Recombinant Sodalis glossinidius Potassium-transporting ATPase C chain (kdpC)

What is Sodalis glossinidius Potassium-transporting ATPase C chain (kdpC)?

Sodalis glossinidius Potassium-transporting ATPase C chain (kdpC) is a membrane protein encoded by the kdpC gene (SG0327) in Sodalis glossinidius, a secondary facultative symbiont found in tsetse flies (Glossina species). The protein functions as part of the potassium-transporting ATPase complex with the enzyme classification EC 3.6.3.12, also known as ATP phosphohydrolase [potassium-transporting] C chain. This complex plays a crucial role in potassium ion homeostasis within the bacterial cell. The full-length protein consists of 192 amino acids with the sequence: MMKLLRPALSVFFLLVLVTAVAYPLVVTGLAQWWFPGAAQGSLVTQDGQPCGSVLIGQTFTRAGYFQGRPSATADTPYNAPASSGSNLAVSNPALDDAVKQRVTALLQANPHADAPVPVELVTASASGLDPHISPAAALWQIPRVAEARHLPQAELRRLVDDNTTRPLLYFIGEPVVNVLKLNMALDARQKG .

How does kdpC contribute to the symbiotic relationship between Sodalis glossinidius and tsetse flies?

Sodalis glossinidius has undergone extensive reductive evolution during its transition to a symbiotic lifestyle within the tsetse fly. The maintenance of the kdpC gene suggests its importance for bacterial survival in this niche. While direct evidence linking kdpC to symbiosis is limited, potassium homeostasis is likely critical for S. glossinidius to survive in the ion-regulated environment of the tsetse. The protein may help the bacterium adapt to fluctuating potassium levels following blood meals consumed by the host. Furthermore, S. glossinidius presence correlates positively with the ability of tsetse flies to be infected by Trypanosoma brucei, the causative agent of human African trypanosomiasis . This suggests that proteins involved in basic physiological functions, such as kdpC, could indirectly impact this tripartite relationship by ensuring symbiont survival and fitness within the host.

What is the evolutionary significance of kdpC retention in the reduced genome of Sodalis glossinidius?

S. glossinidius is characterized by genomic reduction, with more than 1,500 pseudogenes and evidence of ongoing gene loss as it adapts to its symbiotic lifestyle . The retention of functional kdpC in this organism while other genes have been lost indicates strong selective pressure to maintain potassium transport functionality. A methodological approach to investigate this would include:

• Comparative genomic analysis of kdpC across multiple Sodalis species, including the free-living relative Sodalis praecaptivus

• Analysis of selection signatures on kdpC sequences

• Identification of conserved amino acid residues essential for function

• Comparison of the kdp operon structure between symbiotic and free-living Sodalis species

This evolutionary conservation suggests that potassium regulation remains critical for S. glossinidius despite its streamlined symbiotic lifestyle, unlike other metabolic functions that have been lost during adaptation to the host environment.

What are the optimal storage conditions for recombinant Sodalis glossinidius kdpC?

The recombinant Sodalis glossinidius Potassium-transporting ATPase C chain requires careful storage to maintain structural integrity and functional activity. Based on established protocols, the optimal storage conditions are:

The protein should be stored in a Tris-based buffer containing 50% glycerol that has been optimized for this specific protein . It is strongly recommended to create small working aliquots to avoid repeated freezing and thawing, as this can significantly decrease protein stability and activity. For experiments requiring multiple uses, create single-use aliquots rather than subjecting the entire stock to temperature fluctuations.

How can researchers effectively express recombinant Sodalis glossinidius kdpC?

Expression of recombinant Sodalis glossinidius kdpC presents several challenges due to its membrane-associated nature. A methodological approach would include:

• Vector selection: Choose expression vectors with tunable promoters to control expression levels, as overexpression of membrane proteins can be toxic to host cells.

• Host strain optimization: While E. coli is commonly used, the genetic manipulation of Sodalis species using bacteriophage P1-mediated transduction has been demonstrated , potentially allowing for homologous expression.

