Recombinant Staphylococcus saprophyticus subsp. saprophyticus Na (+)/H (+) antiporter subunit C1 (mnhC1)
Description
Introduction to Recombinant S. saprophyticus Na(+)/H(+) Antiporter Subunit C1
The Na(+)/H(+) antiporter subunit C1, designated as mnhC1, is a membrane protein component of the Mnh complex found in Staphylococcus species. In S. saprophyticus, this protein functions as part of a multicomponent system involved in sodium ion excretion and proton uptake, contributing to the maintenance of cytoplasmic pH and ion concentration. The recombinant form of this protein consists of the full-length sequence (115 amino acids) and is typically expressed with an affinity tag, such as a histidine tag, to facilitate purification . S. saprophyticus, unlike its more notorious relative S. aureus, is primarily associated with urinary tract infections, and its adaptation to this niche environment may be partially attributed to its specialized ion transport systems, including the Na(+)/H(+) antiporter complexes .
The Na(+)/H(+) antiporter systems in S. saprophyticus are believed to contribute significantly to alkali tolerance and intracellular pH homeostasis, which are essential for bacterial survival in the urinary tract where pH and ion concentrations can fluctuate considerably . The recombinant expression of mnhC1 has facilitated detailed studies of this protein's properties, providing insights into its role in bacterial physiology and potential as a target for antimicrobial interventions.
Amino Acid Sequence and Protein Structure
The recombinant S. saprophyticus mnhC1 protein consists of 115 amino acids with the following sequence: "MEILMIFVCGILTAMSVYLILSKSLIRIIIGTTLQTHTANLFLITMGGLKKGEVPIYEKGITSYVDPIPQALILTAIVISFSVTAFFLVLAFRSYKELGTDNVESMKGVLEDDRE" . This sequence reflects the hydrophobic nature of this integral membrane protein, which contains multiple transmembrane domains that anchor it within the bacterial cell membrane.
Based on structural predictions and comparisons with related proteins, mnhC1 is classified as a multi-pass membrane protein belonging to the CPA3 antiporters (TC 2.A.63) subunit C family . While the exact three-dimensional structure of S. saprophyticus mnhC1 has not been fully determined, insights can be gained from studies of related transporters in other Staphylococcus species.
Protein Domains and Functional Regions
The mnhC1 protein contains distinct domains that contribute to its function as an ion antiporter. The hydrophobic regions form transmembrane helices that span the bacterial cell membrane, creating channels for ion passage. These transmembrane domains are interconnected by hydrophilic loops that may be involved in substrate recognition and transport regulation.
| Domain Feature | Amino Acid Position | Characteristic |
|---|---|---|
| Transmembrane regions | Multiple segments throughout the protein | Hydrophobic, alpha-helical structures |
| Substrate binding sites | Predicted within transmembrane domains | Charged or polar residues for ion coordination |
| Cytoplasmic domains | N and C termini, interconnecting loops | Involved in regulatory interactions |
This structural arrangement enables mnhC1 to participate in the controlled exchange of Na+ and H+ ions across the bacterial membrane, a process critical for maintaining cellular homeostasis.
Ion Transport Mechanism
The mnhC1 protein functions as part of the Mnh complex, which operates as a Na(+)/H(+) antiporter system. This complex facilitates the exchange of sodium ions (Na+) for protons (H+) across the cell membrane, a process driven by electrochemical gradients . This exchange mechanism is crucial for bacterial survival, particularly in environments with variable pH and high sodium concentrations.
In S. saprophyticus, which frequently colonizes the urinary tract, the Na(+)/H(+) antiporter system plays a vital role in adapting to the highly variable ion content in urine . This adaptation represents a specialized evolutionary response to the bacterium's ecological niche and contributes to its pathogenicity in urinary tract infections.
Role in pH Homeostasis and Stress Response
The Na(+)/H(+) antiporter systems, including those containing mnhC1, are particularly important for maintaining intracellular pH homeostasis through proton uptake . This function becomes especially critical when bacteria encounter alkaline conditions, as the antiporter can help acidify the cytoplasm by importing protons in exchange for sodium ions.
Additionally, these antiporter systems contribute to bacterial stress responses, including resistance to high salt concentrations and oxidative stress. The ability to regulate internal ion concentrations and pH provides S. saprophyticus with resilience against environmental challenges encountered during infection and colonization.
Recombinant Expression Systems
The recombinant S. saprophyticus mnhC1 protein is typically expressed in Escherichia coli expression systems, which offer high yield and relatively straightforward protocols . The gene encoding mnhC1 is cloned into appropriate expression vectors, often incorporating affinity tags such as histidine (His) tags to facilitate subsequent purification.
