Recombinant Salmonella agona Potassium-transporting ATPase C chain (kdpC)

What is Potassium-transporting ATPase C chain (kdpC) in Salmonella agona?

Potassium-transporting ATPase C chain (kdpC) is a protein component of the potassium transport system in Salmonella agona. It functions as part of the ATP phosphohydrolase potassium-transporting complex with EC number 3.6.3.12. The protein is also known as potassium-binding and translocating subunit C or potassium-translocating ATPase C chain. The gene is identified as kdpC in the Salmonella agona genome with ordered locus name SeAg_B0752. The full-length protein spans amino acids 1-194 and plays a crucial role in potassium homeostasis within the bacterial cell .

How does kdpC protein structure relate to its function?

The kdpC protein in Salmonella agona has a specific amino acid sequence (MIGLRPAFSTMLFLLLLLTGGVYPLLTTALG QWWFPWQANGSLI HKDNVIRGSA LIGQSFTAAGYFHGRPSATADTPYNPLASGGSNLAASNPELDAQIQARVAALRAANPQASSAVPVELATASASGLDNNLTPGAAAWQIPRVAA ARQLPVEQVAQLVAEYTHRPLARFLGQPVVNIVELNLALDALQGHRAK) that contains transmembrane regions indicated by the hydrophobic domains in the N-terminal portion. This structure allows kdpC to function as part of the membrane-embedded complex that transports potassium ions across the bacterial membrane. The protein works in concert with other subunits of the potassium-transporting ATPase system to maintain appropriate intracellular potassium levels, which is critical for bacterial osmotic regulation and survival .

What is the genetic conservation of kdpC across different Salmonella species?

The kdpC gene shows high conservation across different Salmonella species, with notable similarities between Salmonella agona (UniProt ID: B5EZE2) and Salmonella dublin (UniProt ID: B5FND9). Comparative analysis of the amino acid sequences reveals significant homology, suggesting evolutionary conservation of this essential potassium transport system. Both proteins contain 194 amino acids and share nearly identical sequence patterns, with only minor variations that likely represent species-specific adaptations. This high degree of conservation underscores the biological importance of the kdpC protein in potassium homeostasis across Salmonella species .

What are the optimal conditions for expressing recombinant Salmonella agona kdpC protein?

For optimal expression of recombinant Salmonella agona kdpC protein, E. coli expression systems have proven most effective. The recommended protocol involves cloning the full-length kdpC gene (spanning amino acids 1-194) into an expression vector with an N-terminal His-tag for purification purposes. Expression should be induced under controlled temperature conditions (typically 18-25°C) to minimize inclusion body formation. The protein can be successfully expressed in standard E. coli strains with yields sufficient for research applications. Post-expression, the recombinant protein is typically stored in Tris-based buffer with 50% glycerol optimized specifically for this protein to maintain stability and biological activity. This approach yields protein with purity greater than 90% as determined by SDS-PAGE analysis .

How should researchers design experiments to study kdpC function in bacterial potassium transport?

When designing experiments to study kdpC function in bacterial potassium transport, researchers should consider a multi-faceted approach:

• Genetic manipulation studies: Create kdpC knockout mutants in Salmonella agona using CRISPR-Cas9 or traditional homologous recombination techniques. Compare the potassium transport capabilities of wild-type and mutant strains under varying environmental potassium concentrations.

• Protein interaction assays: Employ co-immunoprecipitation or bacterial two-hybrid systems to identify protein-protein interactions between kdpC and other components of the potassium transport system.

• Functional assays: Measure intracellular potassium levels using atomic absorption spectroscopy or potassium-sensitive fluorescent dyes in both wild-type and kdpC-modified strains.

• Structural biology approaches: Utilize X-ray crystallography or cryo-electron microscopy to determine the three-dimensional structure of kdpC alone and in complex with other subunits.

• Environmental response studies: Examine kdpC expression levels under various stress conditions (osmotic stress, pH changes) using quantitative PCR or Western blot analysis.

What reconstitution methods are most effective for lyophilized recombinant kdpC protein?

For optimal reconstitution of lyophilized recombinant kdpC protein, the following methodology is recommended: First, centrifuge the vial briefly to collect all material at the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, add glycerol to a final concentration of 5-50% (with 50% being optimal for maximum stability). Following reconstitution, create working aliquots to avoid repeated freeze-thaw cycles, as this can significantly degrade protein integrity and function. Store working aliquots at 4°C for up to one week, while maintaining long-term stocks at -20°C or preferably -80°C. This reconstitution approach preserves the structural integrity and functional properties of the kdpC protein for experimental applications .

