Recombinant Rhodopirellula baltica ATP synthase subunit alpha 1 (atpA1), partial

What is Rhodopirellula baltica and why is it important as a model organism?

Rhodopirellula baltica (R. baltica) SH 1T is a marine bacterium isolated from the Kiel Fjord (Baltic Sea) and taxonomically grouped within the bacterial phylum Planctomycetes. It has emerged as an important model organism for several reasons:

R. baltica is abundant in aquatic habitats and plays a significant role in carbon cycling. The organism possesses several unique features, including peptidoglycan-free proteinaceous cell walls, intracellular compartmentalization, and a distinctive mode of reproduction via budding. This reproductive strategy results in a life cycle comprised of motile and sessile morphotypes, resembling that of Caulobacter crescentus .

The genome of R. baltica has been completely sequenced, revealing several interesting traits including a high number of sulfatase genes, fascinating carbohydrate-active enzymes, and a conspicuous C1-metabolism pathway. Additionally, R. baltica serves as a model organism for aerobic carbohydrate degradation in marine systems, where polysaccharides represent the dominant components of biomass .

What is the role of ATP synthase in bacterial energy metabolism?

ATP synthase (also known as F-ATPase or F1F0-ATPase) plays a crucial role in bacterial energy metabolism by catalyzing the synthesis of ATP from ADP and inorganic phosphate using the energy stored in a transmembrane electrochemical gradient.

The structure of F-ATPase consists of two domains: F1-ATPase contains three catalytic β subunits that perform ATP synthesis (or in some cases hydrolysis), while F0-ATPase translocates protons (or sodium ions) across the membrane down the electrochemical gradient. In coupling proton transport to ATP synthesis, the F0 and F1 domains function as a pair of rotary motors linked by a common central rotor (γεc) and a peripheral stator (bδ) .

The multiple c-subunits (proteolipids) form a ring, and rotation of this c-ring allows protons to be carried between two partial subunit-a channels that lead to opposite sites of the membrane. Each c-subunit typically contains two transmembrane helices connected by a cytoplasmic loop and one protonizable carboxylate group (glutamate or aspartate) .

What are the specific characteristics of ATP synthase subunit alpha in bacteria?

The alpha subunit (encoded by atpA gene) is one of the major components of the F1 portion of ATP synthase. While the search results don't provide specific information about the R. baltica atpA1 subunit, general characteristics of ATP synthase alpha subunits in bacteria include:

• The alpha subunit forms part of the catalytic hexamer (α3β3) in the F1 domain

• It contains nucleotide binding sites that are regulatory rather than catalytic

• It plays a crucial role in the conformational changes required for ATP synthesis

• It interacts with both beta subunits and the central stalk components

In R. baltica, the gene expression studies revealed that genes associated with energy production and conservation (COG class C), which would include ATP synthase genes, show differential regulation throughout the growth phases, suggesting adaptation to changing energy demands during the organism's life cycle .

How is ATP synthase expression regulated during R. baltica's life cycle?

During growth phase transitions in R. baltica, significant changes occur in the expression of genes related to energy production and conservation. Transcriptomic analysis revealed that genes associated with energy production were downregulated in mid-exponential phase (62h) compared to early exponential phase (44h), suggesting lower metabolic activity as nutrient availability decreased .

When comparing transition phase and stationary growth phase (82h) with mid-exponential phase (62h), more genes were regulated in the stationary phase, indicating metabolic adaptations to nutrient limitation. During the stationary phase, especially late stationary phase (240h vs 82h), R. baltica induces genes associated with energy production while repressing others involved in energy production, suggesting complex regulatory mechanisms to maintain energy balance under stress conditions .

This differential regulation likely extends to ATP synthase components, including atpA1, as the organism adapts its energy metabolism to changing environmental conditions.

How does R. baltica adapt its energy metabolism under stress conditions?

