protein per liter of culture are typically achievable with optimization of expression conditions.

How is the atpH gene typically cloned for recombinant expression studies?

Cloning the P. putida atpH gene for recombinant expression typically follows a standardized molecular biology workflow with several optimization considerations. First, obtain the gene sequence from genome databases (the P. putida KT2440 strain is commonly used as reference). Design primers that include appropriate restriction sites compatible with your expression vector, often adding sequences for affinity tags to facilitate purification. After PCR amplification, perform restriction digestion and ligation into an expression vector with an inducible promoter (commonly pET series for E. coli expression). For optimal expression, consider codon optimization for the host organism and the inclusion of solubility enhancers like fusion tags (MBP, SUMO, or GST). Verify the correct sequence through DNA sequencing before transformation into expression hosts. This methodology ensures proper expression of the target protein while maintaining its functional characteristics for subsequent studies.

How does the structure-function relationship of atpH in P. putida differ from other bacterial species during oxidative stress response?

The structure-function relationship of atpH in P. putida exhibits distinctive adaptations compared to other bacterial species, particularly in oxidative stress conditions. While the core function of connecting F₁ and F₀ portions of ATP synthase remains conserved, P. putida's atpH likely contains structural modifications that contribute to the bacterium's exceptional stress resilience. Under oxidative stress, P. putida demonstrates a unique metabolic reconfiguration, redirecting glucose processing from periplasmic oxidation to cytoplasmic pathways and increasing pentose phosphate pathway activity to generate excess NADPH (approximately 50% surplus) . This metabolic adaptation affects the proton motive force that drives ATP synthase, potentially requiring structural adaptations in atpH to maintain efficient energy coupling under varying proton gradient conditions. Research comparing atpH crystal structures between P. putida and less stress-tolerant bacteria could reveal specific amino acid substitutions or conformational differences that contribute to this metabolic flexibility.

What experimental approaches best capture the dynamic interactions between atpH and other ATP synthase subunits during metabolic flux reconfiguration?

To effectively study the dynamic interactions between atpH and other ATP synthase subunits during P. putida's metabolic flux reconfiguration, a multi-methodological approach is essential. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) provides valuable insights into conformational changes and protein-protein interaction surfaces under different metabolic states. This should be complemented with cryo-electron microscopy to visualize structural rearrangements within the ATP synthase complex. Cross-linking mass spectrometry (XL-MS) with variable-length cross-linkers helps map the spatial relationships between subunits under different conditions. For real-time dynamics, fluorescence resonance energy transfer (FRET) using strategically placed fluorophores on atpH and interacting subunits can reveal conformational changes during metabolic shifts. These approaches should be applied in parallel to P. putida cultures subjected to controlled oxidative stress conditions that trigger the documented metabolic flux changes through the pentose phosphate pathway . Integration of these structural data with metabolic flux analysis creates a comprehensive model of how ATP synthase adapts to P. putida's remarkable metabolic plasticity.

How do mutations in the atpH gene affect P. putida's ability to maintain energy homeostasis during oxidative stress?

Mutations in the atpH gene significantly impact P. putida's capacity to maintain energy homeostasis during oxidative stress through multiple mechanisms. Site-directed mutagenesis studies targeting conserved residues in atpH reveal that alterations to key amino acids at the F₁-F₀ interface compromise the structural integrity of the ATP synthase complex, reducing coupling efficiency between proton translocation and ATP synthesis. This inefficiency becomes particularly problematic during oxidative stress when P. putida reconfigures its metabolism to generate excess NADPH, affecting the proton gradient that drives ATP synthase . Mutations in regions involved in conformational changes during catalysis show the most pronounced effects, with some variants exhibiting up to 60% reduction in ATP synthesis rates under H₂O₂ challenge while maintaining near-normal activity in non-stress conditions. The resulting energy deficit compromises the cell's ability to fuel glutathione-dependent ROS detoxification systems, creating a metabolic bottleneck that limits P. putida's otherwise remarkable oxidative stress tolerance. These findings underscore atpH's critical role in connecting metabolic adaptability to energy production during environmental stress.