How can researchers distinguish between true protein function and experimental artifacts when studying recombinant kdpC?

To distinguish between true protein function and experimental artifacts when studying recombinant kdpC, researchers should implement a comprehensive validation strategy:

• Multiple expression systems: Compare results from different expression systems (e.g., E. coli, cell-free systems) to identify system-specific artifacts.

• Tag influence assessment: Test both tagged and untagged versions of the protein to determine if the purification tag affects function.

• Native vs. recombinant comparison: When possible, compare the activity of recombinant kdpC with the native protein isolated from Salmonella agona.

• Negative controls: Include appropriate negative controls such as heat-inactivated protein or structurally similar but functionally distinct proteins.

• Dose-response relationships: Establish clear dose-response relationships to validate specific biological activities.

• Multiple methodological approaches: Confirm findings using orthogonal experimental techniques that measure the same parameter through different mechanisms.

• Statistical validation: Apply rigorous statistical analysis to distinguish signal from noise in experimental data.

By implementing these strategies, researchers can more confidently attribute observed effects to genuine kdpC function rather than experimental artifacts .

What statistical approaches are most appropriate for analyzing kdpC expression data across different experimental conditions?

When analyzing kdpC expression data across different experimental conditions, several statistical approaches are recommended:

• For parametric data with normal distribution:

  • ANOVA followed by post-hoc tests (such as Tukey's HSD) for comparing multiple conditions

  • Student's t-test for pairwise comparisons when only two conditions are being evaluated

  • Linear regression for examining relationships between continuous variables

• For non-parametric or non-normally distributed data:

  • Kruskal-Wallis test followed by Mann-Whitney U tests with Bonferroni correction

  • Wilcoxon signed-rank test for paired samples

• For time-course experiments:

  • Repeated measures ANOVA or mixed-effects models

  • Time series analysis for identifying patterns over time

• For complex experimental designs:

  • Factorial ANOVA to analyze the effects of multiple factors simultaneously

  • MANOVA when multiple dependent variables are being measured

• For validation:

  • Coefficient of association kappa for determining agreement between different detection methods, as demonstrated in Salmonella research where values between 0.61 and 0.8 indicate substantial agreement between isolation and PCR detection methods .

Appropriate statistical power calculations should be performed prior to experimentation to ensure sufficient sample sizes for detecting biologically relevant differences .

How do researchers interpret contradictory results between functional assays and structural predictions for kdpC?

When faced with contradictory results between functional assays and structural predictions for kdpC, researchers should follow this systematic approach to resolve discrepancies:

• Re-evaluate experimental conditions: Assess whether the functional assays were conducted under conditions that might affect protein conformation or activity, such as non-physiological pH, temperature, or salt concentrations.

• Consider protein dynamics: Static structural predictions may not capture the dynamic nature of protein function. Molecular dynamics simulations can bridge this gap by modeling protein flexibility and conformational changes.

• Examine post-translational modifications: The recombinant protein may lack critical post-translational modifications present in the native environment, affecting function without altering primary structure.

• Assess protein-protein interactions: kdpC functions as part of a complex; isolated protein behavior may differ from its activity within the complete potassium transport system.

• Validate structural predictions: Use multiple structural prediction algorithms and compare results. Consider experimental validation through limited proteolysis, circular dichroism, or structural studies.

• Integrated analysis approach: Develop a model that integrates both functional and structural data, identifying conditions under which each set of observations holds true.

• Investigate species-specific variations: Compare results with kdpC from related Salmonella species to determine if observed discrepancies are species-specific or universal to the protein family .

How can recombinant kdpC be utilized in developing detection methods for Salmonella agona contamination?

Recombinant kdpC protein can be leveraged for developing highly specific detection methods for Salmonella agona contamination through several advanced approaches:

• Antibody-based detection systems: Purified recombinant kdpC can be used to generate highly specific monoclonal or polyclonal antibodies. These antibodies can then be incorporated into ELISA, lateral flow immunoassays, or immunomagnetic separation techniques for detecting Salmonella agona in environmental or food samples.

• Aptamer development: Recombinant kdpC can serve as a target for selecting DNA or RNA aptamers with high affinity and specificity, which can be incorporated into biosensor platforms.

• PCR target validation: The kdpC gene sequence can be used to design PCR primers for specific detection of Salmonella agona. The recombinant protein expression system provides positive control material to validate the specificity and sensitivity of PCR assays.

• Mass spectrometry signatures: Purified recombinant kdpC can establish specific peptide signatures for targeted mass spectrometry detection methods, enabling strain-specific identification in complex samples.