R. baltica employs several strategies to adapt its energy metabolism under stress conditions:

In response to nutrient limitation and increased cell density during transition phase, R. baltica cells adapt metabolically by inducing stress-related genes. Specifically, genes coding for glutathione peroxidase (RB2244), thioredoxin (RB12160), bacterioferritin comigratory protein (RB12362), universal stress protein (uspE, RB4742), and chaperones (e.g., RB8966) are upregulated. Additionally, diverse dehydrogenases, hydrolases, and reductases are differentially regulated to facilitate metabolic adaptation and prepare for long-term survival under unfavorable conditions .

During the stationary phase, R. baltica increases expression of genes involved in ubiquinone biosynthesis (RB2748, RB2749, and RB2750), which may compensate for lower oxygen availability, similar to responses observed in Agrobacter tumefaciens and Rhodobacter sphaeroides .

These adaptations in energy metabolism likely influence ATP synthase activity and expression, including the atpA1 subunit, to maintain ATP production under varying conditions.

What post-translational modifications might affect R. baltica ATP synthase activity?

While the search results don't specifically address post-translational modifications of R. baltica ATP synthase, research on other bacterial ATP synthases suggests several potential modifications:

• Phosphorylation: Phosphorylation of ATP synthase subunits can regulate activity in response to cellular energy status

• Disulfide bond formation: Under oxidative stress, disulfide bonds may form between cysteine residues, affecting enzyme conformation and activity

• Acetylation: Lysine acetylation has been observed in bacterial ATP synthases and may influence activity

• Proteolytic processing: Some subunits may undergo proteolytic cleavage during maturation

The stress response mechanisms identified in R. baltica, including upregulation of thioredoxin and chaperones , suggest that redox-based modifications and protein folding quality control may influence ATP synthase activity during the organism's adaptation to environmental changes.

What expression systems are optimal for producing recombinant R. baltica atpA1?

For recombinant expression of R. baltica atpA1, several expression systems could be considered based on general principles of recombinant protein expression:

Table 1: Comparison of Expression Systems for Recombinant R. baltica atpA1

The search results provide a relevant example of successful heterologous expression: a bacterial phosphatase from R. baltica was successfully expressed in both yeast (S. cerevisiae) and mammalian cells, where it localized correctly to mitochondria and retained functionality . This suggests that R. baltica proteins can be successfully expressed in heterologous systems while maintaining their function.

What purification strategy should be employed for recombinant R. baltica atpA1?

Based on general principles for purifying recombinant ATP synthase components, a multi-step purification strategy is recommended:

• Affinity Chromatography: Use of a fusion tag (His6, FLAG, GST) for initial capture

  • His6-tag is often preferred due to small size and minimal impact on protein function

  • Consider placement at N- or C-terminus based on structural predictions

• Ion Exchange Chromatography: Separation based on charge differences

  • Consider theoretical pI of atpA1 to select appropriate resin type and pH conditions

• Size Exclusion Chromatography: Final polishing step to obtain homogeneous protein

  • Also useful for assessing oligomeric state of the purified protein

• Special Considerations for ATP Synthase Subunits:

  • Buffer optimization to maintain stability (typically including ATP or ADP)

  • Addition of glycerol (10-20%) to prevent aggregation

  • Careful selection of detergents if membrane-associated regions are present

The search results demonstrate that recombinant proteins from R. baltica can be successfully purified and characterized, as shown with the PTPMT1-like phosphatase that was purified and demonstrated to have enzymatic activity in vitro .

How can the functionality of recombinant R. baltica atpA1 be assessed?