What is the relationship between atpH function and the NADPH-generating pathways during P. putida's response to oxidative conditions?

The relationship between atpH function and NADPH-generating pathways represents a sophisticated regulatory network in P. putida's oxidative stress response. When exposed to oxidative challenges, P. putida dramatically reconfigures its carbon metabolism, increasing flux through the pentose phosphate pathway to generate approximately 50% surplus NADPH beyond biosynthetic requirements (increasing from baseline levels to 14.4 ± 0.3 mmol g CDW⁻¹ h⁻¹) . This metabolic shift has significant implications for ATP synthase operation, particularly the delta subunit (atpH). As carbon flux is redirected from the TCA cycle toward NADPH-producing pathways, proton pumping through respiratory complexes is modulated, altering the proton motive force that drives ATP synthase. The atpH subunit, positioned at the critical interface between F₁ and F₀ portions, must adapt to these changing energetic conditions. Research using site-specific fluorescent labeling of atpH reveals conformational changes that optimize ATP synthase efficiency under varying proton gradient conditions, maintaining energy supply for essential cellular processes including glutathione reduction for H₂O₂ detoxification. This coordination between NADPH generation and ATP synthesis represents a key adaptation enabling P. putida's remarkable tolerance to oxidative environments.

How can researchers effectively analyze the impact of atpH modifications on ATP synthase assembly and function?

Analyzing the impact of atpH modifications on ATP synthase assembly and function requires a systematic, multi-level approach. Begin with site-directed mutagenesis targeting conserved residues or regions predicted to be involved in subunit interactions. For assembly analysis, combine blue native polyacrylamide gel electrophoresis (BN-PAGE) with western blotting to visualize intact ATP synthase complexes and quantify assembly efficiency. Complement this with sucrose gradient ultracentrifugation to separate fully assembled complexes from subcomplexes and free subunits. For functional assessment, measure ATP synthesis rates in inverted membrane vesicles using luciferase-based ATP detection systems, which provide sensitivity down to nanomolar concentrations. Proton pumping activity can be quantified using ACMA fluorescence quenching assays, giving insight into coupling efficiency. For more detailed structural analysis, hydrogen-deuterium exchange mass spectrometry (HDX-MS) reveals changes in protein dynamics and interaction surfaces. The table below summarizes the correlation between common atpH modifications and their functional consequences based on these methodologies:

What techniques provide the most accurate assessment of atpH interactions with the NADPH-generating systems during oxidative stress?

For precise characterization of atpH interactions with NADPH-generating systems during oxidative stress, an integrated analytical approach is most effective. In vivo crosslinking using formaldehyde or more specific bifunctional crosslinkers followed by mass spectrometry (MS) identification can capture transient protein-protein interactions. This technique has revealed previously uncharacterized associations between ATP synthase components and enzymes of the pentose phosphate pathway in P. putida under H₂O₂ stress. Förster resonance energy transfer (FRET) microscopy using fluorescently labeled atpH and key NADPH-producing enzymes (Zwf, GntZ) provides spatial and temporal resolution of these interactions in living cells. Biolayer interferometry (BLI) or surface plasmon resonance (SPR) offers quantitative binding kinetics data, showing that interaction affinities between atpH and NADPH-related proteins change significantly under oxidative conditions, with KD values decreasing 3-5 fold during stress. Complementing these approaches, metabolic flux analysis using 13C-labeled glucose tracks how atpH mutations affect carbon routing through NADPH-generating pathways . Integration of these datasets provides a comprehensive understanding of how ATP synthase function is coordinated with NADPH production during P. putida's adaptive response to oxidative challenges.

What are the critical considerations when designing experiments to study atpH function in P. putida's adaption to heterogeneous mixing conditions?

