Recombinant Myxococcus xanthus NADH-quinone oxidoreductase subunit A (nuoA)

What is the fundamental role of NADH-quinone oxidoreductase subunit A in Myxococcus xanthus?

NADH-quinone oxidoreductase subunit A (nuoA) is a component of the respiratory complex I (NADH:ubiquinone oxidoreductase) in Myxococcus xanthus. This complex plays a critical role in the electron transport chain, transferring electrons from NADH to ubiquinone while contributing to proton translocation across the membrane. In the electron transfer chain, complex I removes electrons from NADH and passes them via enzyme-bound redox centers (FMN and Fe-S clusters) to the electron acceptor ubiquinone. For each pair of electrons transferred, approximately four protons are removed from the matrix, contributing to the proton gradient that drives ATP synthesis .

Within M. xanthus, an obligate aerobic soil bacterium with complex social behaviors, this energy generation system is crucial for supporting the organism's distinctive multicellular lifestyle, including predation, swarming motility, and fruiting body formation .

What are the optimal expression systems for recombinant M. xanthus nuoA production?

For efficient expression of recombinant M. xanthus nuoA protein, E. coli-based expression systems have been demonstrated to be effective. According to the product information from commercial sources, recombinant full-length M. xanthus nuoA (1-123aa) has been successfully expressed in E. coli with N-terminal His-tags .

For laboratory-scale expression, the following methodology is recommended:

• Vector selection: pET-series vectors with T7 promoter systems provide strong, inducible expression.

• Host strain optimization: E. coli strains such as BL21(DE3), C41(DE3), or C43(DE3) are preferred for membrane protein expression. The latter two strains were specifically developed for expressing toxic or membrane proteins.

• Expression conditions:

  • Lower temperatures (16-25°C) after induction improve proper folding

  • Reduced IPTG concentrations (0.1-0.5 mM) can enhance soluble protein yields

  • Extended expression times (16-24 hours) at lower temperatures often yield better results

• Codon optimization: Since M. xanthus has a different codon usage bias compared to E. coli, codon optimization of the nuoA gene sequence may significantly improve expression levels.

What purification strategies yield the highest purity and activity of recombinant nuoA?

Purification of recombinant M. xanthus nuoA requires specialized techniques due to its membrane protein nature. Based on established membrane protein purification protocols and commercial product information , the following strategy is recommended:

• Cell lysis and membrane fraction isolation:

  • Mechanical disruption (sonication, French press, or bead beating)

  • Differential centrifugation to isolate membrane fractions (low-speed centrifugation to remove cell debris, followed by high-speed ultracentrifugation to pellet membranes)

• Membrane solubilization:

  • Detergent screening is crucial (common detergents include DDM, LDAO, or CHAPS)

  • Gentle solubilization at 4°C with optimized detergent:protein ratios

  • Similar to M. xanthus C-factor purification, CHAPS has been shown to be effective for membrane protein solubilization from this organism

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged proteins

  • Gradual imidazole gradient elution to minimize contaminants

• Secondary purification:

  • Size exclusion chromatography to remove aggregates and further purify the protein

  • Ion exchange chromatography (e.g., MonoQ, as used in M. xanthus C-factor purification) can provide additional purification

• Quality control:

  • SDS-PAGE and Western blotting to confirm purity and identity

  • Activity assays to confirm functional state

For storage, buffer optimization is critical. A Tris-based buffer with 50% glycerol has been reported to stabilize the protein, and storage at -20°C or -80°C is recommended for long-term preservation .

What is the role of nuoA in the oxygen sensing and adaptation pathways of M. xanthus?

Recent studies suggest that respiratory chain components in M. xanthus may be integrated with oxygen sensing and adaptation pathways. While direct evidence for nuoA's involvement isn't explicitly stated in the search results, several lines of research point to potential connections:

• NmpRSTU oxygen sensing system:

  • M. xanthus possesses an oxygen-sensing pathway called NmpRSTU, which influences social behaviors and development

  • The NmpU sensor kinase contains a heme-binding domain that detects oxygen levels

  • Mutation of the heme-binding site (H96A) dramatically reduces autophosphorylation activity

• Potential metabolic integration:

  • As a component of the respiratory chain, nuoA function may be regulated in response to oxygen availability

  • Under hypoxic conditions, changes in respiratory chain composition or activity could serve as an adaptation mechanism

• Fruiting body formation connection:

  • During fruiting body formation, M. xanthus cells likely experience oxygen limitation due to high cell density

  • The NmpRSTU system plays a role in fruiting body development, suggesting oxygen sensing is important during this process

  • As a respiratory chain component, nuoA's activity may be modulated during development to optimize energy generation under changing oxygen conditions

To investigate this connection experimentally, researchers could:

• Compare nuoA expression levels under aerobic versus hypoxic conditions

• Analyze the impact of nuoA mutations on adaptation to oxygen limitation

• Assess whether nuoA interacts with components of the NmpRSTU system

• Examine whether nuoA is differentially regulated during fruiting body formation

How does nuoA contribute to the social behaviors and developmental cycle of M. xanthus?

