Recombinant Pelagibacter ubique ATP synthase subunit c (atpE)

What is the function of ATP synthase subunit c (atpE) in Pelagibacter ubique?

ATP synthase subunit c forms the c-ring in the F₀ domain of the ATP synthase complex, which is embedded in the cell membrane. This c-ring plays a crucial role in transmembrane proton translocation, converting the proton motive force into mechanical rotation that drives ATP synthesis. In Pelagibacter ubique, this function is particularly important as this organism has evolved a streamlined genome with minimal metabolic redundancy, making energy efficiency critical for survival .

The atpE protein contains conserved amino acid residues that serve as proton acceptor and donor sites in the proton translocation pathway. As protons move through the membrane domain, they cause rotation of the c-ring, which is mechanically coupled to the central stalk of the F₁ domain, ultimately driving conformational changes that catalyze ATP synthesis from ADP and inorganic phosphate .

How does Pelagibacter ubique ATP synthase compare structurally to other bacterial ATP synthases?

The ATP synthase of α-proteobacteria like P. ubique likely shares similarities with other bacterial systems such as the one from Bacillus PS3, having the same core functions while potentially exhibiting unique regulatory mechanisms adapted to its marine environment and streamlined metabolism . For example, in α-proteobacteria like Paracoccus denitrificans, the ζ subunit has acquired inhibitory and regulatory properties, while the ε subunit has lost those functions that are present in non-α-proteobacterial inhibitory ε subunits .

What are the challenges in recombinant expression of Pelagibacter ubique atpE?

Recombinant expression of proteins from Pelagibacter ubique presents several challenges:

• Codon optimization: P. ubique has a distinctive codon usage pattern due to its AT-rich genome, which may require codon optimization for efficient expression in common host systems like E. coli.

• Membrane protein expression: As a membrane protein, atpE is hydrophobic and may be toxic when overexpressed, potentially requiring specialized expression strategies such as fusion tags or specialized host strains.

• Post-translational modifications: Any native post-translational modifications required for proper function would need to be considered in the recombinant system.

• Functional assembly: The atpE subunit normally functions as part of a multisubunit complex, and expressing it in isolation may present challenges for proper folding and stability .

To overcome these challenges, researchers often employ expression systems with tightly regulated promoters, membrane protein-specific host strains, and fusion partners that enhance solubility or facilitate purification.

How does the proton-pumping mechanism in P. ubique ATP synthase interact with its proteorhodopsin system?

P. ubique utilizes two distinct proton-motive force generation systems: ATP synthase, which uses protons for ATP synthesis, and proteorhodopsin (PR), which functions as a light-driven proton pump. Research has demonstrated that PR in P. ubique is not merely a sensory molecule but actively functions to generate proton motive force under energy-limited conditions .

The interaction between these systems appears to be complementary. Under light conditions, PR can generate a proton gradient that the ATP synthase can utilize for ATP production. Evidence supporting this includes:

• Increased ATP levels in cells exposed to light

• Enhanced transport rates in illuminated cells

• Decreased respiration in the presence of light

This suggests a sophisticated energy management system where P. ubique can substitute light-derived energy for metabolic respiration when needed. This may be particularly important given the organism's streamlined genome and limited metabolic capabilities. For researchers, understanding this interaction is crucial when designing experiments involving recombinant atpE, as light conditions may significantly impact the protein's function in native contexts.

What role does ATP synthase play in the nitrogen metabolism adaptation of P. ubique?

P. ubique has evolved to survive in nutrient-limited marine environments, particularly with respect to nitrogen availability. The ATP synthase complex plays an indirect but critical role in nitrogen assimilation by providing the ATP required for nitrogen acquisition and metabolism.

Under nitrogen limitation conditions, P. ubique upregulates transporters for ammonium (AmtB), taurine (TauA), amino acids (YhdW), and opines (OccT), as well as enzymes for assimilating amine into glutamine (GlnA), glutamate (GltBD), and glycine (AspC) . These processes require substantial energy in the form of ATP, highlighting the importance of efficient ATP synthase function.

The glycine requirement of P. ubique is particularly noteworthy, as the organism appears to utilize the glycine cleavage complex (gcvTHP) to degrade glycine to ammonium, potentially providing a nitrogen source. The AspC enzyme may play a role in producing glycine from glyoxylate . This metabolic relationship suggests that the ATP provided by ATP synthase supports the organism's unusual nitrogen metabolism.

What experimental approaches can distinguish between inhibitory effects on P. ubique ATP synthase compared to other α-proteobacterial ATP synthases?

Based on studies of related α-proteobacteria, researchers investigating inhibitory effects on P. ubique ATP synthase should consider:

• Heterologous reconstitution experiments: Similar to studies with Paracoccus denitrificans, heterologous reconstitution of recombinant subunits can be used to examine inhibitory functions across different α-proteobacteria. This approach allows for direct comparison of inhibitory mechanisms .

