Recombinant Acinetobacter sp. NADH-quinone oxidoreductase subunit I (nuoI)

What is the genomic context of nuoI in Acinetobacter species?

The nuoI gene in Acinetobacter species typically exists within the nuo operon, which encodes the 14 subunits (nuoA through nuoN) of the proton-pumping NADH-quinone oxidoreductase (Complex I). In most Acinetobacter genomes, the nuo genes are arranged in a conserved order. The nuoI gene specifically codes for a critical iron-sulfur cluster-containing subunit that participates in electron transfer within the complex. Genomic analysis of clinical Acinetobacter isolates has revealed that while the respiratory chain components are generally conserved, sequence variations can occur particularly in areas affected by homologous recombination, which has been documented to occur across approximately 20% of Acinetobacter genomes .

How can researchers distinguish between nuoI sequence variants in different Acinetobacter species?

Distinguishing between nuoI sequence variants requires a combination of molecular and bioinformatic approaches:

• Perform whole genome sequencing of Acinetobacter isolates following established protocols similar to those used for tracking epidemic strains

• Align nuoI sequences from multiple isolates using software like MUSCLE or ClustalW

• Construct phylogenetic trees to visualize relationships between variants

• Identify conserved and variable regions that may correlate with specific phenotypes

Genomic analysis of clinical Acinetobacter isolates has demonstrated that homologous recombination contributes significantly to genetic diversity . When analyzing nuoI variants, researchers should be aware that recombination events may affect interpretation of phylogenetic data. Similar considerations should be made as those used when analyzing the widespread recombination observed in epidemic Acinetobacter strains .

What is the relationship between nuoI and energy metabolism in multidrug-resistant Acinetobacter strains?

The relationship between nuoI and energy metabolism in multidrug-resistant (MDR) Acinetobacter strains is complex and may be influenced by several factors:

MDR Acinetobacter strains, particularly those belonging to clonal complex 92 (CC92), often carry multidrug efflux pumps such as abe and ade systems . These energy-dependent transporters require proton motive force, which is generated in part by the NADH-quinone oxidoreductase complex containing the nuoI subunit. Therefore, nuoI function may indirectly support antibiotic resistance mechanisms by providing energy for these efflux systems.

What expression systems are most effective for recombinant nuoI production?

The optimal expression system for recombinant nuoI depends on research objectives and downstream applications:

For functional studies, expression in E. coli C43(DE3) or C41(DE3) strains specifically designed for membrane protein expression may be advantageous. These strains can better accommodate proteins that affect respiratory chain function, minimizing toxic effects during expression.

What purification challenges are specific to recombinant nuoI and how can they be addressed?

Purification of recombinant nuoI presents several challenges:

• Membrane association: Although nuoI is a peripheral membrane subunit, it often co-purifies with membrane components. Solution: Use detergents like n-dodecyl-β-D-maltoside (DDM) or digitonin in purification buffers.

• Iron-sulfur cluster integrity: The iron-sulfur clusters in nuoI are sensitive to oxidation. Solution: Include reducing agents (5-10 mM DTT or 2-5 mM β-mercaptoethanol) in all buffers and work under anaerobic conditions when possible.

• Protein instability: Isolated nuoI may be unstable outside its complex. Solution: Express with interacting partners or optimize buffer conditions (pH 7.0-8.0, 150-300 mM NaCl, 5-10% glycerol).

• Activity loss during purification: Solution: Measure activity at each purification step and minimize exposure to harsh conditions.

When designing purification protocols, researchers should consider the high recombination capacity observed in Acinetobacter species , which may result in strain-specific nuoI variants with different biochemical properties requiring customized purification approaches.

What assays can accurately measure nuoI activity in recombinant systems?

Several complementary approaches can be used to measure recombinant nuoI activity:

• NADH oxidation assay: Monitor NADH oxidation spectrophotometrically at 340 nm in the presence of appropriate electron acceptors like ubiquinone-1 or decylubiquinone.

• Electron paramagnetic resonance (EPR) spectroscopy: Detect and characterize the iron-sulfur clusters in nuoI, providing information about their redox state and environment.

