Recombinant Protochlamydia amoebophila NADH-quinone oxidoreductase subunit I (nuoI)

What is Protochlamydia amoebophila and its significance in research?

Protochlamydia amoebophila is an obligate intracellular symbiont that belongs to the phylum Chlamydiae. It thrives as an endosymbiont within free-living amoebae, specifically Acanthamoeba species. Unlike its pathogenic relatives in the Chlamydiaceae family, P. amoebophila establishes a long-term relationship with its host in which both bacteria and amoebae multiply in a synchronized manner .

The significance of P. amoebophila in research stems from several factors:

• It provides insights into the evolution of host-pathogen interactions, as it represents an ancient lineage that diverged from pathogenic chlamydiae approximately 700 million years ago .

• It serves as a model organism for studying symbiotic relationships and the mechanisms of intracellular survival.

• The study of P. amoebophila contributes to our understanding of metabolic dependencies between symbionts and hosts.

• It possesses unique proteins, such as inclusion membrane proteins, that are involved in host cell interaction and manipulation .

What is NADH-quinone oxidoreductase (Complex I) and what role does it play in bacterial metabolism?

NADH-quinone oxidoreductase, also known as Complex I, is a multisubunit integral membrane enzyme found in the respiratory chains of both bacteria and eukaryotic organelles. It plays a central role in energy metabolism by coupling the oxidation of NADH to the reduction of quinone, while simultaneously pumping protons across the membrane .

The bacterial Complex I typically consists of 14 core subunits (labeled NuoA through NuoN) arranged in an L-shaped structure with two main domains:

• A hydrophilic arm containing the electron transport chain cofactors (FMN and iron-sulfur centers)

• A membrane arm responsible for proton translocation

Complex I functions as a reversible enzyme that can either:

• Catalyze NADH oxidation coupled to proton pumping during aerobic respiration

• Drive NADH synthesis from quinol using the proton motive force, as seen in certain photosynthetic bacteria

According to phylogenomic analysis of 970 bacterial genomes, Complex I is predicted to be present in approximately 52% of bacterial species but is nearly absent in archaea .

What is the specific function of subunit I (nuoI) within the NADH-quinone oxidoreductase complex?

Subunit I (nuoI) is one of the core components of the NADH-quinone oxidoreductase complex. Based on structural and functional studies of Complex I:

Research has identified four subunits (including soluble NuoB/D and transmembrane NuoH/antiporter-like subunits) that compose the core necessary to couple redox activity to ion transport, with nuoI playing a supporting role in this machinery .

What are the most effective methods for recombinant expression of P. amoebophila proteins?

Based on successful approaches with similar proteins, the most effective methods for recombinant expression of P. amoebophila proteins include:

Expression System Optimization:

• Codon harmonization (not just optimization) has been shown to significantly improve expression levels of chlamydial proteins in E. coli .

• Using low copy number vectors with moderate strength promoters rather than high-copy vectors with strong promoters, which can overwhelm the cellular machinery .

• Selection of appropriate leader sequences to guide protein secretion and membrane insertion .

Culture Conditions:

• Utilizing reduced culture temperatures (often 30°C instead of 37°C) during induction .

• Optimizing induction parameters (IPTG concentration, timing, and duration) .

Expression Verification Methods:

• Whole cell flow cytometry antibody binding to confirm surface expression .

• Western blot analysis using specific antibodies against the target protein .

• SDS-PAGE to assess expression levels and protein integrity .

A comparative study for recombinant expression of Chlamydia trachomatis MOMP showed that codon harmonization resulted in approximately 2-fold increase in expression compared to standard codon optimization .

What challenges arise when expressing membrane proteins like nuoI, and how can they be addressed?

Expressing membrane proteins like nuoI presents several challenges:

For integral membrane proteins like NADH-quinone oxidoreductase subunits, targeting expression to the E. coli outer membrane has proven successful, as demonstrated with the Chlamydia MOMP protein .

How can I verify the functional integrity of recombinantly expressed nuoI?

Verification of functional integrity requires multiple complementary approaches:

Structural Integrity Assessment:

• Circular dichroism (CD) spectroscopy to confirm secondary structure elements characteristic of properly folded protein .

• Size exclusion chromatography to verify proper oligomeric state.

• Limited proteolysis to assess conformational stability.

Functional Activity Testing:

• Electron transfer activity measurement using artificial electron acceptors.

• Reconstitution experiments with other Complex I subunits to test interaction capability.