Several approaches can be employed to assess the functionality of recombinant atpA1:

• ATP Binding Assays:

  • Fluorescent ATP analogs to measure binding affinity

  • Isothermal titration calorimetry (ITC) for thermodynamic binding parameters

• ATPase Activity Assays:

  • Colorimetric detection of inorganic phosphate release

  • Coupled enzyme assays linking ATP hydrolysis to NADH oxidation

  • Luciferin/luciferase assays for ATP consumption

• Structural Integrity Assessment:

  • Circular dichroism (CD) spectroscopy for secondary structure analysis

  • Thermal shift assays to evaluate protein stability

  • Limited proteolysis to assess folding quality

• Interaction Studies:

  • Pull-down assays with other ATP synthase subunits

  • Surface plasmon resonance (SPR) for binding kinetics

  • Native gel electrophoresis to assess complex formation

The search results show that functional assessment was successfully applied to the R. baltica PTPMT1-like phosphatase, where in vitro enzymatic activity was demonstrated, and complementation of a yeast mutant confirmed its functionality . Similar approaches could be adapted for atpA1 functional characterization.

What structural features distinguish R. baltica ATP synthase from other bacterial ATP synthases?

While the search results don't provide specific structural information about R. baltica ATP synthase, general structural features of bacterial F-type ATP synthases can be considered:

F-type ATP synthases typically consist of F1 and F0 domains. The F1 domain contains three catalytic β subunits that perform ATP synthesis, while the F0 domain translocates protons across the membrane. The two domains function as rotary motors linked by a central rotor and a peripheral stator .

R. baltica's unique cellular organization, with compartmentalized cells and specialized membranes, might result in adaptations to its ATP synthase structure. The search results mention that R. baltica has a distinctive cell wall composition, which changes during different growth phases . This could potentially influence membrane protein organization, including ATP synthase.

Comparative sequence analysis with related species would be necessary to identify specific structural features that distinguish R. baltica ATP synthase from other bacterial homologs. Such analysis could reveal adaptations related to the marine environment or the organism's unique cellular architecture.

How does the ATP synthase gene organization in R. baltica compare to other bacteria?

The search results provide information about ATP synthase gene clusters in another bacterial species (Candidatus Kuenenia stuttgartiensis) that can serve as a comparative reference:

In C. Kuenenia stuttgartiensis, four ATPase gene clusters were identified:

• F-ATPase-1 contained a complete set of genes similar to E. coli F-ATPase

• F-ATPase-2 and -3 lacked a gene encoding the delta subunit

• V-ATPase-4 contained catalytic subunits (B and A), proteolipid (L), ion channel (I), and parts of the rotor (D) and stator (E)

Expression analysis showed that F-ATPase-1 was highly expressed at both transcriptome and proteome levels, while F-ATPase-2 and -3 showed very low transcription and weren't detected in the proteome. V-ATPase-4 catalytic subunits were detected in the transcriptome, and one peptide was detected in the proteome .

While specific information about R. baltica's ATP synthase gene organization isn't provided in the search results, its genome has been completely sequenced , which would enable comparative genomic analysis to understand the organization and regulation of its ATP synthase genes relative to other bacterial species.

How might the marine environment influence R. baltica ATP synthase properties?

R. baltica's adaptation to the marine environment likely influences its ATP synthase properties in several ways:

• Salt Adaptation: The search results mention salt resistance observed during cultivation of R. baltica . Marine bacteria need to adapt to higher salt concentrations, which could affect membrane properties and the function of membrane proteins like ATP synthase. This may be reflected in amino acid compositions that enhance stability in high-salt conditions.

• pH and Ion Composition: Marine environments have distinct pH and ion composition compared to freshwater or terrestrial environments, which could influence the proton gradient used by ATP synthase and potentially lead to adaptations in the proton channel components.

• Temperature Adaptation: The Baltic Sea, where R. baltica was isolated, experiences temperature fluctuations, which may necessitate adaptations in enzyme kinetics and stability.

• Pressure Considerations: While not directly mentioned in the search results, marine bacteria often face different pressure conditions than terrestrial bacteria, which can affect protein structure and function.

The search results indicate that R. baltica modifies its cell wall composition and membrane properties in response to environmental changes , suggesting that membrane-associated proteins like ATP synthase may have adaptations to function optimally in changing marine conditions.

