Recombinant Prochlorococcus marinus ATP synthase subunit c (atpE)

What is the significance of ATP synthase subunit c (atpE) in Prochlorococcus marinus?

ATP synthase subunit c (atpE) plays a crucial role in energy metabolism in Prochlorococcus marinus, a globally abundant marine cyanobacterium. This protein is part of the F0 complex of ATP synthase that forms the proton channel through the membrane, enabling ATP synthesis via chemiosmosis. In Prochlorococcus, which dominates phytoplankton populations in large regions of the oligotrophic ocean, efficient energy production is essential for survival in nutrient-poor environments . The protein's structure and function are particularly interesting given Prochlorococcus' remarkable ecological success despite having a relatively small genome (2.4 MB) compared to other prolific secondary metabolite producers .

How does one clone and express recombinant Prochlorococcus marinus atpE in E. coli?

The expression of recombinant Prochlorococcus proteins requires careful optimization due to their unique codon usage and membrane protein characteristics. A methodological approach includes:

• Gene synthesis or PCR amplification of the atpE gene with appropriate restriction sites

• Cloning into an expression vector with an N-terminal His6-tag for purification

• Transformation into an E. coli expression strain (similar to methods used for other Prochlorococcus proteins described in the literature)

• Expression induction with IPTG at lower temperatures (16-20°C) to enhance proper folding

• Membrane fraction isolation followed by detergent solubilization

• Affinity purification using nickel-nitrilotriacetic acid (Ni-NTA) resin

This approach is similar to methodologies used for other Prochlorococcus membrane proteins and has been successfully employed for heterologous expression of various proteins from this organism as described for ProcA peptides and ProcM enzyme .

What purification strategies work best for recombinant Prochlorococcus marinus atpE?

Purification of recombinant Prochlorococcus marinus atpE requires specialized approaches due to its hydrophobic nature as a membrane protein component. The recommended purification strategy includes:

This strategy draws from successful membrane protein purification approaches and has been adapted based on known properties of cyanobacterial ATP synthase components .

How does the structure-function relationship of Prochlorococcus marinus atpE differ from that of other cyanobacteria?

Prochlorococcus marinus atpE exhibits unique structural adaptations that may contribute to its function in extreme oligotrophic environments. While the core structure remains conserved, key differences include:

• Amino acid substitutions that may optimize proton conductance at low energy states

• Potential adaptations for operation at varying light intensities, corresponding to the diurnal cycles experienced in marine environments

• Structural modifications that may enhance interaction with other ATP synthase subunits in Prochlorococcus' streamlined genome context

These adaptations likely reflect Prochlorococcus' evolutionary optimization for survival in nutrient-poor conditions where it relies on interactions with heterotrophic bacteria rather than forming resting cells during starvation periods . Comparative structural analysis through homology modeling suggests that while the core c-ring structure is preserved, subtle variations may contribute to the unique ecological niche occupied by Prochlorococcus.

What site-directed mutagenesis approaches are most effective for studying atpE function in Prochlorococcus marinus?

For effective site-directed mutagenesis of Prochlorococcus marinus atpE, researchers should consider established techniques that have been successful with similar cyanobacterial proteins:

• Homologous recombineering using the pJV75amber episomal plasmid system (expressing the recombinase protein gp61 from Che9c) has shown success in related systems . This approach requires:

  • Design of single-stranded DNA oligonucleotides (41-70 bp) with the mutation centrally positioned

  • Induction of recombinase expression (e.g., with acetamide at 0.2%)

  • Transformation with the mutagenic oligonucleotide

  • Selection and verification through PCR and sequencing

• CRISPR-Cas9 mediated editing, which offers advantages for difficult-to-transform species:

  • Design of guide RNAs targeting the atpE locus

  • Creation of a repair template carrying the desired mutation

  • Co-transformation with both components

  • Screening of transformants by sequence analysis

The choice between these methodologies depends on the specific research question and the genetic tractability of the Prochlorococcus strain being studied. For analyzing key functional residues, as has been done with the Ile66Val mutation in other systems, the homologous recombineering approach has demonstrated reliability .

How can researchers assess the impact of environmental factors on Prochlorococcus marinus atpE expression and function?

To assess environmental impacts on Prochlorococcus marinus atpE expression and function, researchers should implement a multi-faceted approach:

This approach incorporates the knowledge that Prochlorococcus distributions are strongly influenced by temperature and light availability , while recognizing that these cyanobacteria rely on interactions with heterotrophic bacteria for survival during nutrient starvation rather than forming resting cells . Single-cell techniques like NanoSIMS are particularly valuable for measuring metabolic activities under different conditions, allowing researchers to connect atpE function to the ecological success of Prochlorococcus in oligotrophic environments.

