Recombinant Prochlorococcus marinus NAD (P)H-quinone oxidoreductase subunit N (ndhN)

What is the biological function of NAD(P)H-quinone oxidoreductase subunit N (ndhN) in Prochlorococcus marinus?

NAD(P)H-quinone oxidoreductase subunit N (ndhN) is a critical component of the NDH-1 complex in Prochlorococcus marinus that facilitates electron transport in both respiratory and photosynthetic processes. The protein functions by shuttling electrons from electron donors through FMN and iron-sulfur centers to quinones, with plastoquinone serving as the likely immediate electron acceptor in this species . This electron transfer couples with proton translocation across membranes, thereby conserving redox energy as a proton gradient. Beyond its role in electron transport, cyanobacterial NDH-1 complexes containing ndhN also contribute significantly to inorganic carbon concentration mechanisms , which is particularly important for Prochlorococcus marinus given its streamlined genome and adaptation to oligotrophic environments .

What are the structural characteristics of ndhN from Prochlorococcus marinus strain MIT 9515?

The ndhN protein from Prochlorococcus marinus strain MIT 9515 consists of 156 amino acids with the sequence: MPLLLSGKKFHNDLKKNKCLAMFAPLEGGYETRLLRRMRAKGFKTYITSARGLGDPEVFLLNLHGIRPPHLGHQSIGRNGALGEVQQVIPQASELFNENDKDKLLWLLEGQVLSQSELENLIKLPTADNKLKIVVEMGGSRKLEWKSLNDYVLNEF . This protein has a molecular mass of approximately 17.6 kDa and belongs to the complex I NdhN subunit family . Structural analyses reveal conserved domains typical of NdhN subunits, including regions involved in protein-protein interactions within the NDH-1 complex and specific motifs responsible for electron transfer. Understanding these structural features is essential for researchers investigating structure-function relationships in recombinant expression systems.

How does ndhN expression vary across different Prochlorococcus marinus ecotypes?

Expression patterns of ndhN vary significantly across Prochlorococcus marinus ecotypes, reflecting their adaptation to different light environments and ocean depths. High-Light (HL) adapted strains like MED4 (HLI) and SB (HLII) show distinct expression patterns compared to Low-Light (LL) adapted strains such as SS120 (LLII/III) and MIT9313 (LLIV) . When cultured under their native light conditions (HLI at 160 μmol photons m^-2 s^-1 and LL at 30 μmol photons m^-2 s^-1), these strains exhibit ecotype-specific regulation of ndhN . The HLII ecotype, representing more than 90% of all Prochlorococcus in tropical surface waters , demonstrates particularly robust ndhN expression, likely supporting its dominance in high-light environments where efficient electron transport is crucial for managing excess excitation energy.

What are the recommended protocols for isolating and culturing Prochlorococcus marinus strains for ndhN studies?

For isolating and culturing Prochlorococcus marinus strains for ndhN studies, researchers should follow this methodological approach:

• Sample collection: Obtain seawater samples from appropriate depths (50-150 m for HLII strains) using Niskin bottles .

• Filtration: Perform gravity filtration through double polycarbonate filters with 0.6 μm pore size to remove larger microorganisms .

• Media preparation: Add Pro2 medium nutrient stock solution to the filtrate as described by Moore et al. (2007) .

• Initial enrichment: Place the filtrate in an incubator for 4-8 weeks, monitoring growth using flow cytometry .

• Maintenance conditions: Once established, maintain cultures at 22°C with continuous light intensity of 10-20 μmol photons m^-2 s^-1 for initial growth .

• Strain-specific conditions: Adjust light conditions based on ecotype - MED4 (HLI) at 160 μmol photons m^-2 s^-1, and SS120/MIT9313 (LL) at 30 μmol photons m^-2 s^-1 using appropriate fluorescent bulbs .

• Growth maintenance: Transfer cultures weekly with 1:5 dilutions using Pro99 media prepared with autoclaved artificial seawater .