• Expression conditions: Consider the following parameters:

  • Induction temperature: Lower temperatures (16-25°C) often improve membrane protein folding

  • Induction time: Extended expression periods at lower inducer concentrations

  • Media composition: Use media containing additional potassium to stabilize the protein

• Solubilization strategy: For functional studies, gentle detergents like n-dodecyl-β-D-maltoside (DDM) or digitonin should be evaluated for optimal extraction while maintaining native conformation.

• Purification approach: Affinity chromatography using carefully positioned tags that do not interfere with protein function, followed by size exclusion chromatography to ensure homogeneity.

When expressing in S. glossinidius itself, researchers should consider using the defined medium SGM11, which has been developed specifically for this organism and provides better growth conditions than complex media alone .

What analytical techniques are most suitable for assessing kdpC activity?

Analyzing the functional activity of Potassium-transporting ATPase C chain requires techniques that can measure both binding and transport activities:

• ATPase activity assays:

  • Colorimetric phosphate release assays (Malachite green method)

  • Coupled enzyme assays with pyruvate kinase and lactate dehydrogenase

  • Radioactive ATP hydrolysis measurements

• Potassium transport measurement:

  • Fluorescent potassium indicators (PBFI, Asante Potassium Green)

  • Rubidium-86 uptake as a potassium analog

  • Patch-clamp electrophysiology in reconstituted systems

• Binding studies:

  • Isothermal titration calorimetry (ITC) to determine binding affinity

  • Surface plasmon resonance (SPR) for real-time binding kinetics

  • Microscale thermophoresis (MST) for determining binding constants

• Structural assessment:

  • Circular dichroism (CD) spectroscopy to confirm proper folding

  • Limited proteolysis to assess conformational states

  • Hydrogen-deuterium exchange mass spectrometry to map ligand-binding sites

Each approach should be optimized specifically for kdpC, taking into account its membrane protein nature and the requirement for appropriate detergent environments or membrane reconstitution systems.

How might researchers investigate the relationship between kdpC function and trypanosome establishment in the tsetse fly?

The presence of Sodalis glossinidius correlates positively with the ability of tsetse flies to be infected by Trypanosoma brucei . Investigating the potential role of kdpC in this relationship requires a multifaceted approach:

• Genetic manipulation strategies:

  • Generate kdpC knockout or knockdown strains using P1 phage transduction

  • Create point mutations in critical functional residues

  • Develop conditional expression systems to modulate kdpC levels

• Infection experiments:

  • Compare trypanosome establishment rates in tsetse flies with wild-type versus modified S. glossinidius

  • Analyze spatial distribution of bacteria in relation to trypanosomes

  • Perform time-course studies to determine critical periods where kdpC function might influence infection

• Mechanistic investigations:

  • Examine potassium levels in different tsetse tissues during trypanosome infection

  • Analyze expression changes in kdpC during different stages of trypanosome establishment

  • Investigate potential direct interactions between bacterial and parasite proteins

• Systems biology approach:

  • Metabolomic comparison of infection microenvironments with different kdpC variants

  • Transcriptomic analysis to identify coordinated responses

  • Network modeling to predict potential interaction pathways

This comprehensive research strategy would help determine whether kdpC plays a direct role in facilitating trypanosome establishment or if its contribution is limited to maintaining symbiont fitness within the host.

How do potassium ion gradients maintained by kdpC affect other aspects of Sodalis glossinidius physiology?

Potassium is the most abundant intracellular cation and affects numerous cellular processes. Research methodologies to investigate the broader impact of kdpC-maintained potassium gradients include:

• Membrane potential studies:

  • Fluorescent voltage-sensitive dye measurements

  • Patch-clamp electrophysiology

  • Computational modeling of ion movements across membranes

• Gene expression analysis:

  • RNA-seq to identify genes co-regulated with kdpC

  • ChIP-seq to identify transcription factors responding to potassium levels

  • Ribosome profiling to assess translational responses

• Stress response characterization:

  • Osmotic challenge experiments with kdpC variants

  • pH perturbation tolerance in relation to potassium transport

  • Oxidative stress response linked to potassium homeostasis

• Cellular ultrastructure analysis:

  • Electron microscopy to assess membrane integrity under potassium limitation

  • Subcellular localization of kdpC under different physiological conditions

  • Morphological changes associated with potassium transport dysfunction

This systematic approach would reveal how the seemingly specialized function of potassium transport cascades into multiple aspects of bacterial physiology, potentially explaining why this function has been preserved during reductive evolution.