What are the best approaches for reconstituting functional Prochlorococcus marinus ATP synthase from recombinant components?

Reconstituting functional ATP synthase from recombinant components requires careful attention to protein-lipid interactions and subunit stoichiometry. The recommended methodology includes:

• Expression and purification of individual ATP synthase subunits including atpE

• Sequential reconstitution starting with the membrane-embedded F0 complex:

  • Incorporation of c-ring (atpE subunits) into liposomes composed of E. coli polar lipids and DOPC (70:30 ratio)

  • Addition of a and b subunits with appropriate detergent solubilization

  • Incorporation of F1 components (α, β, γ, δ, ε) in the correct stoichiometry

• Validation of function through:

  • ATP hydrolysis assays (reverse function)

  • ATP synthesis measurement using artificially imposed proton gradients

  • Structural verification through negative-stain electron microscopy

This methodological approach has been adapted from successful reconstitution studies with other bacterial ATP synthases and considers the unique properties of Prochlorococcus proteins, which may reflect adaptations to their ecological niche in nutrient-poor, high-light environments .

How can researchers identify and characterize potential inhibitors of Prochlorococcus marinus atpE?

To identify and characterize potential inhibitors of Prochlorococcus marinus atpE, researchers should implement a systematic screening and validation pipeline:

• Initial screening approaches:

  • In silico molecular docking of compound libraries targeting the c-ring structure

  • High-throughput binding assays using fluorescently labeled atpE

  • Thermal shift assays to identify compounds that alter protein stability

• Functional validation methodologies:

  • ATP synthesis inhibition assays using reconstituted proteoliposomes

  • Proton leakage measurements in the presence of potential inhibitors

  • Growth inhibition studies using Prochlorococcus cultures

• Structural characterization of binding:

  • Hydrogen-deuterium exchange mass spectrometry to identify binding regions

  • X-ray crystallography or cryo-EM of atpE-inhibitor complexes

This approach draws parallels to studies of atpE inhibitors in other systems, such as bedaquiline (BDQ) resistance in Mycobacterium tuberculosis where mutations like Ile66Val affect inhibitor binding . Understanding such structure-function relationships in Prochlorococcus marinus atpE could provide insights into its unique adaptations and potential vulnerability to specific inhibitors.

What techniques are most effective for studying the assembly and oligomerization of atpE in Prochlorococcus marinus?

Studying the assembly and oligomerization of atpE in Prochlorococcus marinus requires specialized biophysical and biochemical techniques:

These techniques can reveal how Prochlorococcus marinus atpE assembly might differ from that of other organisms, potentially reflecting adaptations that enable this cyanobacterium to thrive in oligotrophic environments where it has evolved unique metabolic strategies, including dependencies on heterotrophic bacteria for survival during nutrient limitation .

How does Prochlorococcus marinus atpE differ from that of other marine cyanobacteria like Synechococcus?

Prochlorococcus marinus atpE shows notable differences from its counterpart in Synechococcus, reflecting the distinct ecological niches these marine cyanobacteria occupy:

• Sequence divergence: Comparative genomic analysis reveals that while the core functional residues are conserved between these cyanobacteria, Prochlorococcus marinus atpE exhibits adaptations potentially related to its dominance in more oligotrophic regions.

• Expression patterns: Unlike Synechococcus, which shows more consistent atpE expression across conditions, Prochlorococcus demonstrates more variable expression patterns correlated with light intensity and nutrient availability.

• Functional efficiency: Biochemical studies suggest Prochlorococcus atpE may be optimized for function at lower ATP/ADP ratios, consistent with its adaptation to nutrient-limited environments.

These differences likely contribute to the distinct global distributions of these organisms, with Prochlorococcus dominating in warm oligotrophic gyres of the Indian and western Pacific Oceans, while Synechococcus shows broader temperature tolerance . The unique properties of Prochlorococcus atpE may be integral to its remarkable ecological success despite having a streamlined genome compared to other secondary metabolite producers .

What insights can be gained from studying mutations in Prochlorococcus marinus atpE compared to those documented in other bacteria?

Comparative mutation analysis of Prochlorococcus marinus atpE provides valuable insights into both functional conservation and divergence:

• Conservation of critical residues: Mutations analogous to the Ile66Val mutation documented in Mycobacterium tuberculosis atpE could be introduced in Prochlorococcus to determine if similar functional impacts occur, indicating evolutionary conservation of core ATP synthase mechanisms.

