Recombinant Agrobacterium vitis Potassium-transporting ATPase C chain (kdpC)

What is the KdpFABC complex and what role does KdpC play within it?

The KdpFABC complex is a unique chimeric K+ uptake system found in bacteria including Agrobacterium vitis. Unlike typical P-type ATPases, KdpFABC combines features of an active pump with the high selectivity of an ion channel. The complex consists of four subunits: KdpF, KdpA, KdpB, and KdpC.

While ATP hydrolysis is accomplished by the P-type ATPase subunit KdpB, KdpC plays a crucial regulatory role . Based on structural analyses, KdpC appears to function similar to β subunits found in Na+/K+ ATPase and gastric H+ ATPase, likely enhancing K+ affinity of the complex . KdpC's position near the selectivity filter suggests it contributes to the complex's high potassium specificity and affinity.

How is the kdpC gene identified and isolated from Agrobacterium vitis?

Identification and isolation of the kdpC gene from A. vitis typically involves:

• Genome mining: Utilizing the complete genome sequences of A. vitis strains (such as S4) which have been sequenced and annotated .

• PCR amplification: Designing primers based on conserved regions of kdpC sequences from related species or strains.

• Sequence verification: Confirming the isolated gene through sequencing and comparison with databases.

For molecular detection and quantification, researchers can adapt methods similar to those used for other A. vitis genes. For instance, droplet digital PCR (ddPCR) has been successfully used to detect and quantify A. vitis by targeting genes like virA, pehA, and virD2 . Similar techniques can be applied to kdpC with specific primers.

The optimal PCR conditions for A. vitis gene amplification typically involve annealing temperatures around 59-60°C, as demonstrated for related genes . DNA isolation from bacterial cultures or plant tissues infected with A. vitis requires optimized protocols that ensure high purity for downstream applications.

What expression systems are most effective for producing recombinant KdpC protein?

For recombinant expression of KdpC from A. vitis, several expression systems have been employed with varying degrees of success:

• E. coli expression systems: The most common approach uses BL21(DE3) or similar strains with pET vector systems under IPTG induction. This system is advantageous for initial characterization but may face challenges with membrane protein folding.

• Homologous expression: Expression in other Agrobacterium strains can provide more native-like post-translational modifications.

• Cell-free expression systems: Useful for toxic proteins or those that affect host cell viability.

Expression optimization typically involves:

• Temperature modulation (often lowered to 16-18°C for membrane proteins)

• Induction strength variation (IPTG concentration)

• Addition of specific membrane mimetics when required

• Fusion tags (His6, MBP, GST) selection for purification and solubility enhancement

Purification typically employs affinity chromatography followed by size exclusion or ion exchange steps. For membrane-associated proteins like KdpC, detergent selection during extraction and purification is critical for maintaining native structure and function.

How does the structure of KdpC contribute to the unique transport mechanism of the KdpFABC complex?

Recent cryo-EM structures of the KdpFABC complex at 3.7Å and 4.0Å resolution have provided insights into how this unique complex functions . These structures revealed the complex in both E1 and E2 states of the transport cycle.

KdpC's position near the selectivity filter in KdpA appears to be crucial for potassium transport. Structural data suggests that KdpC remains relatively static during conformational changes associated with the transport cycle, indicating its role may be in stabilizing the complex and enhancing potassium affinity rather than directly participating in the conformational changes .

The transport mechanism appears to involve two half-channels along KdpA and KdpB, uniting features of P-type ATPases with ion channels. KdpC likely enhances this process by:

• Stabilizing the selectivity filter in KdpA

• Maintaining proper positioning of the channel elements

• Contributing to the high affinity and selectivity for K+ ions

This mechanism explains how KdpFABC can pump potassium ions against concentration gradients as high as 10^4 , making it particularly valuable in low-potassium environments.

What methodological approaches are most effective for studying the interaction between KdpC and other subunits?

For studying KdpC interactions with other KdpFABC subunits, researchers employ multiple complementary approaches:

• Cryo-EM analysis: Currently the leading method for structural characterization of the entire complex, having yielded structures at 3.7Å resolution . This technique preserves the complex in a near-native state.