• Artificial electron acceptor assays: Use ferricyanide, 2,6-dichlorophenolindophenol (DCIP), or other artificial electron acceptors to measure electron transfer activity.

• Reconstitution assays: Incorporate purified nuoI into liposomes or nanodiscs with other Complex I subunits to measure activity in a more native-like environment.

• Oxygen consumption assays: In reconstituted systems or whole cells expressing recombinant nuoI, measure oxygen consumption rates using a Clark-type electrode.

When designing activity assays, it's important to consider that nuoI functions as part of a larger complex. The genomic diversity observed in Acinetobacter species, including the extensive recombination documented in clinical isolates , may result in functional variations that can be detected through these assays.

How does nuoI contribute to Acinetobacter metabolic adaptation in different environments?

NADH-quinone oxidoreductase subunit I (nuoI) plays a critical role in Acinetobacter metabolic adaptation through several mechanisms:

In clinical isolates, the nuoI function may be particularly important for adaptation to the hospital environment, where antimicrobial pressure is high. Genomic studies of Acinetobacter outbreaks have demonstrated that epidemic lineages like European Clone II (EC II) show evidence of ongoing adaptation to hospital environments , which may include optimizations of energy metabolism through modifications of respiratory chain components like nuoI.

Can nuoI serve as a potential drug target for combating multidrug-resistant Acinetobacter infections?

The potential of nuoI as a drug target for combating multidrug-resistant Acinetobacter infections merits serious consideration for several reasons:

• Essential function: As a component of the respiratory chain, nuoI is critical for energy production, making it an essential gene in most growth conditions.

• Structural uniqueness: Bacterial Complex I has structural differences from human mitochondrial Complex I, potentially allowing for selective inhibition.

• Link to resistance mechanisms: Energy-dependent efflux pumps like abe and ade systems, which are present in all Acinetobacter baumannii isolates studied , require the proton motive force generated in part by Complex I.

• Limited bypass mechanisms: While alternative NADH dehydrogenases exist in some bacteria, they may not fully compensate for Complex I inhibition.

Researchers should consider that in clonal complex 92 (CC92) strains of A. baumannii, which show 100% multidrug resistance rates , targeting energy metabolism may provide a strategy to overcome existing resistance mechanisms. Target validation studies should include genetic knockdown/knockout of nuoI and assessment of resulting changes in antibiotic susceptibility.

How does nuoI expression correlate with virulence factors in pathogenic Acinetobacter strains?

The correlation between nuoI expression and virulence factors in pathogenic Acinetobacter strains involves complex regulatory networks:

• Co-regulation with virulence genes: Under certain stress conditions, nuoI may be co-regulated with virulence factors through global regulators.

• Energy provision for virulence factor production: Many virulence factors require significant energy for synthesis and export, linking nuoI function to virulence capability.

• Adaptation to host environments: Modulation of respiratory chain function via nuoI may support adaptation to different host niches.

• Biofilm formation: Energy metabolism through Complex I contributes to biofilm formation capacity, a key virulence trait.

Research methodologies to investigate these correlations should include:

• Transcriptomic analysis comparing nuoI expression with virulence gene expression under different conditions

• Metabolic flux analysis in wild-type and nuoI-modified strains

• Assessment of virulence factor production in strains with altered nuoI expression

The genomic plasticity of Acinetobacter species, particularly their capacity for homologous recombination as documented in outbreak strains , may contribute to variability in the relationship between nuoI function and virulence traits.

How has recombination affected the evolution of nuoI in Acinetobacter species?

Recombination has significantly influenced the evolution of nuoI in Acinetobacter species, contributing to genetic diversity and potentially functional adaptation:

• Homologous recombination: Genomic studies have demonstrated that approximately 20% of the Acinetobacter genome is subject to recombination , potentially including regions containing respiratory chain components like nuoI.

• Selective pressure: As a component of the essential respiratory machinery, nuoI is subject to selective pressures that may drive recombination events that optimize its function.