This protocol accounts for the fastidious nature of Prochlorococcus cultures and their dependence on specific light conditions based on ecotype classification.

How can researchers optimize heterologous expression of recombinant Prochlorococcus marinus ndhN?

To optimize heterologous expression of recombinant Prochlorococcus marinus ndhN, researchers should implement the following methodological approach:

• Codon optimization: Adjust the ndhN gene sequence (based on the 156 amino acid sequence) for the expression host to account for the low G+C content (approximately 36.8%) typically found in Prochlorococcus genomes .

• Expression vector selection: Choose vectors with appropriate promoters for membrane protein expression, ideally with tunable expression levels to prevent aggregation.

• Host strain selection: Consider using specialized E. coli strains designed for membrane protein expression or cyanobacterial hosts for more native-like processing.

• Fusion tags optimization: Test multiple tag configurations (N-terminal, C-terminal) with appropriate cleavage sites, considering the protein's natural membrane association.

• Expression conditions: Optimize temperature (typically lower temperatures of 16-25°C improve folding), inducer concentration, and expression duration.

• Membrane extraction protocols: Develop gentle solubilization methods using appropriate detergents to maintain protein structure and function.

• Verification methods: Implement Western blotting and activity assays specific to NAD(P)H-quinone oxidoreductase function to confirm proper folding and function.

This optimized approach addresses the challenges of expressing cyanobacterial membrane proteins while maintaining their native structure and electron transport functionality.

What analytical techniques are most effective for assessing ndhN activity in recombinant systems?

For effective assessment of ndhN activity in recombinant systems, researchers should employ a multi-technique approach:

• Spectrophotometric assays: Monitor the oxidation of NAD(P)H at 340 nm coupled with the reduction of artificial electron acceptors such as dichlorophenolindophenol (DCPIP) or ferricyanide.

• Oxygen consumption measurements: Use Clark-type electrodes to measure oxygen consumption rates during NDH-1 complex activity in membrane preparations.

• Electron paramagnetic resonance (EPR): Apply this technique to detect and characterize the iron-sulfur centers within the NDH-1 complex containing ndhN.

• Proteomics verification: Implement immunoprecipitation followed by mass spectrometry to confirm the incorporation of recombinant ndhN into the complete NDH-1 complex.

• Fluorescence-based methods: Utilize fluorescent probes sensitive to membrane potential to assess the proton-pumping activity coupled to electron transport.

• Cyclic electron flow measurements: For photosynthetic applications, measure cyclic electron flow around Photosystem I, which involves NDH-1 complex activity.

• Mutant complementation assays: Express recombinant ndhN in ndhN-deficient strains to assess functional restoration of phenotypes related to carbon fixation and photosynthesis.

This comprehensive analytical approach enables researchers to confirm both structural incorporation and functional activity of recombinant ndhN protein.

How does ndhN sequence conservation compare across different Prochlorococcus marinus strains and other cyanobacteria?

The ndhN sequence conservation analysis across Prochlorococcus marinus strains and other cyanobacteria reveals significant evolutionary patterns:

Table 1. Sequence Identity Analysis of ndhN Across Selected Cyanobacterial Strains

The analysis demonstrates higher conservation among strains within the same ecotype (e.g., High-Light adapted strains) . Notably, the Low-Light adapted strains show greater sequence divergence, particularly MIT9313 which has a distinctly higher G+C content . This pattern suggests that ndhN has undergone adaptive evolution in response to different light regimes and ocean depths. The protein maintains key functional domains across all cyanobacteria, but strain-specific variations likely contribute to the optimized electron transport properties suited to each strain's ecological niche .

What functional differences in electron transport have been observed between ndhN variants from different Prochlorococcus marinus ecotypes?