• Cross-linking studies: Chemical cross-linking followed by mass spectrometry can identify interaction interfaces between KdpC and other subunits.

• FRET (Förster Resonance Energy Transfer): By tagging KdpC and potential interaction partners with fluorophores, researchers can monitor real-time interactions in living cells.

• Yeast two-hybrid screening: Though this has limitations for membrane proteins, modified systems can be employed to identify protein interactions, similar to approaches used for studying Agrobacterium virulence proteins .

• Co-immunoprecipitation: Using antibodies against KdpC to pull down the entire complex and identify interacting partners.

The most informative approach combines structural data with functional assays following targeted mutations at predicted interaction interfaces. This provides both static structural information and dynamic functional insights.

How does environmental stress affect expression and function of the KdpC subunit in A. vitis?

Environmental stress significantly impacts KdpC expression and function in A. vitis, particularly as this bacterium must maintain potassium homeostasis under varied conditions, including the acidic plant wound environment .

Research methodologies to study these effects include:

• qRT-PCR analysis: Monitoring kdpC gene expression under different stress conditions (pH, osmotic stress, potassium limitation).

• Reporter gene fusions: Linking the kdpC promoter to reporter genes like GFP or luciferase to visualize expression patterns.

• Proteomics: Using mass spectrometry to quantify protein levels under different conditions.

Similar to the virG gene in Agrobacterium, which has an acid-inducible promoter , the kdpC gene may also respond to environmental pH changes. This is particularly relevant as the plant wound environment in which Agrobacterium functions is typically acidic, with pH affecting various virulence and homeostasis systems .

What are the structural and functional differences between KdpC in A. vitis and homologous proteins in other bacterial species?

The KdpC subunit shows structural conservation across bacterial species, yet displays species-specific adaptations that may reflect environmental niches and physiological requirements:

Comparative analysis reveals:

• Sequence conservation: Core functional domains show high conservation, while peripheral regions display greater variability.

• Size variations: KdpC proteins range from approximately 190-230 amino acids across species, with A. vitis KdpC being closer to the larger end of this spectrum.

• Functional adaptation: While the general role in enhancing K+ affinity appears conserved , species-specific interactions with other Kdp subunits may vary.

• Regulatory elements: Promoter regions and transcriptional control mechanisms show greater variation, reflecting different environmental response patterns.

Research methodologies for these comparative analyses typically include:

• Multiple sequence alignments

• Homology modeling

• Heterologous expression and complementation studies

• Evolutionary rate analysis of conserved domains

The evolutionary pattern suggests that while the KdpFABC complex as a whole represents a chimeric system combining features of channels and pumps , the KdpC subunit has likely co-evolved to optimize this unique transport mechanism for specific bacterial lifestyles.

How can structural knowledge of KdpC be applied to develop anti-Agrobacterium strategies for plant disease control?

Understanding KdpC structure and function provides potential targets for controlling A. vitis infections in grapevines. Crown gall disease causes significant economic losses, estimated at US$46,500 per 0.4-ha vineyard over a 6-year period , making new control strategies valuable.

Potential research approaches include:

• Structure-based inhibitor design: Using the resolved structures of KdpFABC to identify small molecules that specifically bind to KdpC or its interaction interfaces.

• Peptide mimetics: Designing peptides that mimic KdpC interaction regions and disrupt complex assembly.

• Genetic approaches: Developing plant-expressed RNA interference constructs targeting kdpC expression in the pathogen.

Methodological considerations:

• Virtual screening against the KdpC structure to identify candidate inhibitors

• Fragment-based drug design focusing on critical functional regions

• In vitro transport assays to validate inhibitor efficacy

• Plant infection models to test efficacy in reducing A. vitis virulence

This approach leverages the critical nature of potassium homeostasis for bacterial survival, particularly in the competitive plant wound environment. By targeting KdpC, which appears crucial for the high-affinity potassium uptake , it may be possible to reduce bacterial survival under the low-potassium conditions often encountered during infection.

What are the optimal conditions for expressing and purifying recombinant KdpC for structural studies?

Optimizing expression and purification of recombinant KdpC requires careful consideration of multiple parameters:

Expression system optimization:

• Vector selection: pET-based vectors with tightly controlled induction are preferred for toxic membrane proteins.

