Recombinant Shewanella halifaxensis Na (+)-translocating NADH-quinone reductase subunit D
Description
Introduction to Na(+)-translocating NADH-quinone reductase
Na(+)-translocating NADH-quinone reductase represents a unique class of respiratory enzymes found predominantly in marine and pathogenic bacteria. Unlike the more common H(+)-translocating NADH:quinone oxidoreductases (Complex I), the Na(+)-NQR complex utilizes sodium ions instead of protons to establish an electrochemical gradient across the bacterial cell membrane . This mechanism is particularly important for bacteria inhabiting marine environments where sodium concentrations are naturally elevated.
Shewanella halifaxensis is a gram-negative, facultatively anaerobic bacterium that possesses versatile respiratory capabilities. Like other members of the Shewanella genus, it has adapted to thrive in deep-sea environments, where the Na(+)-NQR complex contributes significantly to its bioenergetic processes. The complete Na(+)-NQR complex typically consists of six subunits (NqrA through NqrF), with NqrD being one of the integral membrane components essential for the enzyme's function .
Functional Role in Bacterial Energy Metabolism
Based on studies of Na(+)-NQR complexes from related bacterial species, particularly Vibrio cholerae, the NqrD subunit plays a crucial role in the sodium ion translocation mechanism of the enzyme complex . As an integral membrane component, NqrD likely contributes to the formation of the sodium channel through which ions are pumped from the cytoplasmic to the periplasmic side of the bacterial membrane.
The complete Na(+)-NQR complex catalyzes the oxidation of NADH and the reduction of quinones while simultaneously pumping sodium ions across the membrane. This process can be represented by the following reaction:
NADH + Q + n Na(+)in → NAD(+) + QH2 + n Na(+)out
where Q represents quinone, QH2 represents reduced quinone (quinol), and n represents the number of sodium ions transported per reaction cycle .
When reconstituted into liposomes, the Na(+)-NQR complex has been shown to generate both a sodium gradient and an electrical potential (ΔΨ) across the membrane . This capability underscores its function as a primary sodium pump, contributing to the electrochemical gradient that drives various cellular processes in the bacterial cell, including ATP synthesis, solute transport, and flagellar rotation.
Unlike some other subunits of the Na(+)-NQR complex (such as NqrB and NqrC, which contain covalently bound flavins), NqrD appears to function primarily in a structural capacity, potentially contributing to the formation of the sodium ion channel rather than directly participating in electron transfer reactions .
Expression and Purification Methods
Recombinant Shewanella halifaxensis Na(+)-translocating NADH-quinone reductase subunit D is typically produced using an E. coli expression system . The protein is expressed with an N-terminal histidine tag, which facilitates its purification via affinity chromatography. Following expression and extraction from bacterial membranes, the protein is purified to a high degree of homogeneity, with purity greater than 90% as determined by SDS-PAGE analysis .
Table 2: Production and Purification Parameters
| Parameter | Description |
|---|---|
| Expression Host | E. coli |
| Protein Tag | N-terminal His tag |
| Extraction Method | Likely detergent solubilization (based on membrane protein nature) |
| Purification Method | Affinity chromatography (His-tag) |
| Final Form | Lyophilized powder |
| Purity | >90% by SDS-PAGE |
Research on Na(+)-NQR complexes from Vibrio cholerae has demonstrated successful expression of the complete six-subunit complex, with the incorporation of a six-histidine tag on the carboxy terminus of the NqrF subunit . This approach has allowed for the purification of the entire complex by affinity chromatography in a highly active form. Similar strategies might be applicable to the Shewanella halifaxensis Na(+)-NQR complex, potentially including the NqrD subunit as part of the complete enzyme complex.