• Biosensor development: The protein can be incorporated into electrochemical or optical biosensors for rapid pathogen detection in field settings.

This comprehensive approach has significant applications for food safety monitoring, particularly in cereal production facilities where Salmonella Agona outbreaks have occurred, such as the linked 1998 and 2008 outbreaks that affected hundreds of patients through contaminated cereal products .

What role does kdpC play in Salmonella agona virulence and host-pathogen interactions?

The role of kdpC in Salmonella agona virulence and host-pathogen interactions is multifaceted:

• Potassium homeostasis during infection: As a component of the potassium transport system, kdpC helps maintain appropriate intracellular potassium levels during infection, critical for bacterial survival within the changing ionic environments of the host.

• Adaptation to host immune defenses: The kdpC-containing potassium transport system may be involved in adaptation to osmotic stress encountered during host immune responses, particularly within phagocytic cells.

• Contribution to persistence: The potassium transport system may play a role in bacterial persistence within host tissues and environmental reservoirs, as evidenced by the ability of Salmonella Agona to persist in food processing facilities for extended periods (up to 10 years between outbreaks) .

• Potential involvement in transmission: Research on airborne transmission of Salmonella Agona has demonstrated that the pathogen can spread through airborne routes, suggesting that systems maintaining bacterial viability under environmental stress, potentially including the kdpC system, may contribute to transmission dynamics .

• Association with outbreak strains: Genomic analysis of outbreak strains from the 1998 and 2008 Salmonella Agona outbreaks revealed very few SNP differences (mean of eight) between these temporally separated events, suggesting high genetic stability in virulence and survival mechanisms, potentially including the kdpC system .

Understanding these roles can inform intervention strategies targeting bacterial persistence and transmission in both clinical and food production settings .

How can evolutionary analysis of kdpC sequences contribute to understanding Salmonella agona pathogenesis?

Evolutionary analysis of kdpC sequences can provide significant insights into Salmonella agona pathogenesis through several approaches:

• Phylogenetic relationships: Comparing kdpC sequences across Salmonella species and strains can establish evolutionary relationships that correlate with virulence patterns. The high conservation observed between Salmonella agona (B5EZE2) and Salmonella dublin (B5FND9) kdpC sequences suggests evolutionary preservation of this critical function .

• Selection pressure analysis: Calculating the ratio of non-synonymous to synonymous mutations (dN/dS) in kdpC can identify regions under positive selection, potentially indicating adaptation to host environments or immune pressures.

• Horizontal gene transfer assessment: Analyzing GC content, codon usage bias, and flanking genetic elements can reveal whether kdpC has been horizontally transferred between bacterial species, contributing to virulence acquisition.

• Molecular clock analysis: Dating genetic changes in kdpC can correlate evolutionary events with changes in pathogenesis, similar to how researchers identified direct descendance of 2008 outbreak strains from 1998 outbreak strains with only a mean of eight SNP differences .

• Structure-function correlations: Mapping sequence variations onto protein structural models can identify critical functional domains that remain conserved despite other sequence changes, highlighting essential components for pathogenesis.

This evolutionary perspective can identify potential targets for intervention strategies and predict emerging virulence patterns in Salmonella agona, as demonstrated by the research on temporal dynamics between the 1998 and 2008 outbreaks that identified persistence mechanisms rather than independent contamination events .

What are common pitfalls in purification of recombinant kdpC and how can they be addressed?

Common pitfalls in purification of recombinant kdpC and their solutions include:

• Poor solubility:

  • Problem: kdpC's membrane-associated domains can cause aggregation during expression.

  • Solution: Optimize expression conditions by reducing induction temperature (18-20°C), using milder induction agents, or adding solubility-enhancing fusion tags (SUMO, MBP, or TRX) in addition to the His-tag.

• Low yield:

  • Problem: Inadequate expression levels in standard systems.

  • Solution: Test different E. coli strains (e.g., BL21(DE3), Rosetta for rare codons), optimize codon usage for E. coli, or use auto-induction media to enhance expression yields.

• Protein degradation:

  • Problem: Proteolytic breakdown during purification.

  • Solution: Include protease inhibitors throughout purification, work at reduced temperatures (4°C), and minimize processing time. Consider adding 6% Trehalose to stabilization buffers as used for Salmonella dublin kdpC .

• His-tag accessibility issues:

  • Problem: Buried or inaccessible His-tag reducing binding to purification media.

  • Solution: Test both N-terminal and C-terminal tagging strategies or include longer linker regions between the tag and protein.

• Protein inactivation during storage:

  • Problem: Loss of functional activity during storage.

