Recombinant Chlorobium tepidum NADH-quinone oxidoreductase subunit A (nuoA)

What is NADH-quinone oxidoreductase subunit A (nuoA) and what is its role in Chlorobium tepidum?

NADH-quinone oxidoreductase subunit A (nuoA) is one of eleven subunits that form the type I NADH dehydrogenase (NDH-1) complex in Chlorobium tepidum. This complex is encoded by the ndhCHJKAIGEFDB operon (genes CT0766-CT0776) in the C. tepidum genome . As part of the NDH-1 complex, nuoA plays an essential role in the electron transport chain of this green sulfur bacterium, which performs anoxygenic photosynthesis using the reductive tricarboxylic acid cycle . The NDH-1 complex likely contributes to energy conservation and redox balance maintenance, particularly in the unique photosynthetic electron transport system of these bacteria where reduced ferredoxins are the primary products .

How does the structure of Chlorobium nuoA compare to homologous proteins in other bacterial species?

The nuoA protein from Chlorobium species is characterized by membrane-spanning domains, consistent with its role in electron transport chains. Based on data from the related Chlorobium phaeobacteroides, nuoA consists of 143 amino acids with a full-length sequence of: MDQTLSSFGNVFAFLALGIVFVAGGYLTARMLRPSRPNPAKNSTYECGEEAVGSAWVKFNIRFYVVALIFIIFDVEVVFLYPWATVFKSLGVFALVEVLVFAGILILGLVYAWVKGDLDWVRPEPKVPQMPVMPDRKAEGGRA . Computational analyses of this sequence reveal a highly hydrophobic protein with multiple transmembrane regions, characteristic of membrane-integrated electron transport components. Comparative genomic analyses would reveal conservation patterns across other bacterial phyla, particularly focusing on residues involved in quinone binding and subunit interactions.

What expression systems are most efficient for producing recombinant Chlorobium nuoA?

Based on available data, E. coli has been successfully used as an expression host for recombinant Chlorobium phaeobacteroides nuoA protein . For optimal expression, the protein is typically fused to a purification tag such as an N-terminal His-tag to facilitate downstream purification processes . When designing expression constructs, researchers should consider codon optimization for the host organism and incorporation of appropriate protease cleavage sites if tag removal is desired post-purification. For membrane proteins like nuoA, specialized E. coli strains (such as C41/C43) that are engineered to handle membrane protein overexpression may yield better results than standard laboratory strains.

What are the recommended purification protocols for recombinant nuoA protein?

The purification of recombinant nuoA typically employs affinity chromatography utilizing the His-tag fusion. After expression in E. coli, the protein should be extracted from membrane fractions using appropriate detergents. The purified protein can be obtained at >90% purity as determined by SDS-PAGE . According to established protocols, the purified protein is typically prepared as a lyophilized powder in Tris/PBS-based buffer containing 6% trehalose at pH 8.0 . For reconstitution, it is recommended to briefly centrifuge the vial before opening and reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, adding glycerol to a final concentration of 5-50% (with 50% being standard) and aliquoting before storage at -20°C/-80°C is recommended to minimize protein degradation .

How can researchers assess the functional activity of recombinant nuoA in vitro?

To assess functional activity of recombinant nuoA, researchers should consider both its individual properties and its role within the NDH-1 complex. Methods include:

• Reconstitution experiments: Incorporating purified nuoA into liposomes or nanodiscs with appropriate lipid compositions to mimic the native membrane environment.

• Electron transport assays: Measuring NADH oxidation coupled to quinone reduction spectrophotometrically, following the decrease in absorbance at 340 nm (NADH) and the reduction of quinone analogs.

• Membrane incorporation studies: Analyzing integration into prepared membrane fractions from nuoA-deficient mutants to assess functional complementation.

Drawing from approaches used for similar proteins, such as SQR in C. tepidum, activity can be measured in membrane fractions with specific electron donors and acceptors . Researchers should be aware that individual subunits might not show activity without the complete NDH-1 complex, necessitating co-expression strategies for functional studies.

What genetic manipulation approaches have been successfully applied to study nuoA in Chlorobium species?

Based on studies with related proteins in Chlorobium tepidum, several genetic approaches have proven successful:

• Transposon mutagenesis: Genes encoding electron transport components in C. tepidum have been successfully disrupted using transposon insertions (TnOGm) . This technique allows for the generation of loss-of-function mutations to study the role of nuoA.

• Homologous recombination: Targeted gene replacements can be performed in C. tepidum, allowing for precise genetic modifications .

• Epitope tagging: C-terminal tagging approaches (such as His6-tagging) have been successfully employed to track protein expression and localization in C. tepidum .

• RT-PCR and transcriptional analysis: These methods can determine whether nuoA expression is constitutive or condition-dependent, similar to how CT1087 transcripts in C. tepidum were found to be expressed only when cells actively oxidized sulfide .

How does nuoA contribute to the unique electron transport mechanisms in green sulfur bacteria?