• Knockout mutant studies: Creation of knockout mutants lacking specific regulatory subunits (like ζ in P. denitrificans) can reveal their role in inhibition. Comparing growth rates and ATP synthesis/hydrolysis activities between wild-type and knockout strains would be informative .

• Inhibition constant (appIC₅₀) measurements: Determining the apparent inhibition constants for various inhibitory subunits can quantify their relative potency. For example, in related systems, the appIC₅₀ of Pd-ζ to inhibit RcF₁F₀-ATPase was 3.76 μM, while the corresponding appIC₅₀ of Js-ζ was 1.12 μM .

• Conformational state analysis: Cryo-EM imaging of the ATP synthase complex in different rotational states can reveal how regulatory subunits (such as ε in Bacillus PS3) adopt different conformations to inhibit ATP hydrolysis while allowing ATP synthesis .

These approaches would help distinguish the unique regulatory features of P. ubique ATP synthase from those of other α-proteobacteria.

What purification strategies are most effective for recombinant P. ubique atpE?

While the search results don't provide specific purification methods for P. ubique atpE, several approaches proven effective for membrane proteins and ATP synthase components can be suggested:

• Detergent solubilization optimization: Test a panel of detergents including n-dodecyl β-D-maltoside (DDM), lauryl maltose neopentyl glycol (LMNG), and digitonin to identify optimal solubilization conditions.

• Affinity chromatography: Utilize fusion tags such as poly-histidine, Strep-tag II, or FLAG tag for initial capture. For example, the Bacillus PS3 ATP synthase was successfully expressed in E. coli with affinity tags that facilitated purification .

• Size exclusion chromatography: This technique can separate the properly folded protein from aggregates and other contaminants while allowing buffer exchange to more stable conditions.

• Lipid nanodisc incorporation: For functional studies, reconstitution into lipid nanodiscs can provide a more native-like membrane environment than detergent micelles.

A typical purification workflow might include:

• Membrane isolation by ultracentrifugation

• Detergent solubilization (starting with 1% DDM)

• IMAC purification using His-tagged protein

• Size exclusion chromatography

• Quality assessment by SDS-PAGE and Western blotting (similar to the approaches used for R. capsulatus F₁-ATPase )

What experimental designs can assess the functional activity of recombinant P. ubique atpE?

Functional characterization of recombinant P. ubique atpE requires assessing its integration into the ATP synthase complex and its participation in proton translocation. Several experimental approaches can be employed:

• ATP hydrolysis assays: Measure the ATPase activity of reconstituted complexes containing recombinant atpE using a coupled enzyme assay that monitors NADH oxidation spectrophotometrically.

• Proton pumping assays: Assess proton translocation using pH-sensitive fluorescent dyes like ACMA (9-amino-6-chloro-2-methoxyacridine) in reconstituted proteoliposomes.

• Rotation assays: Direct observation of c-ring rotation using fluorescent probes attached to the F₁ domain while the F₀ domain is immobilized on a surface.

• Inhibitor sensitivity profiling: Compare the sensitivity of recombinant atpE-containing ATP synthase to known inhibitors like oligomycin or DCCD (dicyclohexylcarbodiimide), which specifically bind to the c-subunit.

• Complementation studies: Test whether the recombinant atpE can functionally complement ATP synthase deficient strains.

These approaches can be calibrated using measurements similar to those used in characterizing ATP synthases from related organisms, such as the heterologous reconstitution studies performed with Paracoccus denitrificans and Rhodobacter capsulatus .

How can researchers study the interaction between light-harvesting systems and ATP synthase in P. ubique?

P. ubique's ability to utilize light via proteorhodopsin (PR) to supplement its energy requirements makes the interaction between light-harvesting systems and ATP synthase particularly interesting. Researchers can investigate this interaction through:

• Comparative growth experiments: Assess growth rates and ATP production in light versus dark conditions, comparing wild-type strains with those expressing recombinant atpE variants.

• Real-time ATP monitoring: Utilize luciferase-based ATP sensors to measure ATP levels in response to light exposure and correlate with ATP synthase activity .

• Membrane potential measurements: Use voltage-sensitive fluorescent dyes to monitor the proton motive force generated by PR and utilized by ATP synthase.

• Isotope labeling studies: Employ stable isotope-labeled substrates to track carbon flux through central metabolism under different light conditions.

• Transcriptional response analysis: Compare transcription levels of ATP synthase genes including atpE under varying light regimes to identify regulatory relationships.

Data from such experiments might be presented in tables similar to this:

These methods would help elucidate how P. ubique integrates light energy capture with ATP synthesis, which is critical for understanding the energy economy of this abundant marine organism .

How do researchers reconcile differences between in vitro and in vivo behaviors of recombinant P. ubique atpE?

