Recombinant Solibacter usitatus Potassium-transporting ATPase C chain (kdpC)

What is the Recombinant Solibacter usitatus Potassium-transporting ATPase C chain (kdpC)?

The Recombinant Solibacter usitatus Potassium-transporting ATPase C chain (kdpC) is a recombinant protein derived from the bacterium Solibacter usitatus (strain Ellin6076). It functions as a crucial component of the high-affinity ATP-driven potassium transport (Kdp) system in bacteria. Specifically, kdpC is the potassium-binding and translocating subunit C of the potassium-transporting ATPase complex, with the enzyme commission number EC 3.6.3.12.

The protein is typically produced as a recombinant protein expressed in E. coli systems to enable detailed biochemical and structural studies. In its native context, kdpC acts as a catalytic chaperone that enhances the ATP-binding affinity of the ATP-hydrolyzing subunit KdpB, facilitating the coupling of ATP hydrolysis with electrogenic transport of potassium ions into the cytoplasm.

What are the basic characteristics of the recombinant kdpC protein?

The recombinant kdpC protein exhibits several defining characteristics that are important for researchers to consider when working with this molecule:

The protein is part of the larger Kdp-ATPase complex, which is essential for bacterial survival, especially under potassium-limiting conditions. This recombinant form allows researchers to study the isolated component while maintaining its functional properties.

Why is Solibacter usitatus an important organism for potassium transport studies?

Solibacter usitatus represents an important model organism for potassium transport studies for several reasons. First, it belongs to the phylum Acidobacteria, which is globally ubiquitous in soil environments . The widespread distribution of this bacterial phylum makes insights from S. usitatus potentially applicable to understanding microbial adaptation across diverse soil ecosystems.

S. usitatus has been shown to persist in various environmental conditions, adapting to changes in temperature, pH, and oxygen levels . This adaptability likely involves the regulation of ion transport mechanisms, including potassium homeostasis systems like the Kdp complex. The bacterium's genome has been fully sequenced (strain Ellin6076), providing researchers with a complete genetic framework for studying its transport proteins and their regulation .

Furthermore, S. usitatus demonstrates interesting physiological responses to environmental stressors, including changes in membrane lipid composition . These responses may be coordinated with alterations in ion transport activities, making it an excellent model for studying how potassium transport systems like kdpC function within the broader context of bacterial adaptation mechanisms.

How does the kdpC subunit interact with other components of the Kdp-ATPase complex?

The kdpC subunit functions as an integral component within the larger Kdp-ATPase complex, interacting primarily with the KdpB subunit. As the potassium-binding and translocating component, kdpC serves a specialized function in the quaternary structure of the complex. Research indicates that kdpC acts as a catalytic chaperone that enhances the ATP-binding affinity of KdpB, which is the ATP-hydrolyzing subunit.

This interaction is crucial for coupling ATP hydrolysis with the electrogenic transport of potassium ions into the bacterial cytoplasm. Structurally, kdpC contains specific binding domains that facilitate protein-protein interactions with KdpB and potentially other components of the complex. These binding domains likely undergo conformational changes during the catalytic cycle of the ATPase, coordinating the energy harvested from ATP hydrolysis with the mechanical work of ion transport.

When studying recombinant kdpC, researchers should consider that its functional properties may be influenced by the absence of its natural binding partners. Experimental approaches often require reconstitution with other Kdp-ATPase components to fully recapitulate native activity. Methodological considerations include co-expression systems, pull-down assays, and biophysical techniques such as isothermal titration calorimetry to quantify interaction parameters.

What environmental factors influence the expression and activity of kdpC in Solibacter usitatus?

While specific data on kdpC expression in Solibacter usitatus is limited in the provided search results, we can extrapolate from studies on S. usitatus growth conditions and membrane adaptations. S. usitatus demonstrates significant physiological responses to environmental variables including temperature, pH, oxygen levels, and phosphate concentration .