How can site-directed mutagenesis of R. baltica atpA1 advance our understanding of ATP synthase mechanisms?

Site-directed mutagenesis of R. baltica atpA1 could provide valuable insights into ATP synthase mechanisms through targeted modifications of key residues:

• Catalytic Site Residues: Mutating residues involved in nucleotide binding and hydrolysis to elucidate their specific roles in catalysis

  • Conserved lysine and arginine residues that interact with phosphate groups

  • Residues coordinating magnesium ion binding

• Interface Residues: Modifying residues at subunit interfaces to understand assembly and inter-subunit communication

  • atpA1/beta subunit interface residues to study catalytic cooperativity

  • atpA1/gamma subunit interface residues to investigate rotational coupling

• Marine Environment Adaptations: Targeting unique residues in R. baltica atpA1 compared to non-marine homologs to understand environmental adaptations

  • Residues potentially involved in salt tolerance

  • Unique structural elements that may confer stability in marine conditions

• Experimental Design Approach:

  • Generate a library of single point mutations at conserved and unique sites

  • Express and purify mutant proteins using optimized protocols

  • Characterize effects on structure (using CD spectroscopy, thermal stability assays)

  • Assess functional impacts (ATP binding affinity, hydrolysis rates)

  • Determine effects on assembly using native gel electrophoresis or analytical ultracentrifugation

The search results demonstrate that R. baltica proteins can be successfully expressed in heterologous systems for functional studies , providing a foundation for mutagenesis studies of atpA1.

What insights can comparative genomics provide about the evolution of atpA1 in R. baltica?

Comparative genomics approaches can yield significant insights into the evolution of atpA1 in R. baltica:

• Phylogenetic Analysis: Constructing phylogenetic trees of atpA sequences from diverse species can reveal:

  • The evolutionary relationship of R. baltica atpA1 to homologs in other Planctomycetes

  • Potential horizontal gene transfer events

  • Selective pressures on different domains of the protein

• Synteny Analysis: Examining the genomic context of atpA1 across related species to understand:

  • Conservation of ATP synthase operon structure

  • Gene rearrangements that might affect regulation

  • Acquisition or loss of accessory genes

• Selection Pressure Analysis: Calculating dN/dS ratios to identify:

  • Regions under purifying selection (functionally constrained)

  • Regions under positive selection (potentially adaptations to marine environment)

• Structural Mapping of Conserved Features: Using homology modeling to map conserved and variable regions to structural models to understand:

  • Core functional elements preserved across species

  • Variable regions that might confer species-specific properties

The search results mention that R. baltica has distinctive genomic features and that genome rearrangements occur under stress conditions , suggesting dynamic genome evolution that could affect ATP synthase genes.

How could R. baltica atpA1 be used in synthetic biology applications?

R. baltica atpA1 could be leveraged in several synthetic biology applications:

• Engineered Bioenergetic Systems:

  • Development of salt-tolerant ATP synthases for bioenergy applications

  • Creation of hybrid ATP synthases with optimized properties by combining subunits from different species

  • Engineering ATP synthases with altered ion specificity (H+ vs Na+)

• Biosensors:

  • Development of ATP biosensors based on conformational changes in atpA1

  • Creation of environmental stress biosensors by coupling atpA1 expression to reporter systems

• Model Systems for Studying Membrane Protein Evolution:

  • Using R. baltica atpA1 as a model to understand how membrane proteins adapt to different environments

  • Experimental evolution studies to observe real-time adaptation of ATP synthase components

• Biotechnological Applications:

  • Engineering R. baltica atpA1 for improved ATP production in biotechnological processes

  • Development of stress-resistant biocatalysts for industrial applications

The search results indicate that R. baltica has several features with biotechnological potential , suggesting that its ATP synthase components could be valuable in synthetic biology applications, particularly those requiring function in marine-like conditions or stress tolerance.

How can aggregation issues during recombinant expression of R. baltica atpA1 be addressed?