• Assessment of iron-sulfur center incorporation using electron paramagnetic resonance (EPR) spectroscopy.

Binding Studies:

• Co-immunoprecipitation with partner subunits to verify proper interaction surfaces.

• Surface plasmon resonance (SPR) to quantify binding affinities with complex components.

Cellular Localization:

• Immunofluorescence microscopy to confirm proper cellular localization when expressed in host cells .

• Membrane fractionation followed by western blotting to verify membrane association .

How does the evolutionary history of Complex I in P. amoebophila relate to its symbiotic lifestyle?

The evolutionary history of Complex I in P. amoebophila reflects the organism's adaptation to its symbiotic lifestyle:

• Metabolic Integration: P. amoebophila, like other obligate intracellular bacteria, relies heavily on host metabolism. Analysis of its nucleotide transporters reveals that "these metabolically impaired bacteria are intimately connected with their host cell's metabolism in a surprisingly complex manner" . The presence of Complex I enables efficient energy conversion, crucial for an organism with limited metabolic capabilities.

• Genome Reduction and Conservation: Despite genome reduction typical in obligate intracellular bacteria, P. amoebophila has maintained Complex I genes, suggesting their essential role in its metabolic strategy. This contrasts with some other intracellular pathogens that have lost parts of their respiratory chains .

• Horizontal Gene Transfer: Phylogenomic analyses suggest that Chlamydiae have contributed at least 55 genes to plant genomes, including some metabolic genes . This indicates the ancient nature of chlamydial symbiosis and possible gene exchange during evolution.

• Adaptation to Microaerobic Environment: The Complex I in P. amoebophila likely adapted to function efficiently in the microaerobic environment of the amoeba host cell, potentially showing modifications compared to free-living bacteria.

• Transmission Mode Effects: Research on P. amoebophila shows that transmission modes (horizontal vs. vertical) influence symbiont evolution, with horizontally transmitted strains showing enhanced replication rates . This likely affects the expression and possibly structure of metabolic complexes like NADH-quinone oxidoreductase.

What role might nuoI play in the host-symbiont interaction of P. amoebophila and Acanthamoeba?

The role of nuoI in host-symbiont interaction likely extends beyond its primary function in electron transport:

• Energy Production for Intracellular Survival: As part of Complex I, nuoI contributes to the efficient generation of ATP, enabling P. amoebophila to survive within its host despite limited metabolic capacity .

• Redox Balance Maintenance: The NADH:quinone oxidoreductase complex helps maintain redox balance within the bacterium, which is crucial when adapting to changing conditions within the host cell .

• Metabolic Synchronization: Complex I activity may synchronize bacterial metabolism with host metabolic cycles, facilitating the coordinated multiplication observed between P. amoebophila and its amoeba host .

• Response to Stress Conditions: Complex I components may be regulated in response to host-induced stress, allowing adaptation to the intracellular environment.

• Possible Signaling Role: Beyond energy metabolism, research on other bacterial systems suggests that components of respiratory complexes can influence regulatory pathways that affect host-pathogen interactions .

Experimental evidence indicates that P. amoebophila establishes long-term stable relationships with its host, suggesting that its metabolic systems, including Complex I, have evolved to minimize negative impacts on host fitness .

How does the structure and function of P. amoebophila nuoI compare to homologous proteins in other bacteria, particularly pathogenic Chlamydiaceae?

The structure and function of P. amoebophila nuoI likely shows both conservation and divergence when compared to other bacteria:

Studies on inclusion membrane proteins in P. amoebophila show that "this strategy for host cell interaction is conserved among the chlamydiae and is used by chlamydial symbionts and pathogens alike" , suggesting functional conservation despite sequence divergence. A similar pattern may apply to metabolic complexes like NADH-quinone oxidoreductase.

How might the proton-pumping activity of Complex I contribute to P. amoebophila's adaptation to its intracellular niche?

The proton-pumping activity of Complex I contributes significantly to P. amoebophila's intracellular adaptation:

• Energy Efficiency: The proton gradient generated by Complex I can be utilized for ATP synthesis, enabling more efficient energy production in the nutrient-limited intracellular environment .

• Metabolic Flexibility: Complex I can operate in reverse direction under certain conditions, allowing P. amoebophila to adapt to changing energy states within the host cell .

• pH Homeostasis: Proton translocation contributes to pH regulation within the bacterial cell and possibly within the inclusion, creating optimal conditions for bacterial enzymes .