How can bacteriophage P1 transduction be optimized for genetic studies of kdpC in Sodalis glossinidius?

Bacteriophage P1 has been demonstrated to infect, lysogenize, and promote transduction in Sodalis species, including S. glossinidius . Optimizing this system for kdpC manipulation requires:

• Host strain preparation:

  • Culture S. glossinidius in SGM11 defined medium to achieve optimal physiological state

  • Determine growth phase most amenable to P1 infection

  • Assess calcium requirements for efficient phage adsorption

• Phage propagation optimization:

  • Determine optimal multiplicity of infection (MOI)

  • Establish lysate preparation protocols specific for S. glossinidius

  • Develop enrichment methods for transducing particles

• Target gene engineering:

  • Design kdpC modifications with appropriate selectable markers

  • Create homology regions optimized for S. glossinidius recombination machinery

  • Develop screening strategies to identify successful transductants

• Verification methods:

  • PCR-based confirmation of genetic modifications

  • Functional assays to confirm phenotypic changes

  • Whole-genome sequencing to rule out off-target effects

The relatively slow growth of S. glossinidius compared to E. coli necessitates protocol modifications, including extended incubation times and careful optimization of selection conditions to avoid false positives.

What complementary genetic approaches can be used alongside P1 transduction for comprehensive kdpC functional studies?

While P1 transduction provides a valuable tool for genetic manipulation of S. glossinidius, a comprehensive approach would incorporate multiple complementary techniques:

• CRISPR-Cas9 genome editing:

  • Design guide RNAs specific to kdpC targets

  • Develop delivery systems compatible with S. glossinidius

  • Optimize homology-directed repair for precise modifications

• Conditional expression systems:

  • Implement tetracycline-inducible promoters

  • Develop riboswitches responsive to small molecules

  • Create temperature-sensitive variants for temporal control

• Reporter gene fusions:

  • Transcriptional fusions to monitor kdpC expression

  • Translational fusions to track protein localization

  • Split reporter systems to analyze protein-protein interactions

• Heterologous expression systems:

  • Express S. glossinidius kdpC in model organisms

  • Create chimeric proteins to isolate functional domains

  • Develop surrogate hosts for high-throughput screening

• RNA-based approaches:

  • Antisense RNA for targeted knockdown

  • CRISPR interference for transcriptional repression

  • RNA thermometers for conditional expression

This multi-faceted genetic toolbox would enable researchers to address different aspects of kdpC function with appropriate methodologies tailored to specific experimental questions.

How can researchers develop a high-throughput screening system to identify compounds that modulate kdpC activity?

Developing screens for compounds that affect kdpC function could lead to tools for manipulating symbiont-host interactions and potentially impact trypanosome infection. A methodological approach would include:

• Reporter system development:

  • Growth-based selection in potassium-limited media

  • Fluorescent reporters linked to potassium-responsive promoters

  • FRET-based sensors to detect conformational changes

• Assay miniaturization:

  • Microplate-based activity assays

  • Droplet microfluidics for single-cell analysis

  • Biosensor integration for real-time monitoring

• Compound library selection:

  • Natural product extracts from relevant ecological sources

  • Focused libraries targeting ATPases or transport proteins

  • Fragment-based approaches for novel scaffold identification

• Validation cascade:

  • Primary screens with high sensitivity

  • Secondary assays with increased specificity

  • Tertiary confirmation in physiologically relevant contexts

• Data analysis pipeline:

  • Machine learning algorithms to identify activity patterns

  • Structure-activity relationship development

  • Network pharmacology to predict off-target effects

This screening platform would enable the identification of chemical probes for kdpC research and potentially lead to compounds that could modulate the tripartite relationship between tsetse flies, S. glossinidius, and trypanosomes.

