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

What is NADH-quinone oxidoreductase subunit A (nuoA) in Chlorobium limicola?

NADH-quinone oxidoreductase subunit A (nuoA) is a key component of the respiratory chain complex I in Chlorobium limicola. This protein is encoded by the nuoA gene (locus name: Clim_0844) and functions as part of the NADH dehydrogenase complex that catalyzes electron transfer from NADH to quinones, contributing to energy conservation in this green sulfur bacterium. The protein has several alternative designations including NADH dehydrogenase I subunit A, NDH-1 subunit A, and NUO1, with the enzyme classification EC 1.6.99.5 .

How should recombinant Chlorobium limicola nuoA be stored and handled?

Proper storage and handling are crucial for maintaining the activity of recombinant nuoA protein. The recommended storage conditions are:

Important handling considerations include avoiding repeated freeze-thaw cycles as this can significantly reduce protein activity and integrity. When working with the protein, prepare small working aliquots to minimize freeze-thaw cycles .

What expression systems are suitable for producing recombinant Chlorobium limicola nuoA?

While the search results don't specifically address expression systems for nuoA, general principles for membrane protein expression can be applied. When selecting an expression system for nuoA, researchers should consider:

• Bacterial expression systems (E. coli) for initial screening

• Cell-free expression systems for potentially toxic membrane proteins

• Eukaryotic systems for complex folding requirements

• Codon optimization for the expression host

• Fusion tags to enhance solubility and facilitate purification

The selection of an appropriate expression system should be guided by the specific research objectives and the intended use of the recombinant protein.

How does nuoA function relate to energy metabolism in Chlorobium limicola?

NADH-quinone oxidoreductase containing nuoA plays a pivotal role in the bioenergetics of Chlorobium limicola. As a subunit of complex I, nuoA contributes to:

• Proton translocation across the membrane, generating proton motive force

• Electron transfer from NADH to the quinone pool

• Integration with photosynthetic electron transport chains

Understanding nuoA function provides insights into the unique energy conservation mechanisms in green sulfur bacteria like Chlorobium limicola, which can utilize various electron donors including reduced sulfur compounds depending on environmental conditions .

What are the potential interactions between nuoA function and vitamin B12 metabolism in Chlorobium limicola?

Research indicates complex metabolic interconnections in Chlorobium limicola. While direct interactions between nuoA and vitamin B12 metabolism aren't explicitly documented in the search results, we can analyze potential relationships:

Vitamin B12 deficiency in Chlorobium limicola leads to:

• Reduction in specific bacteriochlorophyll (Bchl) content (Bchl c reduced to 20%, Bchl a to 42%)

• Complete absence of chlorosomes, the light-harvesting structures

• Alterations in cellular ultrastructure with accumulation of granular structures

These changes would likely impact electron transport chain components including NADH-quinone oxidoreductase. The restoration of normal cellular structures and pigment content after vitamin B12 addition suggests a regulatory role of B12 in photosynthetic apparatus assembly, which would indirectly affect nuoA function through altered energy metabolism pathways .

What methods can be used to study the interaction of nuoA with other complex I subunits?

To investigate interactions between nuoA and other complex I subunits, researchers can employ several complementary approaches:

• Co-immunoprecipitation with antibodies specific to nuoA or other subunits

• Cross-linking studies followed by mass spectrometry analysis

• Bacterial two-hybrid or yeast two-hybrid screening

• Blue native PAGE to isolate intact complexes

• Cryo-electron microscopy to determine structural arrangements

• Genetic approaches including site-directed mutagenesis to identify interaction domains

These methods can reveal both direct physical interactions and functional relationships between nuoA and other components of the respiratory apparatus.

How can genetic transformation be optimized for studying nuoA function in Chlorobium limicola?