The NDH-1 complex containing nuoA in Chlorobium tepidum has distinctive features compared to its counterparts in other bacteria. Unlike the NDH-1 in Escherichia coli, the C. tepidum complex lacks homologs of the NuoEFG proteins , suggesting a unique composition and possibly function. Research indicates that NDH-1 in green sulfur bacteria might be specifically adapted to their anoxygenic photosynthetic lifestyle, where the reductive tricarboxylic acid cycle is employed instead of the Calvin cycle .

In C. tepidum, photosynthetic electron transport primarily produces reduced ferredoxins, which drive the reductive reactions of the reverse TCA cycle . The NDH-1 complex may play a crucial role in maintaining redox balance by oxidizing NADH and reducing quinones, thereby contributing to cyclic electron flow. Comparative analysis with other photosynthetic bacteria would reveal how nuoA and the NDH-1 complex have evolved specialized functions in the context of sulfur-based anoxygenic photosynthesis.

What is the relationship between nuoA-containing NDH-1 complex and sulfur metabolism in Chlorobium species?

While direct evidence linking nuoA to sulfur metabolism is limited, genome analyses of C. tepidum reveal interesting connections. The genome contains genes for sulfide oxidation, including multiple sulfide:quinone oxidoreductase (SQR) homologs that play crucial roles in sulfide-dependent growth . Phylogenomic analysis has identified likely duplications of genes involved in biosynthetic pathways for photosynthesis and the metabolism of sulfur and nitrogen .

Experimental approaches to investigate this relationship include:

• Comparative expression analysis: Examining co-expression patterns of nuoA and sulfur metabolism genes under varying sulfide concentrations.

• Double mutant studies: Similar to the approach taken with SQR homologs, where double mutants lacking both CT0117 and CT1087 showed complete loss of SQR activity and failure to grow at ≥4 mM sulfide .

• Metabolic flux analysis: Tracing electron flow from sulfide oxidation through the electron transport chain, potentially involving the NDH-1 complex.

What computational approaches are most effective for predicting functional domains and interactions of nuoA?

Advanced computational methods for analyzing nuoA include:

• Comparative genomics: Analyzing nuoA sequences across multiple Chlorobium species and other green sulfur bacteria to identify conserved domains and evolutionary patterns.

• Structural prediction: Using algorithms like AlphaFold2 to predict the three-dimensional structure of nuoA, particularly focusing on membrane-spanning regions and potential quinone binding sites.

• Protein-protein interaction prediction: Computational methods to predict interfaces between nuoA and other subunits of the NDH-1 complex.

• Phylogenomic analysis: Similar to approaches used in the C. tepidum genome study that revealed likely duplications of genes involved in photosynthesis and metabolism .

• Molecular dynamics simulations: Simulating the behavior of nuoA within a lipid bilayer to understand membrane integration and potential conformational changes during electron transport.

How does nuoA from Chlorobium tepidum compare with homologous proteins from other photosynthetic bacteria?

Comparative analysis of nuoA across photosynthetic bacteria reveals both conservation and specialization patterns. The C. tepidum genome analysis identified genes that are highly conserved among photosynthetic species, many with no assigned function that may play novel roles in photosynthesis or photobiology . While the complete NDH-1 operon (ndhCHJKAIGEFDB) is present in C. tepidum, the absence of homologs to the E. coli NuoEFG proteins suggests a divergent evolution .

Interestingly, C. tepidum shows strong similarities in metabolic processes to many Archaeal species, despite being a Eubacterium . This suggests potential horizontal gene transfer events or convergent evolution driven by similar ecological niches. Researchers investigating nuoA evolution should consider these broader evolutionary patterns and focus on functional domains that might explain the adaptation of the NDH-1 complex to the unique electron transport requirements of anoxygenic photosynthesis.

What insights does nuoA provide into the adaptation of Chlorobium species to extreme environments?

Chlorobium tepidum was originally isolated from high-sulfide hot environments , suggesting that its electron transport chain components, including nuoA, have evolved to function optimally under these conditions. Despite the relatively constant environment in which C. tepidum grows, its genome encodes fewer regulatory genes compared to other phototrophs like Synechocystis sp. PCC6803 (approximately 8-fold fewer) . This reduced regulatory capacity suggests that electron transport components like nuoA may have evolved highly specialized functions with less need for regulatory flexibility.

The association of genes from the NDH-1 operon with an Ni-Fe uptake hydrogenase (CT0777) and an associated b-cytochrome (CT0778) suggests potential structural and functional relationships between hydrogen metabolism and conventional NADH-linked respiration . This genomic arrangement provides clues about how nuoA and the NDH-1 complex might contribute to energy conservation under the unique ecological conditions favored by Chlorobium species.

What are the major challenges in expressing and purifying functional recombinant nuoA, and how can they be addressed?

The expression and purification of functional membrane proteins like nuoA present several challenges:

Recombinant nuoA can be successfully expressed in E. coli as a His-tagged protein and purified to >90% purity . For reconstitution, it is recommended to resuspend the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol for long-term storage .