Temperature appears to be a significant factor influencing S. usitatus physiology, with growth observed between 15°C and 30°C. The bacterium shows distinct responses across this temperature range, with certain membrane components (such as ceramides) showing positive correlation with increasing temperatures . This suggests that membrane-associated proteins like kdpC might similarly be regulated in response to temperature.

pH also influences S. usitatus physiology, with evidence suggesting higher production of certain membrane components at lower pH values . Given that the Kdp-ATPase complex functions in maintaining ion homeostasis, its expression and activity might be particularly important under acidic conditions that create challenging osmotic gradients.

Oxygen availability represents another important variable, with S. usitatus showing cellular adaptations under suboxic conditions (5-10% O₂) . These adaptations include cellular clumping and increased production of extracellular materials, which may reflect broader physiological changes that could include altered potassium transport requirements.

Notably, phosphate concentration appears to significantly impact S. usitatus physiology, with excess phosphate (10 mM) resulting in altered lipid profiles compared to standard conditions (0.2 mM phosphate) . Since phosphate metabolism and ATP availability directly affect ATPase function, phosphate concentration might be particularly relevant for kdpC activity.

How does the recombinant kdpC protein compare structurally and functionally to native kdpC?

When working with recombinant proteins, researchers must consider potential differences from the native form. For recombinant Solibacter usitatus kdpC, several factors may influence its comparability to the native protein:

• Expression System Effects: The recombinant kdpC is typically expressed in E. coli, which may introduce subtle differences in post-translational modifications compared to expression in S. usitatus.

• Protein Folding: Without its natural binding partners (other Kdp-ATPase components), the recombinant kdpC may adopt slightly different conformational states than in the native complex.

• Purification Impact: Purification procedures, including the use of tags and harsh buffer conditions, may affect protein structure and activity.

• Reconstitution Requirements: To achieve functional activity comparable to native conditions, the recombinant kdpC typically requires reconstitution in appropriate lipid environments and/or with other complex components.

Methodologically, researchers can address these potential differences through several approaches:

• Conducting comparative structural analyses using techniques like circular dichroism or thermal shift assays to assess protein folding

• Performing functional assays under varying conditions to identify optimal activity parameters

• Using complementation studies in kdpC-deficient bacterial strains to verify functional equivalence

• Employing membrane reconstitution systems that mimic the native lipid environment of S. usitatus

The recombinant protein's reported purity of >85% as determined by SDS-PAGE suggests minimal contamination, but researchers should still verify batch-to-batch consistency through analytical methods like mass spectrometry.

What are the optimal expression and purification methods for recombinant kdpC?

Based on the available information and standard practices for membrane-associated bacterial proteins, the following methodological approach is recommended for optimal expression and purification of recombinant kdpC:

Expression System:

• Host: E. coli BL21(DE3) or similar expression strain

• Vector: pET series with T7 promoter and appropriate tags (His-tag is commonly used)

• Induction: IPTG at 0.2-1.0 mM, with induction at OD₆₀₀ of 0.6-0.8

• Temperature: Lower induction temperatures (16-25°C) often yield better folding for membrane proteins

• Duration: Extended expression period (16-24 hours) at lower temperatures

Purification Protocol:

• Cell lysis: French press or sonication in buffer containing glycerol and protease inhibitors

• Membrane fraction isolation: Differential centrifugation

• Solubilization: Mild detergents (DDM, LDAO, or Triton X-100)

• Affinity chromatography: Ni-NTA for His-tagged constructs

• Size exclusion chromatography: For final purification and buffer exchange

• Quality assessment: SDS-PAGE analysis to confirm >85% purity

Storage Conditions:

• Short-term: 4°C in buffer with detergent and glycerol

• Long-term: -20°C or -80°C with cryoprotectants

• Recommended concentration: 0.1-1.0 mg/mL in sterile water with glycerol

For functional studies, researchers should consider reconstitution into liposomes or nanodiscs to recreate the membrane environment. The choice of lipid composition may significantly impact protein activity, potentially requiring optimization based on the native lipid environment of S. usitatus.

What assays can be used to evaluate the functional activity of recombinant kdpC?

Evaluating the functional activity of recombinant kdpC requires consideration of its role in potassium transport and ATPase activity. The following assays are recommended:

ATPase Activity Assays:

• Colorimetric Phosphate Release Assay: Measures inorganic phosphate released during ATP hydrolysis using malachite green or molybdate-based detection.