Research Applications and Significance
Recombinant Shewanella halifaxensis Na(+)-translocating NADH-quinone reductase subunit D has several important applications in scientific research:
Functional Assays
When incorporated into liposomes or other membrane models, the protein can be used to study its role in sodium transport and membrane potential generation. Studies on the Na(+)-NQR complex from Vibrio cholerae have demonstrated that the reconstituted enzyme generates both a sodium gradient and an electrical potential across liposomal membranes . Similar approaches could be applied to study the function of the Shewanella halifaxensis enzyme.
Comparative Biochemistry
The NqrD subunit from Shewanella halifaxensis can be compared with homologous proteins from other bacterial species to understand evolutionary adaptations and functional conservation. Such comparative studies can provide insights into how different bacteria have adapted their energy metabolism to various environmental conditions, particularly in marine and deep-sea habitats.
Antibacterial Drug Development
Given that Na(+)-NQR is absent in mammalian cells, this enzyme complex represents a potential target for antibacterial drugs. Studies using recombinant NqrD and other subunits can help identify compounds that specifically inhibit the Na(+)-NQR complex, potentially leading to the development of novel antibiotics against marine and pathogenic bacteria.
Future Research Directions
Future research on the Shewanella halifaxensis Na(+)-translocating NADH-quinone reductase subunit D may focus on several key areas:
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Determination of the high-resolution three-dimensional structure of the NqrD subunit and its position within the complete Na(+)-NQR complex.
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Investigation of the specific amino acid residues involved in sodium ion binding and translocation, potentially through site-directed mutagenesis studies.
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Comparative analysis of NqrD subunits from different Shewanella species to understand adaptive variations in response to different environmental conditions.
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Exploration of potential inhibitors specific to the Na(+)-NQR complex, which could lead to the development of new antibacterial agents.
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Studies on the regulation of nqrD gene expression in response to environmental factors such as salinity, temperature, and oxygen availability.
Product Specs
Note: While we will prioritize shipping the format currently in stock, we understand that you may have specific requirements. Please indicate any preferred format in your order remarks, and we will accommodate your request to the best of our ability.
Note: All our proteins are shipped with standard blue ice packs by default. If dry ice shipment is required, please inform us in advance as additional charges will apply.
Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C. Lyophilized forms typically have a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: shl:Shal_3184
STRING: 458817.Shal_3184
Q&A
What is Na(+)-translocating NADH-quinone reductase subunit D and what is its role in Shewanella halifaxensis?
Na(+)-translocating NADH-quinone reductase subunit D (NqrD) is a critical membrane-bound component of the Na(+)-translocating NADH:quinone oxidoreductase (NQR) complex in Shewanella halifaxensis. The NQR complex functions as a redox-driven sodium pump operating in the bacterial respiratory chain, catalyzing electron transfer from NADH to ubiquinone coupled with Na+ translocation across the cell membrane . NqrD specifically contributes to the membrane-bound section of the complex where it works alongside NqrE to ligate an Fe center within the membrane part of the NQR complex . This functionality is essential for the energy metabolism of S. halifaxensis, particularly in its marine environment where sodium concentration is high.
What are the optimal expression systems for producing recombinant S. halifaxensis NqrD?
The expression of recombinant S. halifaxensis NqrD presents unique challenges due to its membrane-associated nature. Based on successful approaches with similar proteins, E. coli expression systems using specialized vectors that include affinity tags (typically His-tags) have proven effective . When designing expression systems, researchers should consider:
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Expression vector selection: pET-based vectors with T7 promoters offer strong induction control
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Host strain selection: E. coli strains such as BL21(DE3) or C43(DE3), with the latter being particularly suitable for membrane proteins
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Growth conditions: Lower temperatures (16-20°C) post-induction to facilitate proper folding
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Induction parameters: Lower IPTG concentrations (0.1-0.5 mM) to prevent formation of inclusion bodies
For membrane proteins like NqrD, solubilization using appropriate detergents during extraction from the cell membrane is critical. A comparative analysis of expression efficiency across different systems should be performed to optimize yield and functional integrity .
What purification strategies yield the highest purity and activity for recombinant S. halifaxensis NqrD?