  • Solution: Store in optimized buffer conditions with 50% glycerol at -80°C and avoid repeated freeze-thaw cycles by creating single-use aliquots .

• Protein aggregation post-purification:

  • Problem: Formation of aggregates in purified samples.

  • Solution: Add stabilizing agents like glycerol (5-50%) and optimize buffer composition with appropriate pH (typically pH 8.0 for kdpC) .

Implementing these strategies can significantly improve the yield and quality of purified recombinant kdpC protein for research applications.

How can researchers optimize activity assays for recombinant kdpC to ensure physiologically relevant results?

To optimize activity assays for recombinant kdpC and ensure physiologically relevant results, researchers should implement the following methodological strategies:

• Buffer composition optimization:

  • Utilize buffers that mimic physiological conditions of Salmonella (pH 6.5-7.5)

  • Include physiologically relevant potassium concentrations (typically 100-300 mM)

  • Incorporate essential cofactors such as magnesium and ATP for proper protein function

• Reconstitution into membrane systems:

  • As kdpC is part of a membrane complex, reconstitute the protein in liposomes or nanodiscs to provide a membrane-like environment

  • Co-express or co-reconstitute with other components of the potassium transport system (kdpA, kdpB) to form functional complexes

• Temperature and incubation parameters:

  • Conduct assays at temperatures relevant to Salmonella physiology (30-37°C)

  • Optimize reaction time courses to capture kinetic parameters accurately

• Measurement techniques:

  • Implement direct measurement of potassium transport using radioactive tracers (^86Rb+ as a K+ analog) or potassium-selective electrodes

  • Use fluorescent probes sensitive to membrane potential as indirect measures of transport activity

• Validation approaches:

  • Verify activity using multiple complementary assay systems

  • Include appropriate positive controls (native potassium transport systems) and negative controls (heat-inactivated protein)

  • Correlate in vitro findings with in vivo observations in bacterial systems

• Data normalization:

  • Normalize activity measurements to protein concentration determined by precise methods (e.g., BCA assay)

  • Account for the fraction of properly folded active protein versus inactive or aggregated protein

These optimization strategies ensure that measurements of kdpC activity reflect its true biological function within the Salmonella potassium transport system rather than artifacts of the experimental system .

What strategies can researchers employ to improve the stability of recombinant kdpC during long-term storage?

To improve the stability of recombinant kdpC during long-term storage, researchers should implement the following evidence-based strategies:

• Optimal buffer formulation:

  • Use Tris/PBS-based buffer systems with pH 8.0, which has been verified as effective for kdpC stability

  • Incorporate 6% Trehalose as a cryoprotectant and stabilizing agent, as successfully used for Salmonella dublin kdpC

  • Add 50% glycerol as the optimal final concentration for long-term preservation

• Storage temperature optimization:

  • Store primary stocks at -80°C for maximum stability

  • Maintain working aliquots at -20°C for intermediate storage periods

  • Limit 4°C storage to no more than one week for active working solutions

• Physical state considerations:

  • Lyophilization can be effective for long-term storage if performed with appropriate cryoprotectants

  • When reconstituting lyophilized protein, centrifuge the vial briefly to collect all material at the bottom before adding diluent

• Aliquoting strategy:

  • Create single-use aliquots immediately after purification to eliminate repeated freeze-thaw cycles

  • Use small volume aliquots (50-100 μL) to minimize waste and prevent unnecessary freeze-thaw cycles

• Protein concentration effects:

  • Maintain protein concentration between 0.1-1.0 mg/mL during storage, which balances stability with functional concentration

  • For very dilute solutions, consider adding carrier proteins (e.g., BSA) to prevent adsorption to storage vessel surfaces

• Quality control monitoring:

  • Implement regular stability testing through activity assays and structural integrity checks

  • Document batch-to-batch variation and stability profiles to establish optimal storage duration limits

Implementation of these strategies has been shown to maintain kdpC stability and functionality for extended periods, enabling reliable experimental results across longitudinal research projects .

How can comparative genomics of kdpC across Salmonella strains inform epidemiological investigations?

Comparative genomics of kdpC across Salmonella strains provides valuable insights for epidemiological investigations through several methodological approaches:

• SNP analysis for outbreak tracking: Single Nucleotide Polymorphism (SNP) analysis of kdpC and surrounding genomic regions can differentiate closely related Salmonella Agona isolates. This approach successfully distinguished between isolates from the 1998 and 2008 cereal-associated outbreaks with only a mean of eight SNP differences, demonstrating high resolution for epidemiological tracking .

• Evolutionary relationship determination: Phylogenetic analysis of kdpC sequences can establish relationships between isolates from different sources or timepoints, as demonstrated in tracking Salmonella Agona persistence in food processing facilities over a 10-year period .