• Coupled Enzyme Assay: Links ATP hydrolysis to NADH oxidation via pyruvate kinase and lactate dehydrogenase, allowing continuous spectrophotometric monitoring.

• Radioactive ATP Hydrolysis: Uses [γ-³²P]ATP to directly measure released radioactive phosphate.

Potassium Transport Assays:

• Fluorescence-Based Methods: Utilizing potassium-sensitive fluorescent dyes (e.g., PBFI) in reconstituted liposomes.

• Isotope Flux Measurements: Using ⁴²K⁺ or ⁸⁶Rb⁺ (as a K⁺ analog) to track ion movement.

• Electrode-Based Measurements: Potassium-selective electrodes to monitor changes in potassium concentration.

Binding Studies:

• Isothermal Titration Calorimetry (ITC): Measures heat changes associated with potassium binding.

• Surface Plasmon Resonance (SPR): Analyzes binding kinetics with immobilized protein.

• Microscale Thermophoresis (MST): Detects changes in thermophoretic mobility upon ligand binding.

Structural Integrity Assessments:

• Circular Dichroism (CD): Verifies proper secondary structure formation.

• Thermal Shift Assays: Evaluates protein stability under various conditions.

• Limited Proteolysis: Assesses the conformational state of the protein.

For comprehensive functional characterization, kdpC should ideally be reconstituted with other components of the Kdp-ATPase complex (particularly KdpB) to recapitulate the native activity. Researchers should consider controls including inactive mutants and varying potassium concentrations to validate assay specificity.

How can researchers optimize growth conditions for Solibacter usitatus to study native kdpC expression?

Based on information from search result , the following optimized growth conditions are recommended for Solibacter usitatus to study native kdpC expression:

Base Medium and Supplements:

• Modified DSMZ 1266 medium with the addition of 0.67 g/L yeast extract and 2.5 mM glucose

• Buffer with MES (pKa 6.15) and adjust pH with 5M NaOH to desired values

• Standard phosphate concentration of 0.2 mM, with experimental variations to 10 mM possible

Environmental Parameters:

• Temperature range: 15-30°C (optimal growth at 25-30°C)

• pH range: Test both pH 5.5 and 6.5, with evidence suggesting better growth at pH 5.5

• Oxygen conditions: Test both aerobic (21% O₂) and suboxic (5%, 10% O₂) conditions

Culture Setup:

• For aerobic conditions: 25-mL culture tubes with 10 mL of media, shaking at 250 rpm

• For suboxic conditions: 100-mL media bottles with 60 mL of media and gasket-sealed screw-cap lids, continuous gas flushing, and magnetic stirring at 625 rpm

Harvest Method:

• Collect cells in stationary phase by centrifugation (5,000 rpm for 3 min)

• Freeze-dry prior to extraction and analysis

To specifically study kdpC expression, researchers should consider implementing a potassium-limited growth condition to induce the high-affinity potassium transport system. Additionally, quantitative RT-PCR can be used to monitor gene expression levels under various growth conditions, similar to the approach described for other genes in search result .

How can recombinant kdpC be used to study bacterial potassium homeostasis mechanisms?

Recombinant kdpC offers several valuable approaches for investigating bacterial potassium homeostasis mechanisms:

1. Structure-Function Analysis:
The purified recombinant protein allows researchers to perform detailed structural analyses using techniques such as X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy. These structural insights, coupled with site-directed mutagenesis, can elucidate the molecular mechanisms of potassium binding and translocation through the kdpC subunit.

3. Comparative Biochemistry:
The kdpC protein from Solibacter usitatus can be compared with homologs from other bacterial species to identify conserved and divergent features. This comparative approach provides insights into how potassium homeostasis mechanisms have evolved across bacterial taxa and adapted to different environmental niches.

4. Stress Response Investigations:
Combining recombinant protein studies with gene expression analysis under various stress conditions (potassium limitation, osmotic stress, pH fluctuations) can reveal how bacteria regulate their potassium transport machinery in response to environmental challenges. Evidence from S. usitatus suggests that membrane components respond to environmental parameters such as temperature, pH, and oxygen availability , which may indicate parallel regulation of potassium transport systems.