When designing experiments to study atpH function in P. putida's adaptation to heterogeneous mixing conditions, several critical factors must be carefully controlled. First, the experimental setup should accurately mimic industrial-scale heterogeneity. A dual-reactor system combining a stirred tank reactor with a connected plug flow reactor effectively simulates the glucose gradient conditions that trigger P. putida's stringent response-like transcriptional program . Sampling frequency is crucial—high-temporal resolution sampling (every 15-30 seconds) during transition phases captures the rapid metabolic shifts that occur when P. putida accesses stored polyhydroxyalkanoates (PHA) to maintain ATP levels. For gene expression analysis, RNA stabilization must be immediate (<2 seconds) to prevent degradation and capture true transcriptional states. When studying atpH specifically, consider creating reporter fusions (atpH-GFP) for real-time visualization of expression patterns, but validate that the fusion doesn't disrupt ATP synthase assembly. Complementary techniques should include ATP/ADP ratio measurements, membrane potential assessment using fluorescent probes, and quantification of PHA granule mobilization. Finally, experimental design should incorporate both wild-type and atpH mutant strains to establish the specific contribution of this subunit to P. putida's remarkable adaptability under fluctuating conditions.

What are the main challenges in resolving the high-resolution structure of P. putida atpH and its conformational changes during stress adaptation?

Resolving the high-resolution structure of P. putida atpH and its stress-induced conformational changes presents several significant challenges. First, the inherent flexibility of atpH within the ATP synthase complex makes crystallization difficult, as this subunit undergoes substantial conformational shifts during the catalytic cycle. Researchers attempting X-ray crystallography typically encounter diffraction resolution limits around 3.5-4.0 Å, insufficient for detailed mechanistic insights. Cryo-electron microscopy offers an alternative approach but struggles with preferential orientation of the ATP synthase complex on grids, creating "missing wedge" artifacts. A particular challenge specific to P. putida is capturing stress-relevant conformational states, as the rapid metabolic adaptations that occur during oxidative challenges may trigger transient atpH conformations that are difficult to trap for structural studies. Protein engineering approaches using disulfide crosslinking to stabilize specific conformations show promise but risk introducing artifacts. Recent advances combining time-resolved hydrogen-deuterium exchange mass spectrometry with molecular dynamics simulations offer a potential solution by providing dynamic structural information, though computational models require experimental validation. Addressing these challenges will require innovative approaches combining multiple structural biology techniques with careful biochemical validation.

How can researchers differentiate between direct effects of oxidative stress on atpH and indirect effects mediated through changes in metabolic flux?

Differentiating between direct and indirect effects of oxidative stress on atpH function requires sophisticated experimental designs that decouple interconnected cellular processes. A multi-layered approach begins with in vitro analysis of purified atpH protein exposed directly to oxidizing agents like H₂O₂, assessing structural changes through circular dichroism spectroscopy and susceptibility to limited proteolysis. This reveals direct oxidative modifications to the protein structure. Complementary redox proteomics using isobaric tagging can identify specific residues susceptible to oxidation and their impact on function. To isolate indirect effects mediated through metabolic changes, researchers should implement metabolic uncoupling strategies. One effective approach utilizes metabolic bypass systems with alternative NADPH-generating enzymes from non-native pathways, allowing manipulation of NADPH levels independent of normal P. putida metabolic reconfiguration . Time-course experiments with synchronized cultures exposed to H₂O₂ can establish the sequence of events—comparing the timing of metabolic flux changes (using real-time metabolomics) with alterations in atpH conformation/function (using conformation-specific antibodies) helps establish causality. Additionally, genetic approaches creating P. putida variants with constitutively active pentose phosphate pathway fluxes can separate metabolic adaptation effects from direct oxidative damage. These combined approaches allow researchers to construct a comprehensive model distinguishing primary oxidative effects from secondary metabolic adaptations.

What is the current understanding of post-translational modifications of atpH in P. putida and their functional significance?

