Recombinant Xanthomonas oryzae pv. oryzae NADH-quinone oxidoreductase subunit K (nuoK)

What is the structure and function of Xanthomonas oryzae pv. oryzae NADH-quinone oxidoreductase subunit K (nuoK)?

The NADH-quinone oxidoreductase subunit K (nuoK) from Xanthomonas oryzae pv. oryzae (strain MAFF 311018) is a membrane protein component of the respiratory complex I. The protein consists of 101 amino acids with the sequence: MITLGHLLGLGAVLFCISLAGIFLNRKNVIVLLMSIELLLSVNVNFIAFSRELGDTAGQLFVFFILTVAAAEAAIGLAILVTLFRTRRTINVAEVDTLKG . This hydrophobic protein is characterized by multiple transmembrane domains that anchor it within the bacterial membrane.

NuoK functions as an integral component of NADH dehydrogenase I (NDH-1), participating in the electron transport chain by facilitating electron transfer from NADH to quinones. This process is essential for energy metabolism in X. oryzae pv. oryzae, contributing to ATP synthesis through oxidative phosphorylation. The enzyme is classified with EC number 1.6.99.5 and is also known as NADH dehydrogenase I subunit K .

Experimental approaches to study the structure include:

• X-ray crystallography for high-resolution structural analysis

• Cryo-electron microscopy for visualizing membrane protein architecture

• Membrane protein reconstitution in lipid nanodiscs to maintain native conformation

How is the nuoK gene expressed and regulated in Xanthomonas oryzae pv. oryzae?

The nuoK gene (locus tag XOO3057) in X. oryzae pv. oryzae is part of the nuo operon encoding components of the NADH:quinone oxidoreductase complex . Expression regulation involves:

• Metabolic state-dependent regulation: Expression levels vary based on the bacterial metabolic state, with upregulation typically occurring during active respiratory growth.

• Environmental response: Transcriptomic analysis reveals that expression patterns change in response to environmental conditions such as pH, oxygen availability, and nutrient status .

• Stress response: The expression may be modulated during plant infection as part of adaptation to host defenses.

For experimental analysis of nuoK expression, researchers should:

• Employ RT-qPCR to quantify transcript levels under different conditions

• Use reporter gene constructs (e.g., lacZ fusions) to visualize expression patterns

• Apply ChIP-seq to identify transcription factors that bind to the nuo operon promoter

• Perform transcriptomic analysis to identify co-regulated genes in different growth conditions

What are the recommended protocols for isolating and purifying recombinant nuoK protein?

When isolating and purifying recombinant nuoK from X. oryzae pv. oryzae, consider these methodological approaches:

Expression System Selection:

• E. coli heterologous system: Similar to successful expression of other recombinant proteins from X. oryzae

• Cell-free expression systems: Alternative for difficult-to-express membrane proteins

Purification Protocol:

• Cell lysis: Use mechanical disruption (French press or sonication) with a buffer containing protease inhibitors

• Membrane fraction isolation: Ultracentrifugation (100,000 × g for 1 hour)

• Solubilization: Employ mild detergents (DDM, LMNG, or digitonin) to maintain protein structure

• Affinity chromatography: Use His-tag or other affinity tags for initial purification

• Size exclusion chromatography: For final purification and buffer exchange

Storage Recommendations:

• Store in Tris-based buffer with 50% glycerol at -20°C

• For extended storage, maintain at -80°C

• Avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

Validation of purified protein should include SDS-PAGE, Western blotting, and activity assays to confirm identity and functionality.

How does nuoK contribute to the energy metabolism of X. oryzae pv. oryzae?

The nuoK protein plays a critical role in the energy metabolism of X. oryzae pv. oryzae as a component of respiratory complex I. Understanding its contribution requires examination of:

• Electron transport function: NuoK facilitates electron transfer from NADH to quinones in the respiratory chain, similar to the function observed in other bacterial species .

• Proton translocation: As part of complex I, nuoK contributes to the establishment of the proton gradient across the membrane, which drives ATP synthesis.

• Energetic coupling: The protein participates in coupling the redox reaction to proton translocation, though structural studies of bacterial complex I have revealed that uncoupled states can occur where redox reaction is not linked to proton movement .

• Metabolic adaptation: During plant infection, nuoK activity may contribute to bacterial adaptation to changing host environments and nutrient availability.