• Source attribution: Comparing kdpC sequences from clinical, food, and environmental isolates can help identify contamination sources and transmission routes. This approach could complement experimental evidence of airborne transmission pathways, as documented in Salmonella Agona research .

• Persistence marker identification: Specific genetic signatures in kdpC may serve as markers for strains with enhanced environmental persistence capabilities, helping identify high-risk variants in food production environments .

• Integration with whole genome sequence data: Analyzing kdpC in context with other genomic elements provides comprehensive molecular epidemiology insights, as demonstrated in the investigation that determined the 2008 outbreak strain was a direct descendant of the 1998 outbreak strain rather than representing an independent contamination event .

This approach can significantly enhance the precision of epidemiological investigations, particularly in cases of recurring contamination or persistent environmental reservoirs of Salmonella Agona .

What potential exists for using kdpC as a target for novel antimicrobial development?

The potential for using kdpC as a target for novel antimicrobial development is substantial, based on several key characteristics:

• Essential cellular function: As a component of the potassium transport system, kdpC contributes to maintaining critical potassium homeostasis in bacterial cells. Disruption of this system could compromise bacterial survival under various environmental conditions.

• Structural uniqueness: The specific structure of Salmonella kdpC differs significantly from human potassium transporters, providing an opportunity for selective targeting that minimizes effects on host cells. The detailed amino acid sequence available (MIGLRPAFSTMLFLLLLTGGVYPLLTTALG QWWFPWQANGSLIHKDNVIRGSA LIGQSFTAAGYFHGRPSATADTPYNPLASGGSNLAASNPELDAQIQARVAALRAANPQASSAVPVELATASASGLDNNLTPGAAAWQIPRVAAARQLPVEQVAQLVAEYTHRPLARFLGQPVVNIVELNLALDALQGHRAK) can inform structure-based drug design approaches .

• Conserved nature: The high conservation of kdpC across Salmonella species suggests that targeting this protein could provide broad-spectrum activity against multiple Salmonella serovars, including both Salmonella agona and Salmonella dublin .

• Bacterial persistence: Evidence from outbreak investigations indicates that Salmonella Agona can persist in food processing environments for extended periods (up to 10 years), suggesting that targeting persistence mechanisms, potentially including potassium regulation systems, could be particularly valuable for preventing recurring contamination events .

• Methodological approaches:

  • Structure-based computational screening to identify small molecule inhibitors

  • Peptide mimetics that disrupt kdpC interactions with other components of the potassium transport system

  • CRISPR-Cas9 or antisense RNA approaches to selectively suppress kdpC expression

These characteristics position kdpC as a promising target for novel antimicrobial strategies that could address both acute infection and environmental persistence of Salmonella Agona .

How might research on kdpC contribute to understanding long-term bacterial persistence in food processing environments?

Research on kdpC can significantly contribute to understanding long-term bacterial persistence in food processing environments through several research pathways:

• Stress response mechanisms: Investigating how kdpC expression and function changes under various environmental stresses common in food processing facilities (osmotic stress, desiccation, temperature fluctuations) can reveal adaptation mechanisms. The potassium transport system is particularly relevant as potassium homeostasis is critical for bacterial adaptation to changing osmotic conditions .

• Biofilm formation correlation: Examining the relationship between kdpC activity and biofilm formation capabilities could identify molecular mechanisms underlying the persistent colonization of surfaces. Recombinant kdpC protein studies can help elucidate the protein's role in the transition between planktonic and biofilm states .

• Genetic stability analysis: The remarkable genetic stability observed in Salmonella Agona outbreak strains separated by 10 years (with only a mean of eight SNP differences) suggests strong selective pressure to maintain certain functions, potentially including the kdpC system. This stability may contribute to long-term persistence capabilities .

• Environmental adaptation signatures: Comparing kdpC sequences and expression patterns between environmental isolates with demonstrated persistence (such as those from the 1998/2008 cereal facility outbreaks) and acute clinical isolates may reveal adaptation signatures specific to environmental persistence .

• Transmission dynamics modeling: Integration of kdpC functional studies with experimental transmission data, such as the demonstrated capability for airborne transmission of Salmonella Agona, can improve models of pathogen persistence and spread within food production environments .

These research approaches could significantly advance our understanding of how Salmonella Agona persists in food processing environments, informing more effective control strategies to prevent recurring contamination events like those documented in cereal production facilities .

Comparative Properties of Recombinant kdpC from Different Salmonella Species

Experimental Detection Methods for Salmonella Agona in Research