• Ion Balance Regulation: Studies on E. coli and Rhodothermus marinus Complex I revealed Na+ transport in the opposite direction to H+, suggesting a role in maintaining ionic balance . A similar mechanism in P. amoebophila could help adapt to host cell ionic environments.

• Signaling Functions: The proton motive force generated by Complex I may serve as a signal that regulates gene expression related to symbiosis, similar to how membrane potential influences bacterial developmental processes in other systems.

What are the recommended methods for isolating and purifying recombinant nuoI from bacterial expression systems?

Based on successful approaches with similar proteins, recommended methods include:

Cell Lysis and Initial Extraction:

• Harvest bacterial cells by centrifugation (typically 5,000 × g for 15 minutes at 4°C).

• Resuspend cell pellet in appropriate buffer (e.g., 50 mM Tris-HCl pH 8.0, 150 mM NaCl).

• For membrane-associated proteins like nuoI, add appropriate detergents (e.g., 1% DDM, 1% CHAPS, or 0.5% Triton X-100).

• Lyse cells using sonication, French press, or enzymatic methods.

• Remove cellular debris by centrifugation (20,000 × g for 30 minutes at 4°C).

Membrane Fraction Preparation:

• Further centrifuge the supernatant at high speed (100,000 × g for 1 hour at 4°C) to isolate membrane fractions.

• Resuspend membrane pellet in buffer containing mild detergent.

• Solubilize membrane proteins by gentle rotation for 1-2 hours at 4°C.

• Remove insoluble material by centrifugation (100,000 × g for 30 minutes).

Purification Strategy:

• Affinity Chromatography: Use His-tag or other affinity tags to capture the target protein.

• Ion Exchange Chromatography: Further purify based on charge properties.

• Size Exclusion Chromatography: Final polishing step to obtain homogeneous protein.

Critical Considerations:

• Maintain cold temperatures (4°C) throughout the process to preserve protein integrity.

• Include protease inhibitors to prevent degradation.

• For iron-sulfur proteins like nuoI, consider anaerobic purification to maintain redox state.

• Optimize detergent type and concentration for maximum recovery of functional protein.

What bioinformatic approaches are most useful for studying nuoI sequence, structure, and function?

A comprehensive bioinformatic analysis of nuoI should include:

Sequence Analysis:

• Multiple sequence alignment with homologs to identify conserved regions using tools like MUSCLE or Clustal Omega.

• Identification of functional domains and motifs using InterPro, Pfam, or PROSITE.

• Codon usage analysis for optimizing recombinant expression using tools like the Codon Usage Database (http://www.kazusa.or.jp/codon/)[8].

• Phylogenetic analysis to understand evolutionary relationships using RAxML or PhyML .

Structural Prediction:

• Secondary structure prediction using PSIPRED or JPred.

• Homology modeling based on related structures using SWISS-MODEL or Phyre2.

• Iron-sulfur center binding site prediction using specialized tools or machine learning approaches.

• Molecular dynamics simulations to predict functional movements and interactions.

Functional Analysis:

• Protein-protein interaction prediction using STRING or PSICQUIC.

• Metabolic pathway mapping using databases like MetaCyc or PlantCyc .

• Gene co-expression analysis to identify functionally related genes.

Practical Implementation:
Several studies have successfully applied these approaches to related proteins:

• Phylogenomic analysis identified NADH:quinone oxidoreductase in ~52% of surveyed bacterial genomes .

• Comparative sequence analysis revealed conserved domains in membrane proteins from P. amoebophila .

• Bioinformatic screening using secondary structure motifs successfully identified inclusion membrane proteins in P. amoebophila .

How can researchers assess the interaction between nuoI and other subunits of the Complex I machinery?

Assessment of subunit interactions requires multiple complementary approaches:

In Vitro Methods:

• Co-purification Assays: Express and purify multiple subunits together to test stable complex formation.

• Pull-down Assays: Use tagged nuoI to capture interacting partners from cell lysates.

• Surface Plasmon Resonance (SPR): Quantify binding kinetics between nuoI and partner subunits.

• Isothermal Titration Calorimetry (ITC): Determine thermodynamic parameters of subunit interactions.

• Crosslinking Mass Spectrometry: Identify specific residues involved in subunit contacts.

In Vivo Approaches:

• Bacterial Two-Hybrid System: Test binary interactions between nuoI and other subunits.

• Co-immunoprecipitation: Pull down intact complexes from cells expressing tagged subunits.