Experimental approaches to study nuoK's role in energy metabolism include:

• Membrane vesicle preparations to measure NADH oxidation and proton pumping

• Oxygen consumption measurements using oxygen electrodes

• Membrane potential assays using fluorescent dyes

• Mutagenesis studies targeting conserved residues to assess impact on respiratory function

How might nuoK contribute to the pathogenicity of X. oryzae pv. oryzae in rice plants?

The contribution of nuoK to X. oryzae pv. oryzae pathogenicity likely stems from its role in energy metabolism, which indirectly supports virulence mechanisms:

• Energy provision for virulence factors: Energy generated through respiratory processes involving nuoK supports the production and function of virulence factors, including the Type III and Type VI secretion systems identified in X. oryzae .

• Adaptation to host environment: Efficient energy metabolism enables bacteria to adapt to changing conditions within the host plant. Pathogenomic analysis of X. oryzae reveals significant genetic elements contributing to its adaptability .

• Resistance to oxidative stress: Maintaining proper respiratory chain function may help bacteria cope with oxidative stress generated during host defense responses.

• Metabolic flexibility: Energy systems involving nuoK could contribute to the metabolic flexibility observed in X. oryzae strains from different geographic regions, which show considerable genetic divergence .

Research methodology to investigate this relationship should include:

• Creation of nuoK knockout mutants to assess impact on virulence

• Comparative transcriptomics of wild-type and nuoK mutants during infection

• Monitoring bacterial energy status in planta using biosensors

• Analysis of nuoK expression patterns during different infection stages

What potential interactions exist between nuoK and other components of the respiratory chain in X. oryzae?

The nuoK protein in X. oryzae likely engages in complex interactions with other respiratory chain components, similar to those observed in other bacterial systems:

• Intra-complex interactions: Within complex I, nuoK interacts with adjacent subunits to form a membrane-embedded hydrophobic domain. Based on structural studies of bacterial complex I, these interactions involve:

  • Transmembrane helix packing with neighboring subunits

  • Formation of a proton translocation pathway

  • Conformational changes during the catalytic cycle

• Inter-complex interactions: Beyond complex I, potential interactions may occur with:

  • Quinone pool intermediates

  • Components of complex III (cytochrome bc1)

  • Alternative NADH dehydrogenases, similar to those observed in other bacterial species

• Supercomplex formation: In some bacteria, respiratory complexes associate into supercomplexes for improved electron transfer efficiency.

Experimental approaches to investigate these interactions include:

• Crosslinking studies followed by mass spectrometry

• Co-immunoprecipitation with tagged components

• Blue native PAGE to identify complex I assembly

• Förster resonance energy transfer (FRET) for dynamic interaction studies

• Comparative analysis with respiratory complex structures from related bacteria

How do environmental conditions affect nuoK expression and function in X. oryzae during infection?

Environmental conditions significantly impact nuoK expression and function in X. oryzae during the infection process:

• Oxygen availability: Expression patterns likely shift in response to microaerobic conditions encountered within plant tissues.

• pH adaptation: The transcriptomic response of X. oryzae to environmental stimuli indicates pH-dependent gene regulation . The NDH-2B in other bacterial species has shown optimal function at pH 5.5, suggesting respiratory components like nuoK may be similarly affected .

• Nutrient limitation: Host-imposed nutrient restrictions may trigger metabolic reprogramming affecting nuoK expression.

• Plant defense responses: Oxidative burst and antimicrobial compounds produced by the host may influence respiratory chain component expression.

Methodological approaches to study these effects include:

• In vitro culture systems mimicking in planta conditions

• Transcriptomic and proteomic analysis of bacteria isolated from infected plants

• Reporter gene fusions to monitor nuoK expression during infection

• Metabolic flux analysis to track energy pathway utilization under different conditions

Experimental design considerations for such studies should include:

• Careful control of environmental variables

• Adequate biological replication

• Appropriate statistical analysis methods

• Validation using multiple complementary techniques

What are the structural and functional differences between nuoK in X. oryzae and homologous proteins in other bacterial species?

Comparative analysis of nuoK across bacterial species reveals important structural and functional differences:

• Sequence conservation: Alignment analysis shows varying degrees of conservation between X. oryzae nuoK and homologous proteins in other bacteria.

• Membrane topology: While the general transmembrane arrangement is conserved, subtle differences in transmembrane helix length and orientation may affect proton translocation efficiency.

• Quinone interaction sites: Variations in residues surrounding quinone binding sites might reflect adaptation to different quinone types prevalent in various bacteria.

• Regulatory mechanisms: Expression control mechanisms differ between species, with X. oryzae showing specific transcriptional responses to environmental cues .