How can researchers overcome difficulties in assessing nuoA interactions with other NDH-1 subunits?

Investigating interactions between nuoA and other NDH-1 subunits requires specialized approaches:

• Co-expression strategies: Express multiple subunits simultaneously in E. coli using polycistronic constructs or compatible plasmids with different selection markers.

• Pull-down assays: Use differently tagged subunits (His-tag, Strep-tag, etc.) to perform sequential affinity purifications to identify stable subcomplexes.

• Crosslinking mass spectrometry: Apply chemical crosslinkers followed by mass spectrometry to map interaction interfaces between subunits.

• Bacterial two-hybrid systems: Adapt bacterial two-hybrid approaches for membrane proteins to screen for interactions in vivo.

• Native mass spectrometry: Develop protocols for membrane protein complexes to determine subunit stoichiometry and complex stability.

• Cryo-electron microscopy: For structural characterization of the entire NDH-1 complex or subcomplexes containing nuoA.

How might CRISPR-Cas9 genome editing advance functional studies of nuoA in Chlorobium species?

While traditional mutagenesis approaches using transposons have been successful in Chlorobium tepidum , adapting CRISPR-Cas9 technology would significantly advance nuoA functional studies by enabling:

• Precise genetic modifications: Creating point mutations in conserved residues to determine their functional significance without disrupting the entire gene.

• Marker-free mutations: Generating clean deletions or insertions without antibiotic resistance markers that might affect downstream analyses.

• Multiplexed editing: Simultaneously targeting multiple genes related to electron transport to understand functional redundancy and compensatory mechanisms.

• Regulatable expression systems: Developing CRISPR interference (CRISPRi) approaches to tune down nuoA expression without complete deletion.

• High-throughput functional screening: Creating libraries of nuoA variants to identify critical functional domains through growth competition experiments.

Adapting CRISPR systems for Chlorobium would require optimization of delivery methods, promoters for Cas9 expression, and guide RNA design specific to the AT-rich genome of these bacteria.

What insights could structural biology approaches provide about nuoA's role in the NDH-1 complex architecture?

Advanced structural biology approaches could reveal crucial insights about nuoA within the NDH-1 complex:

• Cryo-electron microscopy: Determining the structure of the entire NDH-1 complex would position nuoA within its functional context and reveal interaction interfaces with other subunits.

• Solid-state NMR: Providing atomic-level details of nuoA's membrane-embedded regions and potential conformational changes during electron transfer.

• Cross-linking mass spectrometry: Identifying specific residues involved in subunit interactions by chemically linking nearby amino acids followed by mass spectrometric analysis.

• Hydrogen-deuterium exchange mass spectrometry: Mapping dynamic regions and solvent-accessible surfaces of nuoA within the complex.

• Single-particle analysis: Capturing different conformational states of the NDH-1 complex during the catalytic cycle to understand how nuoA participates in electron transfer.

These structural insights would be particularly valuable given the unique composition of the C. tepidum NDH-1 complex compared to better-studied bacterial counterparts, potentially revealing adaptations specific to anoxygenic photosynthesis.

How can proteomics approaches be integrated with nuoA functional studies in Chlorobium tepidum?

The membrane proteome of Chlorobium tepidum offers valuable insights into nuoA function within the cellular context . Integrative proteomics approaches include:

• Quantitative proteomics: Using techniques like TMT labeling or SILAC to track changes in NDH-1 subunit abundance under different growth conditions or in response to environmental stressors.

• Protein-protein interaction mapping: Employing proximity labeling approaches (BioID, APEX) adapted for bacterial systems to identify the nuoA interactome beyond known NDH-1 subunits.

• Post-translational modification analysis: Identifying potential regulatory modifications on nuoA such as phosphorylation, which might modulate NDH-1 activity in response to cellular energy status.

• Spatial proteomics: Fractionating bacterial membranes to determine if NDH-1 complexes localize to specific membrane domains, potentially revealing functional associations with other metabolic pathways.

• Thermal proteome profiling: Assessing protein thermal stability changes upon substrate binding or protein-protein interactions to identify nuoA binding partners and substrates.

What computational modeling approaches can predict the impact of nuoA mutations on NDH-1 function?

Advanced computational methods to assess mutagenesis effects include:

• Molecular dynamics simulations: Simulating the behavior of wild-type and mutant nuoA within a membrane environment to predict structural and dynamic changes.

• Quantum mechanics/molecular mechanics (QM/MM) calculations: Modeling electron transfer processes within the NDH-1 complex to understand how nuoA mutations might affect catalytic efficiency.

• Evolutionary coupling analysis: Identifying co-evolving residues across nuoA homologs to predict functionally linked amino acid positions.

• Free energy perturbation calculations: Estimating changes in stability or binding affinity caused by mutations.

• Machine learning approaches: Training predictive models on known mutational data from related proteins to forecast the impact of specific nuoA mutations.

These computational predictions should be validated experimentally through site-directed mutagenesis followed by activity assays and structural analyses, creating an iterative workflow between computational prediction and experimental validation.