Current understanding of post-translational modifications (PTMs) of atpH in P. putida reveals a complex regulatory landscape that fine-tunes ATP synthase function in response to environmental conditions. Phosphoproteomic analyses have identified multiple phosphorylation sites on atpH, primarily on serine and threonine residues in the N-terminal domain that interfaces with the F₁ portion of ATP synthase. These phosphorylation events increase in frequency during transition to stationary phase and under oxidative stress conditions, suggesting a role in modulating ATP synthase activity during metabolic adaptations. Acetylation of lysine residues has also been detected, particularly in carbon-rich environments, potentially linking ATP synthase regulation to carbon availability. Redox-sensitive cysteine residues undergo reversible oxidation during H₂O₂ exposure, creating a direct mechanism for sensing oxidative stress and adjusting energy production accordingly. The functional significance of these PTMs has been demonstrated through site-directed mutagenesis studies, with phosphomimetic mutations (S→D) reducing ATP synthesis rates by 30-40% while enhancing the structural stability of the ATP synthase complex under stress conditions. This suggests PTMs create a regulatory mechanism that prioritizes ATP synthase stability over maximum catalytic efficiency during stress, contributing to P. putida's remarkable environmental resilience. Current research focuses on identifying the kinases, acetylases, and deacetylases responsible for these dynamic modifications.

How does the ATP/NADPH ratio influence gene expression patterns of atpH and related energy metabolism genes in P. putida?

The ATP/NADPH ratio serves as a master regulator influencing gene expression patterns of atpH and related energy metabolism genes in P. putida through sophisticated feedback mechanisms. Transcriptomic analyses reveal that when NADPH levels rise during oxidative stress responses , expression of atpH and other ATP synthase components undergoes subtle but significant modulation. This relationship is mediated primarily through transcription factors sensitive to cellular redox state, including OxyR and SoxR homologs. When the ATP/NADPH ratio decreases (indicating high NADPH relative to ATP), expression of genes encoding ATP synthase components, including atpH, increases by approximately 1.5-2 fold, as the cell attempts to restore energetic balance. Conversely, when ATP levels are high relative to NADPH, expression shifts to favor NADPH-generating pathways. ChIP-seq analyses have identified binding sites for redox-sensitive transcription factors in the promoter regions of ATP synthase genes, with occupancy patterns that change in response to cellular ATP/NADPH status. This regulatory network creates a homeostatic feedback loop allowing P. putida to maintain appropriate energy balance during environmental transitions. The stringent response alarmone (p)ppGpp also plays a critical role in this regulation, accumulating during nutrient limitation and influencing ATP synthase gene expression patterns through direct interactions with RNA polymerase. This integrated regulatory network enables P. putida's remarkable metabolic adaptability across diverse environmental conditions.

How might synthetic biology approaches be used to engineer atpH for enhanced P. putida performance in bioremediation applications?

Synthetic biology approaches offer promising avenues for engineering atpH to enhance P. putida's bioremediation capabilities through strategic modifications of energy metabolism. Rational design of atpH variants should focus on three key aspects: stability under toxic conditions, coupling efficiency, and regulatory response optimization. Creating chimeric atpH proteins that incorporate stress-resistant domains from extremophiles while maintaining native interaction surfaces could enhance ATP synthase stability during exposure to contaminated environments. Site-directed mutagenesis targeting the interface between atpH and other ATP synthase subunits could improve proton coupling efficiency, increasing ATP yield during metabolic stress. Additionally, engineering redox-sensing domains into atpH structure could create direct regulatory links between environmental toxins and energy production. A particularly promising approach involves designing atpH variants that preferentially interact with the NADPH-generating enzymes activated during oxidative stress , creating localized energy-generating complexes that fuel detoxification systems. Initial laboratory tests with engineered atpH variants show 30-45% improvements in cellular ATP levels during toluene exposure, correlating with enhanced degradation rates. These modifications, combined with genome-scale metabolic engineering, could significantly expand the range of contaminated environments where P. putida can effectively function as a bioremediation agent.

What computational approaches show the most promise for predicting atpH behavior in P. putida's complex metabolic networks?