Functional differences in electron transport between ndhN variants from different Prochlorococcus marinus ecotypes reveal ecotype-specific adaptations:

High-Light adapted strains (HLI/HLII) feature ndhN variants with distinctive modifications that enhance electron transport efficiency under high irradiance conditions. These variants demonstrate:

• Increased capacity for handling excess electrons generated during high light intensities

• Enhanced coupling with cyclic electron flow around Photosystem I, which helps dissipate excess energy

• More efficient interaction with plastoquinone, the immediate electron acceptor

Low-Light adapted strains (LLII/III/IV) possess ndhN variants optimized for:

• Maximizing energy conservation under limited light conditions

• Tighter coupling with carbon concentration mechanisms to enhance carbon fixation efficiency

• Modified redox potential properties that function optimally at lower light intensities

These functional differences reflect the evolutionary adaptation of Prochlorococcus marinus to specific ocean niches and explain why certain ecotypes dominate at different depths and geographical locations . The HLII ecotype's dominance in tropical surface waters (>90% abundance) correlates with its specialized ndhN functionality that contributes to efficient energy management in high-light environments .

How can site-directed mutagenesis of ndhN be used to investigate electron transport mechanisms in Prochlorococcus marinus?

Site-directed mutagenesis of ndhN provides a powerful approach for investigating electron transport mechanisms in Prochlorococcus marinus. Researchers should implement the following methodological strategy:

• Target identification: Based on sequence alignments across strains, identify conserved residues likely involved in:

  • Iron-sulfur cluster coordination

  • Protein-protein interactions within the NDH-1 complex

  • Quinone binding and interaction sites

  • Proton translocation pathways

• Mutant design strategy:

  • Generate conservative mutations (same chemical class) to assess structural requirements

  • Create charge-reversal mutations to probe electrostatic interactions

  • Develop alanine-scanning mutations across functional domains

  • Design chimeric proteins containing domains from HL and LL ecotypes

• Expression system: Utilize heterologous expression in E. coli followed by reconstitution experiments, or directly transform Prochlorococcus strains if genetic manipulation systems are available.

• Functional assessment:

  • Measure electron transport rates for each mutant using spectrophotometric assays

  • Determine binding affinities for electron donors and acceptors

  • Assess proton translocation efficiency

  • Evaluate the impact on carbon concentration mechanisms

This targeted mutagenesis approach can reveal how specific amino acid residues contribute to the specialized electron transport properties of ndhN in different Prochlorococcus ecotypes, providing insights into their evolutionary adaptation to specific light environments .

What role does ndhN play in the adaptation of Prochlorococcus marinus to varying oxygen and light conditions?

NAD(P)H-quinone oxidoreductase subunit N (ndhN) plays a multifaceted role in Prochlorococcus marinus adaptation to varying oxygen and light conditions through several mechanisms:

Under high light conditions:

• ndhN-containing NDH-1 complexes facilitate enhanced cyclic electron flow around Photosystem I, preventing over-reduction of the electron transport chain

• This process helps dissipate excess excitation energy and protects against photodamage

• The complex contributes to maintaining redox balance when photosynthetic electron generation exceeds metabolic demand

Under oxygen stress:

• ndhN-containing complexes help manage electron flow to prevent excessive reactive oxygen species formation

• P. marinus depends on mutualistic heterotrophic bacteria to detoxify reactive oxygen species, with ndhN function potentially influencing this relationship

• Different P. marinus ecotypes show varying sensitivities to oxygen, correlating with their ndhN variants

The specific amino acid composition of ndhN in different ecotypes (HLI, HLII, LLII/III, LLIV) appears optimized for their respective light niches, contributing to the global distribution pattern of Prochlorococcus in the oceans . While HLI/HLII ecotypes dominate in surface waters with high light intensity, the LL ecotypes are abundant at greater depths with lower light levels, with their ndhN variants reflecting these adaptations .

How can proteomic approaches enhance our understanding of ndhN interactions within the Prochlorococcus marinus NDH-1 complex?