The expression conditions are carefully optimized to maximize protein yield while maintaining proper folding of this membrane protein. This may involve adjustments to growth temperature, induction parameters, and the use of specialized E. coli strains designed for membrane protein expression.
Purification and Quality Control
Purification of recombinant mnhC1 typically involves multiple chromatographic steps, beginning with affinity chromatography that leverages the incorporated His-tag . This may be followed by size exclusion chromatography or ion exchange chromatography to achieve high purity.
The purified protein is typically provided as a lyophilized powder, with purity exceeding 90% as determined by SDS-PAGE analysis . Quality control procedures ensure that the recombinant protein maintains its native structure and function, which is critical for subsequent research applications.
| Purification Step | Method | Purpose |
|---|---|---|
| Initial capture | Affinity chromatography (His-tag) | Selective binding of the tagged protein |
| Intermediate purification | Ion exchange chromatography | Removal of contaminants with different charge properties |
| Polishing | Size exclusion chromatography | Separation based on molecular size, removal of aggregates |
| Analysis | SDS-PAGE, Western blot | Confirmation of purity and identity |
Antibiotic Resistance Research
Transport proteins, including Na(+)/H(+) antiporters, can contribute to bacterial resistance to antimicrobial compounds. While mnhC1 may not directly efflux antibiotics like some specialized transporters, its role in maintaining cellular homeostasis can indirectly contribute to bacterial survival under antibiotic stress.
Research using recombinant mnhC1 can help elucidate how ion transport systems contribute to bacterial resilience and potentially identify strategies to disrupt these systems as part of antimicrobial therapies. The approach used with NorC, where inhibitory antibodies were developed to block transporter function, represents a potential model for similar interventions targeting mnhC1 .
Biotechnological Applications
The recombinant mnhC1 protein may also have applications in biotechnology, particularly in the development of biosensors for detecting specific ions or pH changes. The protein's natural function in ion transport could be harnessed to create sensitive detection systems for environmental or clinical applications.
Additionally, understanding the structure and function of ion transporters like mnhC1 contributes to the broader field of membrane protein engineering, which aims to develop novel proteins with customized transport properties for various applications in medicine and industry.
mnhC1 in Different Staphylococcus Species
The mnhC1 protein has homologs across various Staphylococcus species, with notable examples in S. aureus . Comparative analysis of these proteins reveals both conserved features essential for Na(+)/H(+) antiporter function and species-specific adaptations that may reflect different ecological niches.
For instance, the mnhC1 protein from S. aureus (strain MW2) consists of 113 amino acids and functions as part of a heterooligomeric complex comprising seven subunits: mnhA1, mnhB1, mnhC1, mnhD1, mnhE1, mnhF1, and mnhG1 . This complex structure is likely conserved in S. saprophyticus, suggesting a similar multicomponent transport system.
| Species | Protein Length | Complex Formation | Ecological Niche |
|---|---|---|---|
| S. saprophyticus | 115 amino acids | Mnh complex (predicted) | Primarily urinary tract |
| S. aureus | 113 amino acids | Heterooligomeric Mnh complex | Diverse, including skin, respiratory tract |
Relationship to Other Bacterial Transporters
The mnhC1 protein belongs to the CPA3 antiporters (TC 2.A.63) subunit C family , placing it within a broader context of bacterial ion transporters. This classification provides insights into its evolutionary relationships and functional similarities with transporters from diverse bacterial species.
While distinct from the major facilitator superfamily (MFS) transporters like NorC , mnhC1 shares the fundamental property of facilitating transmembrane movement of ions or small molecules. Understanding these relationships helps contextualize the role of mnhC1 within the complex landscape of bacterial transport systems.
Therapeutic Target Potential
Given the importance of Na(+)/H(+) antiporters for bacterial survival, particularly in specific environments like the urinary tract, mnhC1 and related proteins may represent promising targets for novel antimicrobial strategies. Future research could explore the development of inhibitors specifically targeting these transport systems.
The approach used with NorC, where single-domain camelid antibodies were developed to block the transporter , provides a potential model for similar interventions targeting mnhC1. Such strategies could complement conventional antibiotics and potentially address issues of antimicrobial resistance.
Systems Biology Approaches
Understanding the role of mnhC1 within the broader context of S. saprophyticus physiology and pathogenicity requires integrative approaches that consider multiple cellular processes simultaneously. Systems biology approaches, including transcriptomics, proteomics, and metabolomics, could provide insights into how ion transport systems interact with other cellular components and respond to environmental challenges.
These approaches could reveal previously unrecognized roles for mnhC1 and identify potential synergistic targets for antimicrobial interventions targeting S. saprophyticus infections.
Product Specs
Note: We will prioritize shipping the format we have in stock. However, if you have a specific format requirement, please indicate it during order placement. We will prepare the product according to your request.