• Species-specific variants: Recombination may contribute to the diversity of nuoI sequences observed across the Acinetobacter calcoaceticus-baumannii (ACB) complex.

• Horizontal gene transfer: While the core nuoI function is likely conserved, flanking regions may be more subject to recombination, potentially affecting regulatory elements.

Researchers have observed that in Acinetobacter baumannii, genomic regions showing elevated recombination rates often contain genes encoding surface-exposed proteins or those involved in synthesis of cell-surface molecules . While nuoI is not a surface protein, its role in energy production may indirectly support adaptation of surface structures through complex metabolic networks.

What can comparative genomics reveal about nuoI conservation across different Acinetobacter clonal complexes?

Comparative genomics analysis of nuoI across different Acinetobacter clonal complexes can provide valuable insights:

Methodological approaches for comparative genomics of nuoI include:

• Whole-genome sequencing of diverse isolates, similar to approaches used in outbreak investigations

• Alignment of nuoI sequences and phylogenetic analysis, accounting for recombination

• Analysis of selection signatures (dN/dS ratios) to identify regions under purifying or diversifying selection

• Structural modeling to predict functional consequences of sequence variations

When interpreting comparative genomics data, researchers should consider that non-baumannii Acinetobacter calcoaceticus-baumannii (NB-ACB) complex species show different antimicrobial susceptibility profiles , which may correlate with specific variants or regulatory patterns of nuoI and other respiratory chain components.

How can recombinant nuoI be used to study respiratory chain inhibitors?

Recombinant nuoI can serve as a valuable tool for studying respiratory chain inhibitors through several approaches:

• In vitro binding studies: Purified recombinant nuoI can be used to screen potential inhibitors through binding assays (thermal shift assays, isothermal titration calorimetry, or surface plasmon resonance).

• Structure-based drug design: High-resolution structures of recombinant nuoI can guide rational design of specific inhibitors targeting this subunit.

• Activity assays: NADH oxidation assays with recombinant nuoI (either alone or reconstituted with other Complex I subunits) can assess inhibitor effects on electron transfer.

• Resistance mutation mapping: Recombinant nuoI variants with specific mutations can help map resistance determinants to respiratory chain inhibitors.

• Heterologous expression systems: Expression of Acinetobacter nuoI in model organisms can create platforms for inhibitor screening.

Experimental design considerations should include:

• Appropriate controls to distinguish effects on nuoI from effects on other respiratory chain components

• Comparison of inhibitor effects across nuoI variants from different Acinetobacter strains

• Correlation between in vitro inhibition and effects on whole-cell growth and metabolism

Given the importance of respiratory function in supporting energy-dependent resistance mechanisms like efflux pumps, which are present in all A. baumannii isolates studied , inhibitors identified through these approaches may have potential applications against multidrug-resistant strains.

What are the best approaches for studying nuoI interactions with other respiratory chain components?

Studying interactions between nuoI and other respiratory chain components requires multifaceted approaches:

• Co-immunoprecipitation: Using antibodies against tagged recombinant nuoI to pull down interacting partners, followed by mass spectrometry identification.

• Crosslinking coupled with mass spectrometry: Chemical crosslinking of protein complexes followed by MS/MS analysis to identify interaction interfaces.

• Two-hybrid systems: Modified bacterial or yeast two-hybrid systems adapted for membrane-associated proteins can identify binary interactions.

• Blue native PAGE: Non-denaturing gel electrophoresis to visualize intact complexes containing nuoI and determine subcomplex compositions.

• Cryo-electron microscopy: High-resolution structural analysis of reconstituted complexes containing recombinant nuoI.

• FRET-based approaches: Fluorescently labeled nuoI and potential partners to monitor interactions in vitro or in vivo.

When designing interaction studies, researchers should consider the genomic context of nuoI and potential strain-specific variations. The extensive genomic recombination observed in Acinetobacter species may result in strain-specific interaction patterns that could influence respiratory chain assembly and function.

How can site-directed mutagenesis be applied to study critical residues in nuoI function?