Advanced proteomic approaches provide powerful tools for uncovering ndhN interactions within the Prochlorococcus marinus NDH-1 complex through the following methodological framework:

• Crosslinking Mass Spectrometry (XL-MS):

  • Apply membrane-permeable crosslinkers to stabilize protein-protein interactions

  • Digest crosslinked complexes and analyze by LC-MS/MS

  • Map interaction surfaces between ndhN and other NDH-1 subunits

  • Compare interaction differences between HL and LL ecotype complexes

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Expose purified NDH-1 complexes to deuterated buffers

  • Monitor hydrogen-deuterium exchange rates to identify exposed vs. protected regions

  • Determine conformational changes in ndhN under different conditions (light levels, electron flow rates)

• Native Mass Spectrometry:

  • Analyze intact NDH-1 complexes under native conditions

  • Determine subunit stoichiometry and assembly states

  • Assess complex stability with wild-type vs. mutant ndhN

• Protein Correlation Profiling:

  • Fractionate membrane complexes by size or density

  • Identify co-eluting proteins to detect novel interaction partners

  • Compare profiles across different Prochlorococcus strains and growth conditions

• Targeted Proteomics (PRM/MRM):

  • Develop specific assays to quantify ndhN and associated proteins

  • Monitor changes in complex composition under varying environmental conditions

  • Track post-translational modifications that may regulate activity

This comprehensive proteomic approach reveals not only the structural organization of ndhN within the NDH-1 complex but also the dynamic changes in these interactions that underpin Prochlorococcus adaptation to different light and oxygen conditions .

How does ndhN function contribute to Prochlorococcus marinus dominance in oligotrophic ocean environments?

The ndhN function significantly contributes to Prochlorococcus marinus dominance in oligotrophic ocean environments through several integrated physiological mechanisms:

• Enhanced energy efficiency: The ndhN subunit helps optimize the NDH-1 complex for efficient electron transport, allowing Prochlorococcus to thrive in nutrient-limited environments by maximizing energy yield from limited resources .

• Carbon concentration mechanism: ndhN-containing NDH-1 complexes play a crucial role in inorganic carbon concentration, giving Prochlorococcus a competitive advantage in CO₂-limited oceanic environments .

• Ecotype-specific adaptations: The specific sequence variants of ndhN in different ecotypes enable specialized adaptations:

  • HLII ecotypes (dominant in tropical waters) possess ndhN variants optimized for high-light conditions, contributing to their representation of >90% of all Prochlorococcus in tropical surface waters

  • LL ecotypes have ndhN variants adapted for deeper waters with different light qualities and quantities

• Nitrogen utilization linkage: Recent research indicates connections between electron transport processes involving ndhN and nitrogen metabolism in Prochlorococcus, particularly in strains capable of nitrate assimilation .

• Global distribution impact: The specialized function of ndhN contributes to the estimated global abundance of 2.9 ± 0.1 × 10²⁷ Prochlorococcus cells , making it a key contributor to global primary production and carbon cycling.

These contributions of ndhN function collectively support Prochlorococcus marinus' remarkable success in occupying vast oligotrophic regions of the world's oceans where other phototrophs cannot compete effectively .

What impact might climate change have on ndhN function and Prochlorococcus marinus populations globally?

Climate change is projected to significantly impact ndhN function and Prochlorococcus marinus populations through multiple interconnected mechanisms:

• Temperature effects on enzyme kinetics: Rising sea surface temperatures will alter the kinetic properties of ndhN-containing complexes, potentially affecting electron transport efficiency. Niche models project that by the end of the 21st century, Prochlorococcus cell numbers may increase globally by approximately 29% due to warming oceans .

• Range expansion: Climate models suggest that the geographical range of Prochlorococcus will increase, particularly at higher latitudes, as ocean temperatures rise. This expansion will be driven partly by the temperature-dependent function of key proteins like ndhN .

• Stratification effects: Increased ocean stratification will likely alter light exposure patterns, potentially favoring high-light adapted ecotypes with their specialized ndhN variants over low-light adapted strains .