• Fluorescence Resonance Energy Transfer (FRET): Visualize proximity between fluorescently labeled subunits in living cells.

Structural Methods:

Functional Validation:

• Complementation Assays: Test if nuoI can functionally replace homologs in model organisms.

• Site-directed Mutagenesis: Target predicted interaction sites to disrupt specific subunit contacts.

• Activity Assays: Measure electron transfer activities with different subunit combinations.

Research on Complex I has successfully used these approaches to identify a "common denominator" of four subunits necessary for coupling redox activity to ion transport , providing a model for studying subunit interactions.

What are the major challenges in studying P. amoebophila proteins, and how might they be overcome?

Major Challenges and Solutions:

Experimental evidence suggests that these challenges can be overcome, as demonstrated by successful studies on P. amoebophila inclusion membrane proteins and nucleotide transporters .

How might research on P. amoebophila nuoI contribute to our understanding of bacterial symbiosis?

Research on P. amoebophila nuoI has significant potential to enhance our understanding of bacterial symbiosis:

• Metabolic Integration Models: Understanding how Complex I functions in P. amoebophila could reveal mechanisms by which metabolic activities of symbionts and hosts become integrated during evolution. This builds on findings that P. amoebophila is "intimately connected with their host cell's metabolism in a surprisingly complex manner" .

• Evolution of Symbiosis: P. amoebophila represents an ancient lineage that diverged from pathogenic chlamydiae approximately 700 million years ago . Studying conserved metabolic machinery like Complex I can reveal how ancient prokaryotic-eukaryotic symbioses were established.

• Host-Symbiont Communication: Investigation of how nuoI activity responds to host metabolic states could uncover novel signaling mechanisms between partners in symbiotic relationships.

• Energy Economics of Symbiosis: Quantitative assessment of energy production and consumption through Complex I activity might reveal how symbionts balance their own metabolic needs with contributions to host fitness.

• Transition from Pathogenesis to Symbiosis: Comparative studies of nuoI between P. amoebophila and pathogenic Chlamydiaceae could illuminate how metabolic systems adapt during the evolution from pathogenic to mutualistic lifestyles.

Experimental evolution studies demonstrate that transmission modes influence symbiont evolution, with horizontally transmitted strains showing enhanced replication rates . Similar approaches could reveal how Complex I components adapt to different symbiotic contexts.

What innovative experimental approaches might advance our understanding of nuoI function in P. amoebophila?

Several innovative approaches could significantly advance nuoI research:

Advanced Imaging Technologies:

• Cryo-electron tomography of intact P. amoebophila cells within amoeba hosts to visualize Complex I in its native environment.

• Super-resolution microscopy to track dynamic changes in Complex I distribution during different stages of the symbiont life cycle.

• Label-free imaging techniques (e.g., Raman microscopy) to monitor metabolic activity associated with Complex I function.

Systems Biology Approaches:

• Multi-omics integration (genomics, transcriptomics, proteomics, metabolomics) to model how nuoI expression correlates with global metabolic states.

• Flux analysis using stable isotope labeling to quantify electron flow through Complex I in living symbiont-host systems.

• Network analysis to position Complex I within the broader metabolic network of the symbiont-host system.

Emerging Genetic Technologies:

• CRISPR-Cas9 genome editing adapted for P. amoebophila to create nuoI mutants or tagged variants.

• Conditional expression systems to control nuoI levels in living symbionts.

• Single-cell sequencing techniques to examine heterogeneity in nuoI expression across bacterial populations within hosts.

Synthetic Biology Approaches:

• Reconstitution of minimal functional Complex I assemblies containing nuoI to define essential components.

• Creation of chimeric complexes with subunits from different bacterial sources to test functional conservation.

• Directed evolution of nuoI to identify functional constraints and adaptive potential.

Successful implementation of these approaches would build on established methods such as immunofluorescence analysis and immuno-transmission electron microscopy that have already proven valuable in studying P. amoebophila proteins .

What are the key publications that researchers should review before studying P. amoebophila nuoI?

The following publications provide essential background for studying P. amoebophila nuoI:

• Heinz et al. (2010). "Inclusion Membrane Proteins of Protochlamydia amoebophila UWE25 Reveal a Conserved Mechanism for Host Cell Interaction among the Chlamydiae." Journal of Bacteriology.

• Marreiros et al. (2012). "Study of ion translocation by respiratory complex I. A new insight using (23)Na NMR spectroscopy." Biochimica et Biophysica Acta.