Myxococcus xanthus exhibits complex social behaviors including predation, swarming motility, and fruiting body formation. The contribution of nuoA to these processes can be analyzed from several perspectives:

• Energy metabolism during development:

  • Transcriptome analysis of the 96-hour developmental program revealed sequential expression of genes in distinct modules, with many energy metabolism genes showing specific temporal regulation

  • As part of complex I, nuoA likely plays a crucial role in providing energy during the transition from vegetative growth to development

  • During starvation-induced development, cells must efficiently utilize remaining energy sources, making respiratory chain components like nuoA potentially critical

• Potential role in motility:

  • M. xanthus employs two distinct motility systems: A-motility and S-motility

  • Both motility systems require energy in the form of proton motive force or ATP

  • As a component of the respiratory chain that contributes to proton gradient formation, nuoA indirectly supports motility systems

• Biofilm formation contribution:

  • M. xanthus forms vegetative biofilms that require proper respiratory function

  • Aeration is critical for effective development of submerged biofilms by this obligate aerobe

  • Complex I components like nuoA would be essential for energy generation under the varying oxygen conditions in biofilm structures

• Experimental approaches to study nuoA's role:

  • Generate nuoA knockout mutants and assess their developmental phenotypes

  • Compare developmental transcriptomes to determine if nuoA expression changes during development

  • Examine nuoA mutant behavior under different oxygen tensions to establish connections with the NmpRSTU oxygen sensing system

How does the function of nuoA in M. xanthus compare to homologous proteins in other bacteria?

The NADH-quinone oxidoreductase subunit A is conserved across many bacterial species, but its specific characteristics in M. xanthus can be compared to homologs in other bacteria:

• Structural conservation:

  • The 123-amino acid length of M. xanthus nuoA is consistent with homologs in other bacteria

  • Transmembrane topology is generally conserved, reflecting the fundamental role in complex I structure

• Functional adaptations:

  • In obligate aerobes like M. xanthus, complex I is primarily focused on energy generation

  • In facultative anaerobes, complex I may have additional regulatory features that allow for metabolic switching

• Integration with social behaviors:

  • M. xanthus has unique social behaviors that may require specialized energy management

  • The nuoA function may be integrated with regulatory systems controlling development, predation, and motility

  • This integration could involve specific protein-protein interactions or regulatory mechanisms not present in non-social bacteria

• Comparative genomic analysis:

  • M. xanthus belongs to the deltaproteobacteria, a diverse group with various metabolic strategies

  • Comparing nuoA sequences among myxobacteria versus other bacterial groups could reveal adaptations specific to predatory or social lifestyles

  • Anaeromyxobacter sp. Fw109-5, a close relative of M. xanthus, has homologs of the oxygen-sensing NmpRSTU system , suggesting conservation of respiratory regulation systems within this bacterial group

What are the most common challenges when working with recombinant nuoA and how can they be addressed?

Working with membrane proteins like nuoA presents several technical challenges:

• Low expression yields:

  • Challenge: Membrane protein overexpression can be toxic to host cells

  • Solution: Use specialized expression strains like C41(DE3) or C43(DE3), reduce induction temperature to 16-18°C, and optimize inducer concentration

• Protein misfolding and aggregation:

  • Challenge: Improper folding in heterologous systems

  • Solution: Co-express with chaperones (GroEL/GroES), add solubilizing agents to expression media, or explore cell-free expression systems

• Detergent selection challenges:

  • Challenge: Finding detergents that maintain protein structure and function

  • Solution: Perform detergent screening using thermal stability assays or activity measurements; consider newer amphipathic polymers like SMALPs (styrene-maleic acid lipid particles) for native-like membrane environment preservation

• Maintaining stability during purification:

  • Challenge: Protein degradation during purification steps

  • Solution: Add protease inhibitors, maintain low temperature throughout purification, minimize purification duration

• Activity loss during storage:

  • Challenge: Functional deterioration upon storage

  • Solution: Store in 50% glycerol at -80°C, avoid repeated freeze-thaw cycles, aliquot samples before freezing

What analytical techniques are most informative for studying nuoA structure-function relationships?