Purification of recombinant S. halifaxensis NqrD requires careful consideration of its membrane protein nature. A multi-step purification protocol typically yields the best results:
| Purification Step | Method | Buffer Composition | Comments |
|---|---|---|---|
| Cell Lysis | Sonication or French Press | Tris-HCl (50 mM, pH 8.0), NaCl (300 mM), glycerol (10%) | Inclusion of protease inhibitors critical |
| Membrane Fraction Isolation | Ultracentrifugation | Same as lysis buffer | 100,000×g for 1 hour |
| Membrane Protein Solubilization | Detergent treatment | Tris-HCl buffer with mild detergents (DDM, LDAO) | Optimization of detergent type and concentration needed |
| Affinity Chromatography | Ni-NTA for His-tagged protein | Imidazole gradient in Tris buffer with detergent | Start with low imidazole (20 mM) wash |
| Size Exclusion Chromatography | Superdex 200 | Tris buffer with reduced detergent | Removes aggregates and impurities |
The purified protein should be stored in a stabilizing buffer containing 50% glycerol at -20°C for short-term or -80°C for long-term storage . Repeated freeze-thaw cycles should be avoided, and working aliquots can be kept at 4°C for up to one week .
How can researchers effectively measure the Na+ translocation activity of recombinant S. halifaxensis NqrD?
Measuring Na+ translocation activity of recombinant NqrD requires reconstitution of the protein into proteoliposomes to recreate its native membrane environment. The following methodological approach is recommended:
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Reconstitution into liposomes using purified lipids (typically E. coli polar lipids or a defined mixture)
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Assessment of Na+ transport using:
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Na+-sensitive fluorescent dyes (e.g., SBFI)
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Radioisotope (²²Na+) uptake assays
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Ion-selective electrodes to measure Na+ concentration changes
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When studying NqrD specifically, it's important to note that full functional activity typically requires the complete NQR complex (NqrA-F). Therefore, researchers often need to co-express or reconstitute multiple subunits to observe physiologically relevant activity . Controls using specific inhibitors of the NQR complex, such as HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide) or silver ions, can help confirm the specificity of observed Na+ translocation activity.
What is the role of S. halifaxensis NqrD in bacterial adaptation to cold marine environments?
S. halifaxensis is a psychrophilic (cold-loving) marine bacterium, and its NQR complex, including the NqrD subunit, plays a crucial role in adaptation to cold marine environments . Research indicates several important adaptations:
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Sodium-based bioenergetics: The Na+-translocating NQR complex allows S. halifaxensis to utilize the high sodium content in marine environments as an energy source, which is particularly advantageous in cold conditions where proton-based energy systems may be less efficient .
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Membrane fluidity maintenance: The membrane-embedded NqrD contributes to maintaining appropriate membrane characteristics at low temperatures, with specific amino acid compositions that favor flexibility in cold environments .
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Genomic adaptations: Comparative genomic analysis shows that S. halifaxensis has evolved specific protein structures, including those in the NQR complex, with decreased alanine, proline, and arginine content (p-value <0.01) which increases protein structural flexibility at lower temperatures .
These adaptations make the NQR complex, including NqrD, particularly interesting for research on bacterial cold adaptation mechanisms and marine microbial ecology.
What experimental approaches can be used to investigate the interaction of S. halifaxensis NqrD with other subunits of the NQR complex?
Investigating the interactions between NqrD and other subunits of the NQR complex requires specialized techniques for membrane protein complex analysis:
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Co-immunoprecipitation (Co-IP): Using antibodies against tagged versions of NqrD to pull down interacting partners, followed by mass spectrometry identification.
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Cross-linking studies: Chemical cross-linkers that form covalent bonds between interacting proteins, followed by proteomic analysis to identify interaction sites.
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Förster Resonance Energy Transfer (FRET): Labeling NqrD and other subunits with fluorescent tags to measure proximity and interaction dynamics in reconstituted systems.