• Unique adaptation markers: Certain residues specific to Prochlorococcus marinus atpE likely represent adaptations to its unique ecological niche. Mutational analysis of these sites can reveal their contribution to:

  • Low-light adaptation in deeper water column populations

  • Thermostability differences across ecotypes

  • Interactions with heterotrophic bacterial partners

• Resistance mechanisms: By studying natural variation in atpE across Prochlorococcus ecotypes, researchers can identify potential natural resistance mechanisms that have evolved in response to environmental challenges or competition.

This comparative approach leverages techniques like site-directed mutagenesis through homologous recombineering to generate specific mutations of interest, followed by functional characterization to determine their impact on ATP synthase activity and cellular physiology.

How does atpE expression in Prochlorococcus marinus vary across different ocean regions and depths?

The expression of atpE in Prochlorococcus marinus shows significant variation across oceanic regions and depths, reflecting adaptation to local conditions:

This distribution pattern correlates with the global biogeographic patterns of Prochlorococcus abundance, which reaches maxima in warm oligotrophic gyres of the Indian and western Pacific Oceans . Methodologically, these expression patterns can be assessed through metatranscriptomic analyses of environmental samples, combined with measurements of photosynthetically active radiation (PAR) and temperature at sampling locations. Single-cell approaches using NanoSIMS can further link atpE expression to cellular metabolic activity in these different environments .

How might climate change impact the function and expression of Prochlorococcus marinus atpE?

Climate change is projected to significantly affect Prochlorococcus marinus atpE function and expression through multiple mechanisms:

• Temperature effects: Models project a 29% increase in global Prochlorococcus abundance by the end of the 21st century due to warming oceans . This expansion will likely involve adaptation of atpE function across new temperature ranges, potentially through:

  • Altered amino acid composition affecting c-ring thermostability

  • Changes in expression regulation to maintain ATP homeostasis at higher temperatures

  • Selection for specific atpE variants in expanding populations

• Ocean acidification impacts: Decreased ocean pH may affect the proton gradient that drives ATP synthase, potentially requiring:

  • Adaptations in the proton channel formed by atpE

  • Compensatory expression changes to maintain ATP production efficiency

  • Altered interactions with other ATP synthase subunits

• Methodology for studying these effects includes:

  • Experimental evolution studies under projected climate conditions

  • Functional characterization of atpE from Prochlorococcus ecotypes from varying temperature regimes

  • Computational modeling of proton translocation under different pH scenarios

These changes in atpE function may have cascading effects on ocean ecosystems and biogeochemical cycles due to Prochlorococcus' significant contribution to primary production in oligotrophic regions .

What are the best approaches for studying post-translational modifications of Prochlorococcus marinus atpE?

The study of post-translational modifications (PTMs) of Prochlorococcus marinus atpE requires sophisticated analytical approaches:

• Mass spectrometry-based methods:

  • Bottom-up proteomics: Tryptic digestion followed by LC-MS/MS to identify modified peptides

  • Top-down proteomics: Analysis of intact atpE protein to preserve modification stoichiometry

  • Targeted multiple reaction monitoring (MRM) for quantification of specific modified residues

• Site-specific analysis techniques:

  • Phosphoproteomic enrichment using TiO2 or IMAC for detecting phosphorylation

  • Custom antibodies against predicted modification sites

  • Chemical probes to tag specific modifications (e.g., click chemistry for acetylation)

• Functional correlation methods:

  • Site-directed mutagenesis of modified residues to non-modifiable amino acids

  • Comparison of ATP synthase activity in native vs. demodified states

  • In vitro reconstitution with controlled modification states

This methodological approach considers the unique environmental conditions Prochlorococcus experiences, including nutrient limitation and reliance on heterotrophic bacterial partners , which may drive unique regulatory PTM patterns on atpE to fine-tune ATP synthase function according to environmental conditions.

How can researchers effectively measure the ion conductance properties of recombinant Prochlorococcus marinus atpE?

Measuring ion conductance properties of recombinant Prochlorococcus marinus atpE requires specialized electrophysiological and biophysical approaches:

• Reconstitution systems:

  • Planar lipid bilayers with incorporated purified atpE c-rings

  • Giant unilamellar vesicles (GUVs) containing atpE for patch-clamp studies

  • Solid-supported membranes for capacitive current measurements

• Electrophysiological measurements:

  • Single-channel recordings to determine conductance properties

  • Ion selectivity studies using bi-ionic potential measurements

  • Determination of pH dependence of proton translocation

• Complementary biophysical approaches:

  • Fluorescence-based proton flux assays using pH-sensitive probes

  • Isothermal titration calorimetry to measure binding of ions to the c-ring

  • Molecular dynamics simulations to model ion transport through the c-ring structure

These methodologies allow researchers to understand how Prochlorococcus marinus atpE has adapted to function in oligotrophic marine environments, potentially revealing unique properties that contribute to the ecological success of this globally important cyanobacterium .