• Host strain: C41(DE3) or C43(DE3) E. coli strains, derivatives of BL21(DE3) specifically developed for membrane protein expression, often yield better results than standard strains.

• Expression conditions:

  • Temperature: 18-20°C after induction

  • IPTG concentration: 0.1-0.5 mM

  • Growth media: Terrific Broth supplemented with 1% glucose

  • Induction time: 16-20 hours

Purification protocol:

• Cell lysis: Gentle methods such as enzymatic lysis with lysozyme followed by mild sonication.

• Detergent selection: Critical for membrane protein purification

  • Initial screening: DDM, LMNG, and C12E8 are good starting points

  • Stability assessment: Thermal stability assays with various detergents

• Chromatography sequence:

  • IMAC (Immobilized Metal Affinity Chromatography) using His-tag

  • Ion exchange chromatography

  • Size exclusion chromatography for final polishing

• Buffer optimization:

  • pH 7.5-8.0

  • 150-300 mM NaCl

  • 5-10% glycerol for stability

  • 1-5 mM DTT or TCEP as reducing agent

For structural studies, protein purity >95% is typically required, with yields ranging from 0.5-5 mg per liter of culture depending on optimization. Assessment of proper folding using circular dichroism spectroscopy is recommended before proceeding to structural studies.

How can researchers effectively study the kinetics of K+ transport mediated by the KdpFABC complex?

Studying the kinetics of potassium transport through the KdpFABC complex requires specialized methodologies that can capture the unique features of this high-affinity transport system, which can function against concentration gradients as high as 10^4 .

Experimental approaches:

• Radioisotope flux assays: Using 42K+ or 86Rb+ (as K+ analog) to directly measure transport rates.

• K+-selective electrodes: For real-time monitoring of K+ concentrations.

• Fluorescence-based assays: Using K+-sensitive fluorophores like PBFI or Asante Potassium Green.

• Reconstitution in liposomes: Purified KdpFABC complex reconstituted in proteoliposomes allows precise control of internal and external environments.

• Patch-clamp electrophysiology: For detailed analysis of transport dynamics.

Kinetic parameters to measure:

The alternating access mechanism proposed for KdpFABC, which differs from classical P-type ATPases by having reversed E1/E2 states , requires careful experimental design to properly characterize. Researchers must account for this unique mechanism when designing kinetic studies and interpreting results.

What approaches can be used to investigate the role of KdpC in the plant-pathogen interaction of A. vitis?

Investigating KdpC's role in A. vitis pathogenesis requires integrating molecular, cellular, and plant pathology approaches:

Genetic approaches:

• Gene knockout studies: Creating kdpC deletion mutants in A. vitis and assessing:

  • Bacterial survival in planta

  • Virulence and tumor formation ability

  • Competitive fitness against wild-type strains

• Complementation studies: Reintroducing wild-type or modified kdpC to confirm phenotype specificity.

• Reporter fusion constructs: Tagging kdpC with reporters to track expression during infection.

Plant-based assays:

• Grapevine infection models: Using both standard and tissue culture-based systems to assess pathogen behavior.

• Microscopy techniques: Confocal and electron microscopy to visualize bacteria during infection process.

• Mixed infections: Competing wild-type and kdpC mutant strains to assess fitness contributions.

Molecular approaches:

• Transcriptomics: RNA-seq analysis comparing gene expression in wild-type and kdpC mutants during infection.

• Metabolomics: Assessing potassium levels and related metabolites during infection.

• Protein-protein interaction studies: Identifying plant proteins that may interact with KdpC or be affected by bacterial potassium uptake.

The relationship between potassium homeostasis and virulence in A. vitis could be similar to the connection between bacterial survival in the acidic plant wound environment and efficient vir gene induction, which is essential for transformation . Researchers should consider how potassium availability in the plant microenvironment might influence bacterial gene expression and virulence.

How might computational approaches advance our understanding of KdpC function?