Aggregation is a common challenge when expressing ATP synthase subunits. Several strategies can be employed to address this issue:

• Expression Condition Optimization:

  • Lower induction temperature (16-20°C) to slow protein synthesis and improve folding

  • Reduce inducer concentration to decrease expression rate

  • Use rich media formulations to provide ample resources for protein folding

• Fusion Partners and Solubility Tags:

  • MBP (maltose-binding protein) fusion to enhance solubility

  • SUMO tag to improve folding and enable specific cleavage

  • Thioredoxin fusion to assist disulfide bond formation if relevant

• Co-expression Strategies:

  • Co-express with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) to assist folding

  • Co-express with other ATP synthase subunits that naturally interact with atpA1

  • Include molecular chaperones specific to membrane proteins if atpA1 has membrane-associated regions

• Buffer and Additive Screening:

  • Include stabilizing agents like glycerol, sucrose, or arginine

  • Test various detergents if membrane interactions cause aggregation

  • Optimize pH and ionic strength based on theoretical properties of atpA1

The search results indicate that R. baltica proteins have been successfully expressed in heterologous systems , and the organism's stress response involves chaperones , suggesting that co-expression with appropriate chaperones might be particularly effective.

What strategies can overcome challenges in functional reconstitution of R. baltica ATP synthase complexes?

Functional reconstitution of ATP synthase complexes presents several challenges. Here are strategies to address them:

• Membrane Reconstitution Approaches:

  • Liposome reconstitution using lipid compositions that mimic R. baltica membranes

  • Nanodiscs for stable membrane protein incorporation in a native-like environment

  • Polymer-based systems (amphipols, SMALPs) that maintain membrane protein structure

• Stepwise Assembly Strategies:

  • Reconstitution of subcomplexes (F1 portion) before attempting complete ATP synthase assembly

  • Sequential addition of purified subunits to form functional complexes

  • Co-expression of multiple subunits to promote proper assembly

• Activity Verification Methods:

  • ATP synthesis assays using artificial proton gradients

  • ATPase activity measurements with specific inhibitors as controls

  • Proton pumping assays using pH-sensitive fluorescent dyes

• Optimization for R. baltica-Specific Conditions:

  • Consider salt concentration requirements based on the marine origin of R. baltica

  • Adjust pH to optimal range for R. baltica enzymes

  • Include specific lipids that might be required for function

The search results indicate that R. baltica has unique cell membrane compositions that change during different growth phases , suggesting that lipid composition may be critical for proper reconstitution of its membrane proteins, including ATP synthase complexes.

How can researchers address inconsistent results in ATP hydrolysis/synthesis assays with recombinant R. baltica atpA1?

Inconsistent results in ATP hydrolysis/synthesis assays can be addressed through systematic troubleshooting:

• Sample Quality Control:

  • Verify protein purity using SDS-PAGE and mass spectrometry

  • Assess protein folding using circular dichroism spectroscopy

  • Check for presence of contaminating ATPases using specific inhibitors

• Assay Optimization:

  • Systematically vary assay conditions (pH, temperature, salt concentration)

  • Test different divalent cation concentrations (Mg2+, Ca2+, Mn2+)

  • Include proper controls for background ATP hydrolysis

• Technical Considerations:

  • Ensure equipment calibration (spectrophotometers, pH meters)

  • Prepare fresh reagents and substrates

  • Use multiple detection methods to cross-validate results

• Data Analysis and Reporting:

  • Apply appropriate statistical analysis to identify outliers

  • Report all experimental conditions in detail

  • Consider batch-to-batch variation in protein preparations

Table 2: Systematic Approach to Troubleshooting ATP Hydrolysis Assays

The search results don't provide specific information about ATP hydrolysis assays with R. baltica proteins, but they do indicate that R. baltica adapts its metabolism to different conditions , suggesting that assay conditions should be carefully optimized to reflect the organism's native environment.

What emerging technologies could advance our understanding of R. baltica ATP synthase structure and function?