What structural features distinguish Sodalis glossinidius kdpC from homologous proteins in other bacteria?

Understanding the unique structural attributes of S. glossinidius kdpC requires comparative analysis with homologs from both free-living and symbiotic bacteria:

• Sequence-based analysis:

  • Multiple sequence alignment with homologs from diverse bacterial species

  • Identification of conserved and divergent regions

  • Evolutionary rate analysis to detect adaptively evolving sites

• Structural prediction methods:

  • Homology modeling based on available crystal structures

  • Ab initio modeling of unique regions

  • Molecular dynamics simulations to assess conformational flexibility

• Transmembrane topology analysis:

  • Hydropathy profiling and transmembrane segment prediction

  • Accessibility mapping using substituted cysteine accessibility method

  • Topology validation using reporter fusion approaches

• Functional domain identification:

  • Identification of potassium binding sites

  • Analysis of subunit interaction interfaces

  • Characterization of regulatory domains

The amino acid sequence (MMKLLRPALSVFFLLVLVTAVAYPLVVTGLAQWWFPGAAQGSLVTQDGQPCGSVLIGQTFTRAGYFQGRPSATADTPYNAPASSGSNLAVSNPALDDAVKQRVTALLQANPHADAPVPVELVTASASGLDPHISPAAALWQIPRVAEARHLPQAELRRLVDDNTTRPLLYFIGEPVVNVLKLNMALDARQKG) provides the foundation for these analyses, with particular attention to regions that may reflect adaptation to the symbiotic lifestyle.

What experimental approaches are most effective for determining the structure-function relationship of kdpC?

Elucidating the structure-function relationship of kdpC requires integrating multiple experimental techniques:

• Site-directed mutagenesis studies:

  • Alanine-scanning mutagenesis of conserved residues

  • Charge-reversal mutations at potential ion-coordinating sites

  • Introduction of reporter groups at functional interfaces

• Biophysical characterization:

  • X-ray crystallography or cryo-electron microscopy for structural determination

  • Small-angle X-ray scattering (SAXS) for solution-state conformation

  • Nuclear magnetic resonance (NMR) for dynamic regions

• Functional mapping:

  • Accessibility mapping using membrane-impermeable reagents

  • Cross-linking studies to identify interacting partners

  • Electrophysiological measurements of ion transport

• Computational integration:

  • Molecular dynamics simulations to model ion transport

  • Quantum mechanics/molecular mechanics approaches for catalytic sites

  • Elastic network models to identify allosteric communication

• In vivo validation:

  • Complementation studies with mutant variants

  • Growth phenotypes under varying potassium conditions

  • Competition assays to assess fitness effects

This comprehensive approach would connect structural features to functional roles and potentially reveal adaptations specific to the symbiotic lifestyle of S. glossinidius.

How does the interaction between kdpC and other components of the KDP transport system differ in Sodalis glossinidius compared to free-living bacteria?

The kdpC protein functions as part of the larger KDP potassium transport complex. Investigating its interactions in the context of symbiosis requires:

• Comparative genomic analysis:

  • Examine conservation of kdpA, kdpB, and kdpD genes in S. glossinidius

  • Compare operon organization with free-living relatives like S. praecaptivus

  • Analyze regulatory elements controlling expression

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify interaction partners

  • Bacterial two-hybrid assays to map interaction domains

  • Blue native PAGE to preserve native complex architecture

• Complex assembly analysis:

  • Pulse-chase experiments to track assembly kinetics

  • Subcellular fractionation to identify intermediate complexes

  • Single-molecule approaches to observe assembly in real-time

• Regulatory network mapping:

  • Determine if regulation has been simplified in the symbiont

  • Compare response to potassium limitation with free-living bacteria

  • Identify unique regulatory inputs related to the symbiotic lifestyle