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Blue Native PAGE: Separation of intact membrane protein complexes under non-denaturing conditions to preserve native interactions.
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Cryo-electron microscopy: For structural determination of the entire NQR complex, revealing the position and interactions of NqrD within the assembly.
How does the genetic organization of the nqrD gene in S. halifaxensis compare to other bacteria, and what does this reveal about its evolutionary history?
The nqrD gene in S. halifaxensis (locus tag: Shal_3184) is part of the nqrABCDEF operon encoding the complete Na+-translocating NADH:quinone oxidoreductase complex . Comparative genomic analysis reveals important insights about its evolutionary history:
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Operon structure conservation: The arrangement of nqrABCDEF genes is highly conserved across multiple bacterial species, suggesting strong selective pressure to maintain this organization for proper complex assembly and function .
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Gene neighborhood analysis: In S. halifaxensis, as in many other bacteria, the nqrF gene is followed by apbE, encoding a flavin transferase necessary for covalent FMN attachment to NqrB and NqrC subunits .
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Horizontal gene transfer evidence: Genomic analysis indicates that S. halifaxensis has extensively exchanged genetic material with deep-sea bacterial genomes, suggesting the NQR complex genes may have been acquired or modified through horizontal gene transfer as part of adaptation to marine environments .
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G+C content adaptation: The nqrD gene, like other genes in psychrophilic Shewanella strains, shows decreased G+C content compared to mesophilic counterparts, representing an adaptation to increase protein structural flexibility at low temperatures .
This evolutionary analysis demonstrates how the nqrD gene has been shaped by both selective pressures related to its functional role and environmental adaptations to the cold marine lifestyle of S. halifaxensis.
What genomic approaches can be used to study the phylogenetic relationships of nqrD genes across different Shewanella species?
Several genomic and phylogenetic approaches are recommended for studying nqrD genes across Shewanella species:
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Multiple sequence alignment: Using tools like MUSCLE, MAFFT, or T-Coffee to align nqrD sequences from different Shewanella species and other bacteria.
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Phylogenetic tree construction: Employing maximum likelihood (RAxML, IQ-TREE) or Bayesian inference (MrBayes) methods to generate robust phylogenetic trees.
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Selection pressure analysis: Calculating dN/dS ratios to identify sites under positive, neutral, or purifying selection using tools like PAML or HyPhy.
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Synteny analysis: Examining the conservation of gene order around nqrD across different genomes to identify genomic rearrangements or insertion/deletion events.
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Comparative genomics: Analysis of whole-genome alignments to identify locally collinear blocks (LCBs) as demonstrated in the whole-genome alignment between S. halifaxensis and S. pealeana, which revealed 10 very large genomic segments ranging from 0.26 to 1.09 Mb conserved between these species .
These approaches collectively provide insights into the evolutionary history, selective pressures, and functional constraints acting on nqrD genes across the Shewanella genus and related bacteria.
How can S. halifaxensis NqrD be utilized in research on bacterial bioenergetics and membrane transport systems?
S. halifaxensis NqrD offers unique research opportunities for investigating bacterial bioenergetics and membrane transport systems:
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Alternative respiratory systems: The Na+-translocating NQR complex represents an alternative to the more common H+-translocating NADH:quinone oxidoreductases, providing a model system for studying diverse bioenergetic mechanisms in bacteria .
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Energy conservation in extreme environments: Research on S. halifaxensis NqrD can illuminate how bacteria optimize energy conversion in cold, high-salt environments where conventional proton-motive force generation may be challenging .
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Comparative studies of ion-motive transporters: The NQR complex allows for direct comparison with other primary ion pumps (such as F-type and V-type ATPases) regarding mechanisms of ion selectivity and energy coupling.
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Structure-function relationships: Investigation of NqrD can reveal fundamental principles about how membrane proteins facilitate ion transport across lipid bilayers, particularly in the context of coupling to electron transfer reactions.