• Ito et al. (2012). "Amoebal Endosymbiont Protochlamydia Induces Apoptosis to Human Immortal HEp-2 Cells." PLoS ONE.

• Zhang et al. (2010). "Creation of a Genome-Wide Metabolic Pathway Database for Populus trichocarpa Using a New Approach for Reconstruction and Curation of Metabolic Pathways for Plants." Plant Physiology.

• Spero et al. (2015). "Phylogenomic Analysis and Predicted Physiological Role of the Proton-Translocating NADH:Quinone Oxidoreductase (Complex I) Across Bacteria." mBio.

• Schmitz-Esser et al. (2006). "Novel nucleotide carrier proteins of Protochlamydia amoebophila UWE25." Journal of Bacteriology.

• Wen et al. (2016). "Recombinant expression of Chlamydia trachomatis major outer membrane protein in E. coli outer membrane as a substrate for vaccine research." BMC Microbiology.

• König et al. (2020). "Substrate availability and repeated stress alter the evolution of a mutualism continuum in a bacterial symbiont." PNAS.

• Moustafa et al. (2008). "Chlamydiae Has Contributed at Least 55 Genes to Plantae with Predominantly Plastid Functions." PLoS ONE.

What research tools and databases are most valuable for studying P. amoebophila proteins?

Researchers studying P. amoebophila proteins should utilize the following resources:

Sequence and Structure Databases:

• UniProt/Swiss-Prot: Contains annotated protein sequences, including P. amoebophila proteins

• Protein Data Bank (PDB): For structural data on homologous proteins

• InterPro: For functional domain identification

• CoralHydro: Database for hydropathy analysis useful for membrane protein studies

Genomic Resources:

• NCBI Genome Database: Complete genome sequence of P. amoebophila UWE25

• JGI Integrated Microbial Genomes (IMG): Comparative genomic analysis tools

• KEGG: For metabolic pathway mapping and analysis

Metabolic Databases:

• MetaCyc: Comprehensive database of metabolic pathways and enzymes

• BioCyc: Collection of Pathway/Genome Databases, including chlamydial species

• PlantCyc: Reference database for plant metabolic pathways, relevant for symbiont-plant interactions

Specialized Tools:

• PSORTb: For prediction of protein subcellular localization

• TMHMM/HMMTOP: For transmembrane domain prediction

• SignalP: For signal peptide prediction

• TargetP/Predotar: For organelle targeting prediction

Experimental Resources:

• Addgene: Repository for plasmid vectors useful in recombinant expression

• ATCC/DSMZ: Sources for obtaining reference strains of P. amoebophila and host amoebae

• Proteomics DB: Repository for mass spectrometry-based proteomics data

The MetaCyc database (version 21.1) contains 2,572 base pathways, 10,156 enzymes, and 2,883 referenced organisms , making it a particularly valuable resource for metabolic analysis of P. amoebophila.

How can researchers stay updated with the latest developments in this field?

Researchers can stay updated on P. amoebophila and NADH-quinone oxidoreductase research through:

Scientific Literature Monitoring:

• Set up personalized alerts on platforms like:

  • PubMed/NCBI (using search terms like "Protochlamydia amoebophila," "nuoI," "NADH-quinone oxidoreductase," "Complex I")

  • Google Scholar alerts

  • Web of Science citation alerts

  • Scopus alerts

• Follow key journals:

  • Journal of Bacteriology

  • Molecular Microbiology

  • PLOS Pathogens

  • mBio

  • FEMS Microbiology Reviews

  • Biochimica et Biophysica Acta (BBA) - Bioenergetics

Academic Networking:

• Join relevant scientific societies:

  • American Society for Microbiology (ASM)

  • European Molecular Biology Organization (EMBO)

  • International Society for Microbial Ecology (ISME)

• Attend specialized conferences:

  • Gordon Research Conferences on Microbial Metabolism

  • FEBS Advanced Lecture Courses on Mitochondria and Bioenergetics

  • International Symposium on Phototrophic Prokaryotes

Digital Resources:

• Participate in specialized research communities:

  • ResearchGate groups focused on bacterial symbiosis

  • Microbiology Stack Exchange

  • Specialized Slack or Discord communities

• Follow research institutions with active P. amoebophila research:

  • University of Vienna, Department of Microbiology and Ecosystem Science

  • Swiss Federal Institute of Technology (ETH) Zurich

  • University of British Columbia, Department of Botany

• Utilize preprint servers to access the latest research before formal publication:

  • bioRxiv

  • medRxiv