Note: All of our proteins are shipped with standard blue ice packs. If dry ice shipping is required, please communicate with us in advance as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: ssp:SSP1825
STRING: 342451.SSP1825
Q&A
What is the structural composition of mnhC1 protein?
Recombinant Staphylococcus saprophyticus subsp. saprophyticus Na(+)/H(+) antiporter subunit C1 (mnhC1) is a relatively small membrane protein consisting of 115 amino acids (full length) . The protein's amino acid sequence is: MEILMIFVCGILTAMSVYLILSKSLIRIIIGTTLQTHTANLFLITMGGLKKGEVPIYEKG ITSYVDPIPQALILTAIVISFSVTAFFLVLAFRSYKELGTDNVESMKGVLEDDRE . Based on this sequence, mnhC1 likely contains multiple transmembrane domains characteristic of ion transport proteins. This subunit functions as part of the larger Mnh complex involved in sodium/proton exchange across the bacterial membrane, which is critical for pH homeostasis and ion balance in S. saprophyticus.
What expression systems are most effective for recombinant mnhC1 production?
E. coli expression systems have been successfully used for recombinant production of mnhC1 protein . When expressing membrane proteins like mnhC1, several methodological considerations are essential. Researchers should optimize codon usage for the expression host, consider using expression vectors with inducible promoters (such as pET systems), and evaluate different E. coli strains specialized for membrane protein expression (C41, C43, or Lemo21). The addition of an N-terminal His-tag, as demonstrated in commercial preparations, facilitates purification without significantly altering protein structure or function . For improved solubility and folding, expression at lower temperatures (16-25°C) following induction is recommended, along with the potential use of fusion partners such as MBP or SUMO.
What are the optimal storage and handling conditions for recombinant mnhC1?
The recombinant mnhC1 protein should be stored as a lyophilized powder at -20°C to -80°C upon receipt . For working stocks, aliquoting is necessary to avoid repeated freeze-thaw cycles, which can significantly degrade protein integrity. The protein can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, addition of 5-50% glycerol (final concentration) is recommended before aliquoting and storing at -20°C/-80°C . Short-term working aliquots can be maintained at 4°C for up to one week, but repeated freezing and thawing should be avoided . When preparing the protein for experimental use, brief centrifugation of the vial before opening is recommended to bring contents to the bottom of the tube.
What methodological approaches are recommended for studying mnhC1 ion transport activity?
To investigate the ion transport activity of mnhC1, researchers can employ several complementary techniques:
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Liposome Reconstitution Assays: Purified mnhC1 can be reconstituted into liposomes loaded with pH-sensitive fluorescent dyes (such as BCECF or pyranine). Ion transport can be measured by monitoring fluorescence changes upon addition of Na+ to the external medium.
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Electrophysiological Methods: Patch-clamp techniques or solid-supported membrane electrophysiology can be used to directly measure ion currents across membranes containing reconstituted mnhC1.
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Isotope Flux Assays: Using radioisotopes (22Na+ or tritiated protons) to track ion movement across membranes containing mnhC1.
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pH Gradient Dissipation Measurements: Creating artificial pH gradients across membranes and measuring their dissipation rate in the presence of mnhC1 and various ion concentrations.
For all these approaches, appropriate controls should include protein-free liposomes and liposomes containing known ion transporters with well-characterized activities.
How can site-directed mutagenesis be used to investigate mnhC1 functional domains?
Based on the amino acid sequence of mnhC1 (115 amino acids) , researchers can employ site-directed mutagenesis to systematically identify functional domains:
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Target Selection:
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Conserved charged residues likely involved in ion binding and transport
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Hydrophobic residues within predicted transmembrane domains
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Residues at the interfaces between subunits
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Mutation Design:
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Conservative substitutions (maintaining similar chemical properties) to identify essential residues
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Charge reversal mutations to probe electrostatic interactions
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Alanine scanning across putative functional domains
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Functional Analysis:
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Compare transport activity of wild-type and mutant proteins using transport assays
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Assess protein folding and stability through circular dichroism or thermal shift assays
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Evaluate membrane insertion efficiency using protease protection assays
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| Residue Position | Predicted Function | Suggested Mutations | Expected Effect |
|---|---|---|---|
| Charged residues | Ion coordination | E→Q, D→N, K→R, R→K | Reduced transport activity |
| Hydrophobic core | Membrane anchoring | I→A, L→A, V→A | Impaired membrane insertion |
| C-terminal region | Interaction interface | Truncations, single AA mutations | Disrupted complex formation |
What is the relationship between mnhC1 expression and antibiotic resistance?
While direct evidence linking mnhC1 to antibiotic resistance is not provided in the search results, Na(+)/H(+) antiporters in bacteria can influence susceptibility to antimicrobials through several mechanisms:
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pH-Dependent Antibiotic Activity: By regulating intracellular pH, mnhC1 may indirectly affect the activity of pH-dependent antibiotics.