5. Protein-Protein Interaction Mapping:
Techniques such as cross-linking, co-immunoprecipitation, or bacterial two-hybrid systems using recombinant kdpC can map interaction networks within the Kdp complex and potentially identify novel regulatory proteins that modulate potassium transport activity.

What potential biotechnological applications exist for recombinant kdpC?

Recombinant kdpC offers several promising biotechnological applications based on its function in potassium transport:

1. Biosensor Development:
The potassium-binding properties of kdpC could be exploited to develop biosensors for detecting potassium ions in environmental or biological samples. By coupling kdpC to appropriate signal transduction elements (fluorescent reporters, electrochemical components), researchers could create sensitive and specific potassium detection systems.

2. Agricultural Applications:
Understanding bacterial potassium transport mechanisms through kdpC research could inform strategies for improving plant-microbe interactions in soil. Given that Acidobacteria (including Solibacter usitatus) are globally ubiquitous in soil environments , insights into their potassium acquisition systems could be relevant for agricultural microbiology.

3. Bacterial Growth Control:
The essential nature of potassium transport for bacterial survival under varying environmental conditions suggests that targeting kdpC and related proteins could offer novel approaches for controlling bacterial growth. This could have applications in managing beneficial and detrimental bacterial populations in various contexts.

4. Membrane Protein Engineering:
Research on kdpC contributes to our understanding of membrane protein structure and function. This knowledge can be applied to engineer novel membrane proteins with desired properties for biotechnological applications, such as selective ion transport or sensing capabilities.

5. Drug Development Platforms:
The recombinant expression and purification system for kdpC provides a template for handling other challenging membrane proteins. This methodological approach could be valuable for expressing targets for drug discovery programs, particularly those focused on bacterial ion transport systems.

As noted in search result, the recombinant kdpC protein can be used in various biotechnological applications, including studying potassium transport mechanisms in bacteria, which can provide insights into developing novel biotechnological tools for managing bacterial growth and survival under different conditions.

How does kdpC research contribute to our understanding of bacterial adaptation to environmental stressors?

Research on the kdpC protein significantly enhances our understanding of bacterial adaptation to environmental stressors in several key ways:

1. Potassium Limitation Response:
The Kdp-ATPase system, including kdpC, represents a high-affinity potassium uptake mechanism that is typically induced under potassium-limiting conditions. By studying kdpC function and regulation, researchers gain insights into how bacteria maintain essential cellular processes when faced with nutrient limitation—a common environmental stressor in many ecological niches.

2. Osmotic Stress Adaptation:
Potassium ions play a crucial role in bacterial osmoregulation. The kdpC protein, as part of the potassium transport machinery, contributes to the cell's ability to maintain appropriate internal osmotic pressure under varying external conditions. This adaptive capability is essential for bacterial survival in environments with fluctuating osmolarity.

3. pH Homeostasis Connection:
Research on Solibacter usitatus has shown physiological responses to pH variation , which likely involves coordinated regulation of various ion transport systems, including potassium transporters. The kdpC protein may contribute to maintaining appropriate intracellular pH by influencing the electrochemical gradient across the cell membrane.

4. Integration with Membrane Adaptations:
Evidence from S. usitatus indicates that membrane components (such as ceramides) show distinct patterns of production across temperature, pH, and oxygen gradients . These membrane adaptations likely work in concert with transport proteins like kdpC to maintain cellular homeostasis. Understanding how kdpC function integrates with membrane composition changes provides a more comprehensive picture of bacterial stress response.

5. Environmental Persistence Mechanisms:
Acidobacteria, including S. usitatus, demonstrate remarkable persistence in various soil environments . The functionality of high-affinity potassium transport systems like the Kdp complex, of which kdpC is a component, likely contributes to this environmental resilience by ensuring adequate potassium acquisition under suboptimal conditions.

The research on kdpC thus provides valuable insights into the molecular mechanisms underlying bacterial adaptation and survival in changing environments, contributing to our broader understanding of microbial ecology and evolution.