• Functional redundancy: Unlike some bacterial species such as Staphylococcus aureus which possess alternative NADH:quinone oxidoreductases (NDH-2A and NDH-2B) that can compensate for each other , X. oryzae may have different arrangements of respiratory components.

Methodological approaches for comparative studies:

• Phylogenetic analysis of nuoK sequences across bacterial species

• Homology modeling based on available complex I structures

• Heterologous expression in model organisms followed by functional assays

• Site-directed mutagenesis of conserved versus divergent residues

What considerations should be taken into account when designing experiments to study nuoK function in vivo?

When designing experiments to study nuoK function in vivo, researchers should consider:

• Genetic manipulation approaches:

  • Gene deletion strategies: Consider polar effects on other genes in the nuo operon

  • Complementation strategies: Use inducible promoters to control expression levels

  • Site-directed mutagenesis: Target conserved residues to disrupt specific functions

  • Conditional mutants: Develop systems for temporal control of nuoK expression

• Phenotypic analysis methods:

  • Growth curve analysis under different respiratory conditions

  • Oxygen consumption measurements

  • Membrane potential assays

  • ATP production quantification

  • Virulence assessment in plant infection models

• Experimental design principles:

  • Control all variables except the one being tested

  • Include appropriate positive and negative controls

  • Ensure adequate sample sizes for statistical significance

  • Use multiple independent biological replicates

  • Apply appropriate statistical methods for data analysis

• In planta studies:

  • Consider host variation in susceptibility (e.g., comparing susceptible rice variety PSL2 vs. resistant variety PSL2-Xa21)

  • Monitor bacterial persistence in different plant tissues

  • Account for environmental factors affecting plant-pathogen interactions

A well-designed experimental approach should follow these steps:

• Define clear hypotheses about nuoK function

• Select appropriate genetic and biochemical tools

• Develop robust assay systems

• Implement controls to account for confounding variables

• Use statistical methods to ensure reproducibility and significance

How can researchers optimize heterologous expression systems for producing functional recombinant nuoK protein?

Optimizing heterologous expression of functional recombinant nuoK protein requires addressing several challenges associated with membrane protein expression:

• Expression system selection:

  • E. coli-based systems: BL21(DE3) or C41/C43(DE3) strains designed for membrane protein expression

  • Cell-free expression systems: Avoid issues with toxicity and inclusion body formation

  • Alternative hosts: Consider Pseudomonas species that may provide a more native-like membrane environment

• Vector design considerations:

  • Promoter selection: Tune expression levels using inducible promoters with variable induction strength

  • Fusion tags: N-terminal or C-terminal tags (His, MBP, SUMO) to aid solubility and purification

  • Fusion partners: Consider fusion with GFP to monitor expression and folding

  • Signal sequences: Evaluate the need for signal sequences to ensure proper membrane targeting

• Expression optimization:

  • Temperature: Lower temperatures (16-25°C) often improve membrane protein folding

  • Induction parameters: Optimize inducer concentration and induction timing

  • Media composition: Supplementation with specific lipids or cofactors

  • Growth phase: Induce at optimal cell density

• Purification strategy:

  • Detergent screening: Identify detergents that maintain protein stability and function

  • Buffer optimization: Test various pH ranges and salt concentrations

  • Purification method: Implement multi-step purification to achieve high purity

• Functional validation methods:

  • Activity assays: Develop assays to confirm electron transfer activity

  • Structural analysis: Circular dichroism to verify secondary structure

  • Binding studies: Assess interaction with cofactors and substrates

Success in producing functional nuoK protein has been achieved with other membrane proteins from X. oryzae using approaches similar to those used for recombinant protein expression from other bacterial pathogens .

What methodologies are recommended for analyzing the interaction of nuoK with other respiratory complex components?

To analyze interactions between nuoK and other respiratory complex components, researchers should consider these methodological approaches:

• Structural biology techniques:

  • Cryo-electron microscopy: Provides high-resolution structure of intact respiratory complexes

  • X-ray crystallography: For atomic-level details of protein interactions

  • NMR spectroscopy: For dynamic interaction studies of specific domains

• Protein-protein interaction assays:

  • Co-immunoprecipitation: Pull-down assays using antibodies against nuoK or interaction partners

  • Bacterial two-hybrid system: For in vivo detection of protein interactions

  • Surface plasmon resonance: For quantitative binding kinetics

  • Isothermal titration calorimetry: For thermodynamic parameters of interactions

• Crosslinking approaches:

  • Chemical crosslinking: Coupled with mass spectrometry to identify interaction interfaces

  • Photo-affinity labeling: For capturing transient interactions

  • In vivo crosslinking: To capture interactions in their native environment

• Functional assays:

  • Activity measurements: Compare activities of isolated nuoK versus reconstituted complexes

  • Mutagenesis: Strategic mutations at interaction surfaces to disrupt specific contacts

  • Suppressor mutation analysis: Identify compensatory mutations that restore function

• Advanced imaging:

  • Förster resonance energy transfer (FRET): For real-time monitoring of protein interactions

  • Single-particle tracking: To follow dynamics of complex assembly

What approaches can be used to assess the impact of nuoK mutations on bacterial fitness and virulence?