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Microbial adaptations to environmental challenges: As part of a psychrophilic marine bacterium, NqrD research contributes to understanding microbial adaptation strategies to specific ecological niches .
By isolating and characterizing recombinant NqrD, researchers can better understand these fundamental aspects of bacterial physiology and bioenergetics.
What potential biotechnological applications exist for recombinant S. halifaxensis NqrD and the NQR complex?
Several promising biotechnological applications for recombinant S. halifaxensis NqrD and the NQR complex have emerged:
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Bioremediation technologies: S. halifaxensis can degrade hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), an explosive compound and environmental contaminant . Understanding the relationship between NQR function and RDX degradation pathways could lead to enhanced bioremediation strategies.
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Cold-active enzyme applications: As components of a psychrophilic organism, NqrD and the NQR complex represent potential sources of cold-active enzymes for industrial processes that require low-temperature operation.
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Biosensors for sodium ions: The Na+-binding properties of the NQR complex could be exploited to develop biosensors for monitoring sodium concentrations in environmental or clinical samples.
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Antimicrobial drug targets: The NQR complex influences iron metabolism in bacteria, making it a potential target for novel antibiotics, particularly against marine pathogens or related species like Vibrio cholerae .
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Protein engineering platforms: Understanding the structural adaptations that allow NqrD to function optimally at low temperatures could inform protein engineering efforts to confer cold tolerance to industrial enzymes.
These applications highlight the potential translational impact of fundamental research on S. halifaxensis NqrD and related proteins.
How does the structure and function of S. halifaxensis NqrD relate to antimicrobial resistance mechanisms in marine bacteria?
The relationship between S. halifaxensis NqrD and antimicrobial resistance involves several interconnected aspects:
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Integrative and Conjugative Elements (ICEs): S. halifaxensis harbors an ICE (designated ICE ShаJpn1) that belongs to the SXT/R391 family of ICEs (SRIs) . These elements play a role in the horizontal transfer of antibiotic resistance genes (ARGs) .
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Bioenergetic factors in resistance: The Na+-translocating NQR complex contributes to the cell's energy metabolism, which can indirectly affect antibiotic resistance by influencing the operation of energy-dependent efflux pumps.
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Gene transfer between environments: Research indicates that ICEs in S. halifaxensis share structural similarities with both clinical bacterial ICEs and marine bacterial plasmids, suggesting potential transfer routes for resistance genes between marine and clinical environments .
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"One Health" considerations: The presence of similar genetic elements in environmental bacteria like S. halifaxensis and clinical pathogens highlights the interconnection between environmental and human health concerns regarding antimicrobial resistance .
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Iron metabolism connection: Research has shown that the NQR complex influences iron metabolism in bacteria, which can affect susceptibility to certain antibiotics, making it a potential drug target .
This research area demonstrates how studying fundamental aspects of bacterial physiology, such as the NQR complex, can provide insights into broader public health challenges like antimicrobial resistance.
What are the common challenges in working with recombinant membrane proteins like S. halifaxensis NqrD, and how can researchers overcome them?
Working with membrane proteins like NqrD presents several technical challenges:
| Challenge | Manifestation | Solutions |
|---|---|---|
| Low expression yields | Minimal protein detected in expression systems | Use specialized expression hosts (C43, Lemo21); optimize codon usage; try fusion tags (MBP, SUMO); lower induction temperature |
| Protein misfolding | Formation of inclusion bodies; loss of activity | Reduce expression rate; add chemical chaperones to media; try cell-free expression systems |
| Purification difficulties | Aggregation during extraction; low purity | Optimize detergent selection and concentration; use mild solubilization conditions; employ stepwise purification |
| Stability issues | Rapid activity loss; precipitation during storage | Include stabilizing agents (glycerol, specific lipids); identify optimal detergent-to-protein ratio; optimize buffer conditions |
| Functional reconstitution | Loss of activity after purification | Reconstitute with native lipids; co-express with partner proteins; optimize proteoliposome preparation methods |
For S. halifaxensis NqrD specifically, storage in a Tris-based buffer with 50% glycerol at -20°C (or -80°C for extended periods) helps maintain stability . Furthermore, preventing repeated freeze-thaw cycles and keeping working aliquots at 4°C for up to one week can help preserve protein integrity .