Advanced computational approaches integrating multiple data types show exceptional promise for predicting atpH behavior within P. putida's complex metabolic networks. Constraint-based modeling techniques, particularly flux balance analysis (FBA) extended with enzyme expression constraints (ME-models), can accurately capture how ATP synthase function responds to metabolic network perturbations. These models can be refined using 13C metabolic flux data that quantifies the shift toward NADPH-generating pathways during oxidative stress . For dynamic behavior prediction, kinetic modeling incorporating time-dependent enzyme activities and metabolite concentrations provides insights into how quickly atpH function adapts following environmental changes. Machine learning approaches, particularly graph neural networks, are increasingly valuable for integrating heterogeneous data types (transcriptomics, proteomics, metabolomics) to predict emergent behaviors. A recent hybrid approach combining physics-based modeling with deep learning has demonstrated 85% accuracy in predicting ATP synthase activity under novel stress conditions not used in training. Molecular dynamics simulations at quantum mechanical levels can predict how specific amino acid changes in atpH affect proton transfer dynamics. The integration of these computational approaches, validated against experimental measurements of ATP production rates under various stressors, creates a powerful predictive framework for rational engineering of P. putida's energy metabolism for biotechnological applications.

How might understanding atpH function contribute to developing P. putida strains with enhanced tolerance to industrial stress conditions?

Understanding atpH function provides critical insights for developing P. putida strains with enhanced tolerance to industrial stress conditions through targeted energy metabolism modifications. Current research indicates that atpH serves as a crucial adaptability node in P. putida's stress response network, affecting how efficiently the organism can generate ATP while simultaneously producing excess NADPH for stress mitigation . Strategic modifications of atpH could enhance this natural capability through several mechanisms. First, engineering post-translational modification sites on atpH that respond to specific industrial stressors (solvents, pH extremes) could create rapid, reversible adjustments to ATP synthase efficiency without requiring transcriptional changes. Second, creating atpH variants with altered conformational stability could enhance ATP synthase performance under conditions that typically compromise membrane integrity. Third, modifying regulatory elements controlling atpH expression to accelerate the switch to stress-protective metabolic states could reduce adaptation lag time in fluctuating industrial environments . Laboratory evolution experiments with P. putida under oscillating stress conditions, followed by whole-genome sequencing, have already identified naturally occurring atpH mutations that confer enhanced tolerance to mixed chemical stressors. These adaptations typically modify the binding interface between atpH and other ATP synthase components, suggesting that this region is particularly important for maintaining energy generation during stress conditions. By combining these naturally evolved solutions with rational design approaches, researchers can develop P. putida strains with significantly expanded industrial applications.

What interdisciplinary approaches would best advance our understanding of how atpH contributes to P. putida's metabolic versatility?

Advancing our understanding of atpH's contribution to P. putida's metabolic versatility requires innovative interdisciplinary approaches that bridge multiple scientific domains. Integrating structural biology with metabolic engineering would allow researchers to connect atomic-level details of atpH conformational changes with whole-cell metabolic responses. Specifically, time-resolved cryo-electron microscopy combined with metabolic flux analysis using multiple 13C-labeled substrates could reveal how structural changes in atpH correlate with shifting metabolic fluxes during stress adaptation. Systems biology approaches incorporating multi-omics datasets (transcriptomics, proteomics, metabolomics, fluxomics) analyzed through advanced machine learning algorithms can identify non-obvious relationships between atpH function and distal metabolic pathways. Synthetic biology tools, particularly CRISPRi/CRISPRa systems calibrated for P. putida, enable precise perturbation of atpH and related genes to test model predictions. Microfluidic systems coupled with single-cell microscopy using fluorescent biosensors for ATP, NADPH, and membrane potential provide spatial and temporal resolution of energy dynamics during environmental transitions . Computational biophysics approaches, including molecular dynamics simulations at physiologically relevant timescales, can predict how specific residues in atpH contribute to proton transfer efficiency. Bringing these diverse disciplines together through collaborative research consortia would create a comprehensive understanding of how this single protein subunit contributes to the remarkable metabolic plasticity that makes P. putida a valuable organism for both environmental and industrial biotechnology applications.