Assessing the impact of nuoK mutations on bacterial fitness and virulence requires a multi-faceted experimental approach:

• Generation of mutant strains:

  • Site-directed mutagenesis targeting functional domains

  • Random mutagenesis to identify non-obvious functional residues

  • Construction of deletion mutants (when not lethal)

  • Complementation studies to confirm phenotype specificity

• In vitro fitness assessment:

  • Growth curves under various conditions (different carbon sources, oxygen levels)

  • Competition assays with wild-type bacteria

  • Stress resistance tests (oxidative, pH, temperature)

  • Metabolic profiling to identify altered pathways

• Energy metabolism analysis:

  • Oxygen consumption measurements

  • NADH oxidation rates

  • Membrane potential determination

  • ATP production quantification

• Virulence assessment:

  • Rice plant infection models comparing susceptible and resistant varieties

  • Bacterial colonization and persistence measurements

  • Quantification of disease symptoms

  • Competitive index determinations in planta

• Molecular analysis:

  • Transcriptomic profiling to identify compensatory responses

  • Proteomic analysis to detect changes in protein expression

  • Metabolomic studies to identify altered metabolic states

Experimental design should include:

• Multiple biological replicates

• Appropriate statistical analysis

• Controls to account for growth defects versus specific virulence effects

• Complementation studies to confirm mutation-specific effects

Table 1: Key Properties of Recombinant Xanthomonas oryzae pv. oryzae NADH-quinone oxidoreductase subunit K (nuoK)

Table 2: Comparative Analysis of NADH:quinone oxidoreductase Components Across Bacterial Species

Table 3: Experimental Approaches for nuoK Functional Analysis

What emerging technologies might advance our understanding of nuoK function in X. oryzae?

Several cutting-edge technologies offer promising avenues for deeper insights into nuoK function:

• CRISPR-Cas9 genome editing:

  • Precise modification of nuoK without polar effects

  • Creation of conditional mutants for essential functions

  • Base editing for specific amino acid substitutions

  • CRISPRi for tunable repression of nuoK expression

• Advanced structural biology:

  • Cryo-electron tomography for visualizing respiratory complexes in their native membrane environment

  • Single-particle analysis for capturing different conformational states

  • Integrative structural biology combining multiple techniques

• Systems biology approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Flux balance analysis to model impacts on bacterial metabolism

  • Network analysis to identify regulatory relationships

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize respiratory complex organization

  • Single-molecule tracking to analyze complex assembly dynamics

  • FRET-based sensors to monitor conformational changes

• Synthetic biology tools:

  • Designer respiratory chains with modified nuoK variants

  • Optogenetic control of nuoK expression

  • Biosensors to monitor respiratory activity in real-time

These technologies should be applied with careful experimental design, appropriate controls, and statistical rigor to ensure reliable and reproducible findings .

How can nuoK be targeted for developing novel control strategies against X. oryzae?

The essential role of nuoK in energy metabolism presents potential opportunities for developing targeted control strategies:

• Structure-based inhibitor design:

  • Virtual screening against nuoK structural models

  • Fragment-based drug discovery targeting specific functional domains

  • Peptidomimetics that disrupt protein-protein interactions

• Allosteric modulators:

  • Compounds that lock nuoK in inactive conformations

  • Molecules that disrupt conformational changes required for function

• Complex assembly disruptors:

  • Agents that prevent incorporation of nuoK into respiratory complex I

  • Compounds that destabilize complex I integrity

• Novel delivery systems:

  • Nanoparticle-based delivery of inhibitors

  • Plant-expressed inhibitors activated during infection

• Combination strategies:

  • Targeting nuoK alongside other virulence factors

  • Complementary approaches targeting different aspects of bacterial metabolism

Methodological approaches should include:

• High-throughput screening assays for candidate compounds

• Structural studies of inhibitor binding

• In vitro and in planta efficacy testing

• Assessment of resistance development potential