Several analytical approaches provide valuable insights into nuoA structure-function relationships:

• Site-directed mutagenesis combined with functional assays:

  • Systematically mutate conserved residues to identify those critical for function

  • Similar to the approach used for NmpU where mutation of H96A affected function

  • Assess the impact on complex assembly and activity

• Cryo-electron microscopy (cryo-EM):

  • Allows visualization of complex I architecture including nuoA positioning

  • Can reveal conformational changes upon substrate binding or inhibitor interaction

  • Enables comparison of wild-type versus mutant structures

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Maps protein dynamics and solvent accessibility

  • Identifies regions of nuoA that undergo conformational changes during catalysis

  • Can reveal interaction interfaces with other complex I subunits

• Cross-linking coupled with mass spectrometry:

  • Identifies proximity relationships between nuoA and other subunits

  • Maps the interaction network within complex I

  • Reveals structural changes under different functional states

• Molecular dynamics simulations:

  • Models nuoA behavior within the membrane environment

  • Predicts effects of mutations on protein structure and dynamics

  • Simulates proton transfer pathways involving nuoA

• Nanodiscs or native nanodiscs for functional studies:

  • Provides a more native-like membrane environment than detergent micelles

  • Enables investigation of lipid-protein interactions

  • Maintains functional integrity for more accurate biochemical assays

How might nuoA be involved in the predatory behavior of M. xanthus?

Myxococcus xanthus is known for its predatory behavior, where groups of cells cooperatively lyse and consume prey bacteria. While direct evidence linking nuoA to predation isn't available in the search results, several hypotheses warrant investigation:

• Energy requirements during predation:

  • Predatory rippling behavior requires sustained cell motility

  • As part of complex I, nuoA contributes to energy generation needed for this energy-intensive process

  • During predation, cells must rapidly process nutrients from prey, potentially necessitating increased respiratory chain activity

• Potential connection to outer membrane vesicles (OMVs):

  • M. xanthus utilizes OMVs as vehicles for delivering hydrolytic enzymes and antibiotics to prey microbes

  • The biogenesis and function of these OMVs may have connections to membrane energetics

  • Complex I components like nuoA maintain the proton gradient across the inner membrane, which could indirectly influence outer membrane dynamics

• Adaptations to changing oxygen conditions during predation:

  • During predatory swarming, cells form high-density groups where oxygen availability may be limited

  • The oxygen-sensing NmpRSTU system regulates social behaviors , suggesting respiratory adaptation is important

  • nuoA function may be modulated to optimize energy generation under these changing conditions

• Experimental approaches to investigate:

  • Compare predatory efficiency of wild-type versus nuoA mutant strains

  • Analyze nuoA expression during predation using transcriptomics or reporter constructs

  • Examine the effect of respiratory chain inhibitors on predatory behaviors

What emerging technologies could advance our understanding of nuoA's role in complex I assembly and function?

Several cutting-edge technologies show promise for advancing our understanding of nuoA:

• Cryo-electron tomography of native membranes:

  • Visualizes respiratory complexes in their native membrane environment

  • Reveals the supramolecular organization of respiratory chain components

  • Can be combined with subtomogram averaging for higher resolution details

• In-cell structural biology approaches:

  • FRET-based sensors to monitor complex I assembly in live cells

  • Proximity labeling methods (BioID, APEX) to map the interaction network of nuoA in vivo

  • Single-molecule tracking to observe complex I dynamics in living cells

• High-throughput mutagenesis with deep mutational scanning:

  • Creates comprehensive libraries of nuoA variants

  • Couples functional selection with next-generation sequencing

  • Maps the entire mutational landscape to identify all functional residues

• Integrative structural biology approaches:

  • Combines data from multiple techniques (cryo-EM, HDX-MS, cross-linking MS)

  • Develops computational models incorporating dynamics and functional states

  • Predicts the effects of mutations or drug binding

• In situ cellular imaging technologies:

  • Correlative light and electron microscopy to visualize complex I in cellular context

  • Expansion microscopy for super-resolution visualization of respiratory complexes

  • Focused ion beam-scanning electron microscopy (FIB-SEM) for 3D reconstruction of cellular ultrastructure, similar to techniques used to study M. xanthus membrane structures

Comparative Table of Complex I Components in M. xanthus