Several cutting-edge technologies could significantly advance our understanding of R. baltica ATP synthase:

• Cryo-Electron Microscopy (Cryo-EM):

  • High-resolution structural determination without crystallization

  • Visualization of ATP synthase in different conformational states

  • Structural analysis in more native-like environments

• Single-Molecule Techniques:

  • Fluorescence resonance energy transfer (FRET) to study conformational changes

  • Magnetic tweezers to investigate the mechanical properties of ATP synthase rotation

  • Single-molecule force spectroscopy to measure energy transduction

• Advanced Computational Methods:

  • Molecular dynamics simulations to study conformational dynamics

  • Machine learning approaches to identify patterns in sequence-structure-function relationships

  • Quantum mechanics/molecular mechanics (QM/MM) to study catalytic mechanisms

• Genetic Manipulation Technologies:

  • CRISPR-Cas9 gene editing in R. baltica to study ATP synthase in vivo

  • Development of inducible gene expression systems for R. baltica

  • High-throughput mutagenesis coupled with functional screening

The search results mention the availability of genomic and proteomic data for R. baltica , which provides a foundation for applying these advanced technologies to understand its ATP synthase structure and function.

How might studies of R. baltica atpA1 contribute to our understanding of bioenergetic evolution?

Studies of R. baltica atpA1 could provide valuable insights into bioenergetic evolution:

• Evolutionary Adaptation to Marine Environments:

  • Understanding how ATP synthases adapt to marine conditions could reveal evolutionary mechanisms for environmental adaptation

  • Comparison with terrestrial and extremophile ATP synthases could highlight convergent or divergent evolution

• Planctomycetes-Specific Adaptations:

  • The unique cellular organization of Planctomycetes, including R. baltica, may have driven specific adaptations in their bioenergetic systems

  • Investigating these adaptations could reveal how cellular compartmentalization influences energy conversion machinery

• Ancient Origins of Rotary ATPases:

  • R. baltica belongs to a deeply branching bacterial phylum, and studying its ATP synthase could provide insights into early evolutionary events

  • Comparing with archaeal and eukaryotic ATP synthases could help reconstruct the evolutionary history of these essential enzymes

• Horizontal Gene Transfer and Modular Evolution:

  • Analysis of atpA1 sequences could reveal evidence of horizontal gene transfer events

  • Understanding how different modules of ATP synthase co-evolved could inform theories about the origin and evolution of complex molecular machines

The search results indicate that R. baltica has distinctive genomic features and that its genome rearrangements occur under stress conditions, suggesting dynamic genome evolution that might have influenced its bioenergetic systems.

What potential biotechnological applications might emerge from R. baltica ATP synthase research?

Research on R. baltica ATP synthase could lead to several innovative biotechnological applications:

• Bioenergy Applications:

  • Development of salt-tolerant ATP synthase variants for bioenergy production in non-ideal conditions

  • Engineering ATP synthase for improved efficiency in biotechnological processes

  • Creation of synthetic ATP-generating systems for artificial cells or bioreactors

• Biomedical Applications:

  • Design of ATP synthase inhibitors as potential antimicrobials

  • Development of nanomotors based on the rotary mechanism of ATP synthase

  • Creation of biosensors for detecting cellular energy status

• Industrial Enzyme Applications:

  • Engineering stress-resistant ATPases for industrial processes

  • Development of immobilized ATP synthase systems for ATP regeneration in enzymatic reactions

  • Creation of hybrid enzymes combining functional domains from different species

• Environmental Biotechnology:

  • Applications in bioremediation technologies requiring energy generation in marine environments

  • Development of biosensors for environmental monitoring

  • Creation of synthetic ecosystems with engineered energy metabolism

The search results mention that R. baltica has several features with biotechnological promise, including enzymes for the synthesis of complex organic molecules with possible applications in pharmaceutical, food, or animal-feed industries , suggesting that its ATP synthase components could also have valuable biotechnological applications.