Computational methods offer powerful tools for investigating KdpC structure and function beyond what experimental approaches alone can achieve:

Molecular dynamics (MD) simulations:

• All-atom simulations of KdpC within the KdpFABC complex

• Analysis of conformational changes during the transport cycle

• Identification of water molecules and ions in the transport pathway

• Typical simulation timeframes: 100ns-1μs for standard MD; 10-100μs for enhanced sampling methods

Homology modeling and structure prediction:

• AlphaFold2 and RoseTTAFold predictions of KdpC variants

• Comparative modeling based on existing cryo-EM structures

• Prediction of species-specific structural features

Virtual screening and docking:

• Identification of potential small molecule binding sites

• Screening of compound libraries for potential inhibitors

• Binding energy calculations for structure-based drug design

Network analysis:

• Prediction of allosteric pathways within the KdpFABC complex

• Identification of co-evolving residues indicating functional linkage

• Gene regulatory network analysis integrating expression data

These computational approaches can generate testable hypotheses about structure-function relationships in KdpC, guiding experimental design and interpretation. The recent cryo-EM structures of KdpFABC provide an excellent starting point for such computational investigations.

What is known about post-translational modifications of KdpC and their impact on function?

Post-translational modifications (PTMs) of KdpC remain underexplored but potentially important regulators of function:

Potential PTMs of KdpC:

• Phosphorylation: While ATP hydrolysis occurs at KdpB, regulatory phosphorylation of KdpC could affect complex assembly or function.

• Disulfide bond formation: Cysteine residues in KdpC may form disulfide bonds affecting protein stability and conformation.

• Glycosylation: Though less common in bacteria than eukaryotes, glycosylation could affect KdpC stability or interaction with other proteins.

Methodological approaches:

• Mass spectrometry: LC-MS/MS analysis with enrichment techniques specific for PTMs.

• Site-directed mutagenesis: Changing potential modification sites and assessing functional consequences.

• 2D gel electrophoresis: Detecting charge or mass shifts indicative of modifications.

• Western blotting: Using PTM-specific antibodies if available.

The environmental conditions of the plant wound environment, which is typically acidic , may influence the PTM status of KdpC. Similarly, the activity of bacterial kinases and other modification enzymes may change during plant infection, potentially altering KdpC function during pathogenesis.

How does the interplay between KdpC and other bacterial ion transport systems contribute to A. vitis survival in planta?

The relationship between KdpFABC and other ion transport systems creates a complex network supporting bacterial survival:

Interconnected transport systems:

• Trk/Ktr systems: Lower-affinity K+ transporters that may function alongside KdpFABC.

• P-type ATPases: Other P-type ATPases may share regulatory mechanisms with KdpFABC.

• pH homeostasis systems: The acidic plant wound environment requires coordinated ion transport .

• Osmotic stress response: K+ accumulation is a primary bacterial response to osmotic stress.

Research approaches:

• Multiple knockouts: Creating bacterial strains with mutations in multiple transport systems.

• Transcriptomic profiling: Identifying coordinately regulated transport genes.

• Fluorescent ion indicators: Simultaneous monitoring of multiple ion species in living bacteria.

• Mathematical modeling: Creating predictive models of ion homeostasis networks.

The maintenance of bacterial pH homeostasis in the acidic plant wound environment is essential for both bacterial survival and virulence . The KdpFABC system likely plays a key role in this process, as potassium transport is often coupled to pH regulation. Understanding how KdpC contributes to this integrated system could reveal new targets for controlling A. vitis infections.

What are the most significant unresolved questions regarding KdpC function in A. vitis?

Despite recent advances in understanding KdpFABC structure and function, several critical questions about KdpC remain unresolved:

• Precise contribution to K+ selectivity: While KdpC appears to enhance K+ affinity , the molecular mechanism remains unclear.

• Species-specific adaptations: How KdpC in A. vitis differs from homologs in other bacteria and how these differences relate to the pathogenic lifestyle.

• Regulatory mechanisms: How kdpC expression is controlled during different stages of A. vitis infection cycle.

• Structural dynamics: The specific conformational changes in KdpC during the transport cycle, if any.

• Interaction network: The full range of proteins interacting with KdpC beyond the KdpFABC complex.

Resolving these questions will require integrated approaches combining structural biology, molecular genetics, and infection biology. The unique chimeric nature of the KdpFABC complex makes it particularly important to understand how each component, including KdpC, contributes to its dual channel-like and pump-like properties.