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Membrane Potential Maintenance: Antiporters contribute to membrane potential, which influences the uptake of certain antibiotics.
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Biofilm Association: If mnhC1 contributes to biofilm formation, it may indirectly promote antibiotic tolerance, as biofilms provide physical barriers against antimicrobial agents.
To investigate these potential relationships, researchers should:
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Determine minimum inhibitory concentrations (MICs) for various antibiotics in wild-type versus mnhC1-deficient strains
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Assess antibiotic penetration and activity in biofilms formed by wild-type versus mutant strains
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Evaluate mnhC1 expression levels in response to antibiotic exposure
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Investigate potential interactions between mnhC1 and known antibiotic resistance mechanisms
What techniques are most effective for studying mnhC1 protein-protein interactions?
To investigate how mnhC1 interacts with other proteins, particularly other components of the Mnh complex, researchers can employ multiple complementary approaches:
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Co-immunoprecipitation (Co-IP): Using antibodies against the His-tag of recombinant mnhC1 to pull down protein complexes, followed by mass spectrometry to identify interaction partners.
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Bacterial Two-Hybrid (B2H) Analysis: For screening potential interaction partners in a library or validating specific interactions.
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Surface Plasmon Resonance (SPR): To quantitatively measure binding kinetics between purified mnhC1 and candidate partners.
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Cross-linking Mass Spectrometry: Chemical cross-linking followed by digestion and mass spectrometry to identify interacting regions.
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Förster Resonance Energy Transfer (FRET): For measuring interactions in live cells by tagging mnhC1 and potential partners with different fluorophores.
These approaches should be combined with bioinformatic analysis to predict interaction domains based on the 115-amino acid sequence of mnhC1 .
What are the recommended approaches for crystallizing mnhC1 for structural studies?
Membrane proteins like mnhC1 present significant challenges for crystallization, requiring specialized methodology:
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Protein Preparation:
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Crystallization Strategies:
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Lipidic cubic phase (LCP) crystallization, particularly suitable for small membrane proteins like the 115-amino acid mnhC1
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Detergent screening to identify optimal micelle properties
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Use of lipid bilayer mimetics such as nanodiscs or amphipols
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Antibody fragment co-crystallization to stabilize flexible regions
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Screening Conditions:
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Systematic variation of pH, temperature, and precipitants
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Addition of ligands or substrates to stabilize specific conformations
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Robotic high-throughput screening with specialized membrane protein crystallization kits
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| Crystallization Method | Advantages | Challenges | Applicability to mnhC1 |
|---|---|---|---|
| Vapor diffusion with detergents | Well-established protocols | Limited success with membrane proteins | Moderate |
| Lipidic cubic phase | Native-like lipid environment | Technical complexity | High |
| Bicelle method | Combines detergent and lipid properties | Limited stability | Moderate |
| Antibody co-crystallization | Stabilizes flexible regions | Requires specific antibodies | High for difficult regions |
How does mnhC1 in S. saprophyticus compare with similar antiporters in other bacterial species?
While specific comparative data for mnhC1 is not provided in the search results, a methodological approach to this question would involve:
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Sequence Alignment Analysis: Compare the 115-amino acid sequence of S. saprophyticus mnhC1 with homologous proteins in other staphylococci and more distant bacterial species. Focus on conserved residues that likely represent functional domains.
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Phylogenetic Analysis: Construct phylogenetic trees to understand the evolutionary relationships between mnhC1 and related antiporter subunits.
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Structural Modeling: Create homology models based on crystal structures of related antiporters to predict structural conservation.
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Functional Complementation: Express mnhC1 in other bacterial species with inactivated native antiporters to assess functional conservation.
This comparative approach would be particularly informative given that S. saprophyticus has been shown to acquire gene clusters from other coagulase-negative staphylococci , suggesting potential horizontal gene transfer of functional complexes like ion transporters.
What genomic context surrounds the mnhC1 gene in different S. saprophyticus isolates?
While specific information about the genomic context of mnhC1 is not provided in the search results, we can outline a methodological approach:
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Genome Sequencing and Analysis:
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Mobile Genetic Element Identification:
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Search for insertion sequences, transposons, or other mobile elements near mnhC1
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Evaluate GC content and codon usage as indicators of horizontal gene transfer
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Comparative Genomics:
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Analyze synteny of the mnhC1 region across different S. saprophyticus isolates
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Compare with related staphylococcal species to identify conserved gene neighborhoods
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This approach would determine whether mnhC1 is part of the core genome or if its presence varies between isolates, similar to the variation observed with other genes like the ica cluster in S. saprophyticus .