Based on transformation protocols described for Chlorobium limicola, an optimized approach for nuoA functional studies would include:

• Grow recipient Chlorobium limicola strain to late log phase

• Harvest cells and resuspend in CaCl2 solution (50 mM) under strictly anaerobic conditions

• Prepare transformation mixture with nuoA-containing plasmid DNA

• Incubate the transformation mixture under anaerobic conditions

• Plate transformed cells on selective media

• Verify transformation by:

  • PCR amplification of the nuoA gene

  • Restriction enzyme analysis of isolated plasmids

  • Functional assays for NADH-quinone oxidoreductase activity

This transformation process requires maintaining anaerobic conditions throughout to ensure cell viability and transformation efficiency .

What are the common challenges in purifying active recombinant nuoA and how can they be addressed?

Purification of membrane proteins like nuoA presents several challenges:

Additionally, the hydrophobic nature of nuoA necessitates careful selection of detergents that maintain both protein solubility and native conformation throughout the purification process.

How can the functional activity of purified nuoA be reliably assessed?

Functional assessment of purified nuoA should incorporate multiple complementary approaches:

• Spectrophotometric NADH oxidation assays using artificial electron acceptors

• Reconstitution into proteoliposomes to measure proton pumping activity

• EPR spectroscopy to detect electron transfer through iron-sulfur clusters

• Monitoring quinone reduction using fluorescence quenching techniques

• Complementation studies in nuoA-deficient mutants

When designing activity assays, researchers should consider that isolated nuoA may have different properties than when integrated into the complete complex I structure.

How does thiosulfate metabolism relate to nuoA function in Chlorobium limicola?

The relationship between thiosulfate metabolism and nuoA function represents an interesting area for investigation. Research shows that:

• Certain strains of Chlorobium limicola (designated as forma specialis thiosulfatophilum or Tio+) can utilize thiosulfate as an electron donor

• This ability is conferred by a 14 kb plasmid that is absent in non-thiosulfate-utilizing strains (Tio-)

• Transformation with this plasmid enables previously Tio- strains to utilize thiosulfate

While direct evidence linking nuoA to thiosulfate metabolism isn't presented in the search results, the electron transport chain containing NADH-quinone oxidoreductase would likely interact with electrons derived from thiosulfate oxidation. This suggests potential research questions regarding how alternative electron donors affect the function and regulation of nuoA and other respiratory complex components.

What role might nuoA play in adaptation to different light conditions in Chlorobium limicola?

Green sulfur bacteria like Chlorobium limicola exhibit remarkable adaptations to varying light intensities. Research indicates:

• Low-light adapted strains produce larger and more abundant chlorosomes

• Chlorosome dimensions decrease with increasing light intensities

• Pigment content (bacteriochlorophyll and carotenoids) varies with light conditions

These adaptations would necessitate coordinated regulation of the electron transport chain, including nuoA-containing complexes, to balance energy production with changing light availability. Investigation of nuoA expression and activity under different light regimes could reveal important insights into this regulatory network.

What emerging technologies could advance our understanding of nuoA structure and function?

Several cutting-edge approaches hold promise for nuoA research:

• Cryo-electron microscopy for high-resolution structural analysis of membrane-embedded nuoA

• Single-molecule techniques to observe real-time conformational changes during electron transfer

• Systems biology approaches integrating transcriptomics, proteomics, and metabolomics data

• Advanced computational modeling of electron transfer pathways

• CRISPR-based genome editing for precise manipulation of nuoA in Chlorobium limicola

These technologies could reveal nuoA functional mechanisms at unprecedented resolution and place its role within the broader context of cellular energetics.

How might comparative analysis of nuoA across different species inform our understanding of Chlorobium limicola energy metabolism?

Comparative genomic and functional analyses of nuoA across species could provide valuable evolutionary insights. Researchers could:

• Compare nuoA sequences across diverse photosynthetic and non-photosynthetic bacteria

• Identify conserved functional domains and species-specific adaptations

• Correlate structural variations with different ecological niches and metabolic capabilities

• Conduct heterologous expression studies to test functional conservation

• Use ancestral sequence reconstruction to explore the evolutionary history of complex I

This evolutionary perspective could reveal how nuoA function has been tailored to the specific ecological niche occupied by Chlorobium limicola as a green sulfur bacterium.