Researchers often encounter discrepancies between the behavior of recombinant proteins in vitro and their native function in vivo. For P. ubique atpE, these challenges may include:

• Lipid environment differences: The highly specialized membrane composition of P. ubique may not be replicated in recombinant systems, affecting protein function. Researchers should consider using lipid compositions that mimic the native environment when reconstituting the protein.

• Regulatory interactions: In vivo, atpE function is regulated by interactions with other ATP synthase subunits and environmental factors that may be absent in vitro. These interactions may be studied using techniques like crosslinking mass spectrometry or co-immunoprecipitation.

• Post-translational modifications: Any modifications present in the native system should be verified in recombinant proteins, possibly using mass spectrometry-based proteomics approaches.

• Uncoupling between transcription and translation: As observed in P. ubique under nutrient limitation, there may be uncoupling between transcription and translation levels . This means that mRNA levels may not correlate with protein expression, necessitating both transcriptomic and proteomic analyses to fully understand regulation.

To address these discrepancies, researchers should employ multiple complementary approaches and carefully control experimental conditions to match those found in the native marine environment.

What considerations are important when interpreting evolutionary conservation of atpE in SAR11 clade organisms?

The evolutionary conservation of atpE within the SAR11 clade provides insights into adaptation strategies in these abundant marine bacteria. When interpreting conservation patterns, researchers should consider:

• Genomic streamlining context: P. ubique has undergone extensive genome streamlining, retaining only essential functions. Conservation of atpE sequence elements likely indicates fundamental functional importance rather than regulatory complexity .

• Coevolution with other ATP synthase subunits: The evolution of atpE should be analyzed in the context of other ATP synthase subunits, particularly focusing on interface residues that mediate subunit interactions.

• Environmental adaptation signatures: Sequence variations across SAR11 strains from different oceanic regions may reflect adaptation to specific environmental conditions like temperature, pH, or salinity.

• Comparison with other α-proteobacteria: As seen with the regulatory ζ subunit in Paracoccus denitrificans, α-proteobacteria may have evolved distinct regulatory mechanisms compared to other bacteria . Researchers should examine whether atpE has undergone similar specialized evolution within the SAR11 clade.

• Function-structure relationships: Conservation analyses should consider not just sequence similarity but structural and functional conservation, as convergent evolution may produce similar functions through different sequences.

These considerations help researchers interpret whether observed conservation patterns represent maintenance of core functionality or adaptation to specific ecological niches.

How might synthetic biology approaches improve recombinant production of functional P. ubique atpE?

Synthetic biology offers several promising approaches to enhance the recombinant production of functional P. ubique atpE:

• Codon harmonization: Rather than simple codon optimization, codon harmonization preserves the translation rhythm of the original organism, potentially improving folding of membrane proteins like atpE.

• Minimal expression systems: Using simplified genetic backgrounds or cell-free expression systems could reduce competing cellular processes and improve yield.

• Nanodisk technology: Direct expression into synthetic lipid environments using nanodisks could improve proper folding and stability of the hydrophobic atpE protein.

• Directed evolution: Application of directed evolution techniques could select for variants with improved expression and stability while maintaining functional properties.

• Co-expression strategies: Simultaneous expression of multiple ATP synthase subunits might promote proper complex assembly and stabilize individual components like atpE.

These approaches could potentially overcome the challenges associated with producing membrane proteins from organisms with specialized cellular environments like P. ubique.

What is the potential role of P. ubique ATP synthase in understanding energy conservation in nutrient-limited marine environments?

Understanding P. ubique ATP synthase function has broader implications for marine ecology given the organism's abundance in nutrient-limited oceans:

• Energy efficiency mechanisms: P. ubique's streamlined genome suggests highly optimized energy conservation strategies. Its ATP synthase may reveal specialized adaptations for function at minimal energy expenditure .

• Integration with alternative energy sources: The documented interaction between proteorhodopsin and ATP synthase in P. ubique represents a sophisticated energy management system that allows the organism to substitute light-derived energy for metabolic respiration when available .

• Adaptation to nitrogen limitation: P. ubique's response to nitrogen limitation involves regulated expression of transporters and metabolic enzymes that require ATP, highlighting the interconnection between energy metabolism and nutrient acquisition .

• Ecological modeling: Understanding the energetics of P. ubique ATP synthase could improve models of marine carbon and nitrogen cycling, as SAR11 bacteria constitute approximately 25% of all plankton cells in the oceans.

• Comparative analyses: Comparing ATP synthase efficiency across marine bacteria adapted to different oceanic regions could reveal how energy conservation mechanisms contribute to niche specialization.

Research in this area could provide fundamental insights into how Earth's most abundant organisms maintain cellular functions with minimal resources, potentially informing strategies for engineering energy-efficient biological systems.