What analytical methods are most effective for verifying the structural integrity and functional activity of purified recombinant S. halifaxensis NqrD?
Multiple analytical approaches should be employed to verify the structural integrity and functional activity of purified NqrD:
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Structural integrity verification:
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Circular Dichroism (CD) spectroscopy to assess secondary structure content
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Thermal stability assays (thermal shift assays or differential scanning calorimetry)
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Limited proteolysis to identify properly folded domains
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Mass spectrometry to confirm protein identity and post-translational modifications
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Functional activity assessment:
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Homogeneity and oligomeric state determination:
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Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS)
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Analytical ultracentrifugation
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Native PAGE analysis
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Negative-stain electron microscopy
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These complementary approaches provide a comprehensive assessment of protein quality and suitability for downstream structural and functional studies.
What are the most promising research directions for understanding the role of S. halifaxensis NqrD in cold adaptation and RDX degradation?
Several high-priority research directions could advance understanding of S. halifaxensis NqrD:
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Structure-function studies: Determining the high-resolution structure of NqrD within the complete NQR complex would provide insights into how this protein contributes to Na+ translocation and electron transport, particularly in cold environments. Cryo-EM approaches combined with computational modeling are promising avenues.
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Comparative genomics and proteomics: Expanding comparative studies between psychrophilic and mesophilic Shewanella species to identify specific adaptations in NqrD that facilitate function at low temperatures. This should include analysis of amino acid composition, flexibility, and hydrophobicity patterns.
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Energy coupling mechanisms: Investigating how NqrD and the NQR complex couple Na+ translocation to electron transfer, particularly under cold conditions where enzyme kinetics are generally slower. This could involve development of real-time activity assays compatible with low-temperature measurements.
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RDX degradation pathways: Exploring the potential link between the NQR complex and RDX degradation machinery, potentially through metabolic flux analysis and gene expression studies under RDX exposure conditions. S. halifaxensis is one of the first anaerobic bacteria known to degrade RDX, making this relationship particularly interesting .
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Evolutionary adaptation studies: Investigating the evolutionary history of the NQR complex in marine bacteria, focusing on how horizontal gene transfer and environmental adaptation have shaped its structure and function in different ecological niches.
These research directions would contribute significantly to both fundamental microbiology and potential biotechnological applications.
How might emerging technologies in structural biology and biophysics advance our understanding of S. halifaxensis NqrD and the NQR complex?
Emerging technologies offer exciting opportunities to deepen our understanding of NqrD:
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Cryo-electron microscopy (cryo-EM): Recent advances in single-particle cryo-EM and tomography could enable determination of the complete NQR complex structure in different functional states, revealing the dynamic conformational changes during ion translocation.
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Mass photometry: This emerging technique allows label-free visualization of membrane protein complexes and their assembly processes, potentially providing insights into how NqrD incorporates into the larger NQR complex.
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Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This approach could map conformational dynamics and solvent accessibility of different regions of NqrD under various conditions, providing insights into structural changes during function.
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Time-resolved spectroscopy: Advanced spectroscopic techniques could track electron transfer events through the NQR complex in real-time, correlating them with Na+ translocation steps.
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Artificial intelligence approaches: Machine learning algorithms could predict structure-function relationships and identify potential sites for targeted mutagenesis to understand NqrD function better.
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Nanodiscs and native-like membrane mimetics: These systems provide more physiologically relevant environments for membrane proteins compared to detergent micelles, potentially revealing functional aspects not observable in conventional systems.
Application of these technologies to the study of S. halifaxensis NqrD would significantly advance our understanding of this important membrane protein and its role in bacterial bioenergetics.