Site-directed mutagenesis provides a powerful approach to dissect nuoI structure-function relationships:

• Target selection strategy:

  • Iron-sulfur cluster coordination sites (cysteine residues)

  • Conserved charged residues potentially involved in electron transfer

  • Residues at interfaces with other Complex I subunits

  • Residues differing between Acinetobacter strains with varying resistance profiles

• Mutagenesis workflow:

  • Design primers containing desired mutations using overlap extension PCR methods

  • Introduce mutations into expression vectors using standard molecular biology techniques

  • Verify mutations by sequencing before proceeding with expression

  • Express and purify mutant proteins using identical conditions to wild-type

• Functional characterization:

  • Compare enzyme kinetics between wild-type and mutant proteins

  • Assess structural integrity through CD spectroscopy or thermal stability assays

  • Evaluate electron transfer capacity using spectroscopic methods

  • Test assembly into larger subcomplexes using blue native PAGE

• In vivo validation:

  • Complement Acinetobacter nuoI deletion strains with mutant variants

  • Assess effects on growth, respiration, and antimicrobial susceptibility

When interpreting mutagenesis results, researchers should consider that nuoI functions within a complex biological context. The extensive recombination and genetic diversity observed across Acinetobacter strains may provide natural variants that can inform targeted mutagenesis approaches.

What are the optimal conditions for measuring electron transport activity of recombinant nuoI?

Optimizing conditions for measuring electron transport activity of recombinant nuoI requires careful consideration of multiple parameters:

Methodological considerations:

• Prepare all reagents fresh and degas buffers to minimize oxidative damage

• Include appropriate controls including heat-inactivated enzyme

• Measure initial rates before substrate depletion occurs

• Consider using a stopped-flow apparatus for rapid kinetics measurements

• Use multiple detection methods (spectrophotometric, fluorometric, polarographic) for validation

When establishing assay conditions, researchers should be aware that different Acinetobacter strains, particularly those from different clonal complexes like CC92 vs. non-CC92 , may show variations in optimal conditions reflecting their adaptation to different environments.

How does Acinetobacter nuoI differ from equivalent subunits in other pathogenic bacteria?

Comparative analysis of Acinetobacter nuoI with equivalent subunits from other pathogenic bacteria reveals important differences:

Research approaches for comparative studies:

• Sequence alignment and phylogenetic analysis of nuoI across diverse bacterial species

• Heterologous expression of Acinetobacter nuoI in model bacterial systems lacking native Complex I

• Comparative structural modeling to identify unique features of Acinetobacter nuoI

• Cross-species complementation studies to assess functional conservation

These comparative studies can leverage insights from genomic investigations of Acinetobacter strains to understand how respiratory chain components may contribute to the unique metabolic adaptability and antimicrobial resistance profiles of different bacterial pathogens.

Can heterologous expression of Acinetobacter nuoI complement mutations in model organisms?

Heterologous expression of Acinetobacter nuoI in model organisms with nuoI mutations offers valuable insights into functional conservation and species-specific adaptations:

• Expression in E. coli nuoI mutants:

  • Transformation with Acinetobacter nuoI expression vectors

  • Assessment of respiratory function restoration

  • Growth rate comparison under different carbon sources

  • Measurement of proton-pumping efficiency

• Methodological considerations:

  • Optimize codon usage for host organism expression

  • Use inducible promoters to control expression levels

  • Include appropriate targeting sequences if necessary

  • Consider co-expression with interacting partners

• Expected outcomes:

  • Complete complementation suggests high functional conservation

  • Partial complementation indicates species-specific adaptations

  • No complementation may reveal incompatibility with host Complex I

• Applications:

  • Development of model systems for inhibitor screening

  • Structure-function studies in well-characterized backgrounds

  • Investigation of species-specific nuoI variants from different Acinetobacter strains

When interpreting complementation studies, researchers should consider the extensive genetic diversity observed within Acinetobacter species . nuoI variants from different clonal complexes or species within the ACB complex may show different complementation capabilities, potentially correlating with their metabolic adaptations and resistance profiles.