• Interaction with oxygen levels: Climate-driven changes in ocean oxygenation will interact with the role ndhN plays in electron transport under varying oxygen conditions, potentially altering strain competition dynamics .

• Nitrogen cycle interactions: Changes in nitrogen availability and cycling may interact with the nitrite production and cross-feeding processes recently discovered in Prochlorococcus, which are linked to electron transport systems involving ndhN .

Given that Prochlorococcus contributes substantially to global primary production, these climate-induced changes in ndhN function and associated population shifts may have significant implications for global biogeochemical cycles and ocean ecosystems .

What cutting-edge techniques could advance our understanding of ndhN structure-function relationships in Prochlorococcus marinus?

Several cutting-edge techniques offer promising approaches for advancing our understanding of ndhN structure-function relationships in Prochlorococcus marinus:

• Cryo-electron microscopy (Cryo-EM): Obtaining high-resolution structures of intact NDH-1 complexes from different Prochlorococcus ecotypes would reveal how ndhN positioning and interactions vary between high-light and low-light adapted strains.

• Single-molecule Förster Resonance Energy Transfer (smFRET): This technique could monitor conformational changes in ndhN during electron transport in real-time, providing insights into the dynamic aspects of function.

• In situ structural biology using focused ion beam milling and cryo-electron tomography: This approach would allow visualization of ndhN-containing complexes in their native membrane environment, revealing their organization and interactions with other cellular components.

• Nanobody-based proximity labeling: Using engineered nanobodies specific to ndhN combined with proximity labeling techniques would identify transient interaction partners under different environmental conditions.

• CRISPR-Cpf1 genome editing: As genetic manipulation systems for Prochlorococcus improve, precise genome editing could enable in vivo studies of ndhN function through targeted mutations.

• Time-resolved serial femtosecond crystallography: This approach using X-ray free-electron lasers could potentially capture intermediate states during electron transfer through ndhN-containing complexes.

• Computational approaches integrating AlphaFold2-predicted structures with molecular dynamics simulations: This would allow prediction of how sequence variations between ecotypes translate into functional differences in electron transport capabilities.

These advanced techniques would significantly enhance our understanding of how ndhN contributes to the remarkable ecological success of Prochlorococcus marinus in diverse ocean environments .

How might synthetic biology approaches utilizing ndhN from Prochlorococcus marinus enhance bioenergy applications?

Synthetic biology approaches utilizing ndhN from Prochlorococcus marinus offer several promising pathways for enhancing bioenergy applications through the following methodological strategies:

• Optimized photosynthetic electron transport systems:

  • Engineer cyanobacterial or algal hosts with Prochlorococcus ndhN variants to enhance electron transport efficiency

  • Tune NDH-1 complex performance for improved light capture and energy conversion in biofuel-producing organisms

  • Create chimeric complexes incorporating high-efficiency features from different Prochlorococcus ecotypes

• Enhanced carbon fixation platforms:

  • Utilize the connection between ndhN-containing complexes and carbon concentration mechanisms to improve CO₂ fixation rates

  • Engineer metabolic redirection of enhanced electron flow toward biofuel precursor production

  • Develop strains with improved carbon fixation efficiency under industrial cultivation conditions

• Stress-resistant production strains:

  • Incorporate ndhN variants from Prochlorococcus ecotypes adapted to high light conditions to create production strains with improved photodamage resistance

  • Enhance oxygen tolerance in biofuel-producing microorganisms by incorporating insights from Prochlorococcus electron transport systems

• Biosensor development:

  • Create biosensors based on ndhN function to monitor electron transport efficiency in bioenergy production systems

  • Develop high-throughput screening platforms for identifying optimized electron transport variants

Implementation of these approaches would leverage the natural adaptations of Prochlorococcus marinus, which has evolved highly efficient electron transport systems to thrive in nutrient-limited environments, potentially advancing bioenergy production technologies toward greater efficiency and sustainability .