Recombinant Herpetosiphon aurantiacus Potassium-transporting ATPase C chain (kdpC)

What is the Potassium-transporting ATPase C chain (kdpC) in Herpetosiphon aurantiacus?

Potassium-transporting ATPase C chain (kdpC) is a component of the KdpFABC complex, which functions as a high-affinity potassium uptake system in Herpetosiphon aurantiacus. The protein consists of 193 amino acids with the sequence MRTFFRPALAAIIIFSVLTGVIYPALVTVIAQVTFPGQANGSLIEQAGQQRGSSLIGQQFDQPEYFWGRLSATGPVPYNAAASSGSNYGPLNPALAEAVQARIDALKAADPSNQLPIPVDLVTASASGLDPEISPAAANYQVQRVAAARGLAVEQVQQLVEQHTSQRTLGVLGEPRVNVLQLNIALDQIKSLD . This protein is classified as an ATP phosphohydrolase with EC number 3.6.3.12 and serves as the potassium-binding and translocating subunit within the larger transport complex. The gene encoding kdpC in H. aurantiacus is identified as Haur_2278 in genome annotations .

To study this protein effectively, researchers should consider its membrane-associated nature, which necessitates specialized approaches for expression, purification, and functional characterization. Expression systems that accommodate membrane proteins, such as modified E. coli strains or eukaryotic systems, may be required for optimal recombinant production.

What is the taxonomic classification and biological context of Herpetosiphon aurantiacus?

Herpetosiphon aurantiacus belongs to:

• Domain: Bacteria

• Phylum: Chloroflexi

• Class: Chloroflexia

• Order: Herpetosiphonales

• Family: Herpetosiphonaceae

• Genus: Herpetosiphon

• Species: aurantiacus

The type strain of Herpetosiphon aurantiacus is designated as 114-95 (also cataloged as DSM 785 and ATCC 23779), isolated from lake water in Birch Lake, Minnesota, USA . H. aurantiacus is a filamentous, gliding bacterium that can reach up to 500 μm in length and is enclosed within a sheath . It is aerobic, growing optimally at 28°C, and forms swarming colonies with orange to red pigmentation due to carotenoid production .

Methodologically, researchers should culture this organism at 30°C using Medium 67 as recommended by culture collections . The complete genome of H. aurantiacus type strain 114-95T is 6.79 Mbp with 5,577 protein-encoding genes and two circular plasmids , providing ample genomic resources for researchers studying specific gene functions including kdpC.

How does the kdpC protein contribute to potassium homeostasis in bacteria?

The kdpC protein plays a critical role in potassium homeostasis by functioning as an integral part of the high-affinity potassium uptake system. Methodologically, to investigate this function:

• Growth experiments in potassium-limited media can demonstrate the importance of the Kdp system

• Gene expression studies can show upregulation of kdpC under potassium limitation

• Electrophysiological approaches can measure potassium transport rates in reconstituted systems

For experimental design, researchers should consider:

• Including appropriate positive and negative controls (e.g., potassium-replete conditions vs. potassium-limited conditions)

• Using genetic approaches (knockouts, complementation) to confirm specific roles

• Implementing real-time monitoring of intracellular potassium levels using fluorescent probes or ion-selective electrodes

The precise coordination of kdpC with other components of the Kdp system ensures that bacteria maintain appropriate intracellular potassium concentrations even in environments where potassium is scarce.

How does the structure of kdpC relate to its function in potassium transport?

Analysis of the kdpC amino acid sequence reveals several key structural domains with specific functional implications:

To investigate structure-function relationships experimentally, researchers should consider:

• Using site-directed mutagenesis to create point mutations at conserved residues

• Performing complementation studies with mutant variants in kdpC-deficient strains

• Applying structural techniques such as X-ray crystallography or cryo-electron microscopy to the purified protein or reconstituted complex

• Conducting molecular dynamics simulations to predict conformational changes during transport cycles

These approaches can reveal how specific structural elements contribute to potassium binding, transport, and regulation within the complete KdpFABC complex .

What is the relationship between potassium transport systems and predatory behavior in Herpetosiphon aurantiacus?

Herpetosiphon aurantiacus exhibits "wolf pack" predation against a variety of microorganisms including Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus species, Enterococcus faecalis, Bacillus subtilis, and Candida albicans . While direct experimental evidence linking kdpC specifically to predation is limited, several potential connections can be investigated:

• Ion homeostasis during predation: The predatory lifestyle may create unique challenges for maintaining intracellular potassium levels, particularly during the osmotic changes that might occur when prey cells are lysed.

• Energy requirements: As an ATP-dependent transport system, the Kdp complex represents an energy investment that must be balanced against the nutritional benefits gained through predation.

• Signaling function: Potassium gradients might serve as signaling mechanisms that coordinate predatory behaviors within H. aurantiacus communities.

Methodologically, researchers could investigate these connections by:

• Performing transcriptomic or proteomic analyses during predation events to detect changes in kdpC expression

• Creating and characterizing kdpC knockout strains to assess effects on predatory efficiency

• Measuring potassium fluxes during predation using ion-selective electrodes or fluorescent indicators

• Comparing potassium transport systems between predatory and non-predatory bacterial species

Such studies would contribute to our understanding of the physiological basis of bacterial predation .

How does the kdpC gene from Herpetosiphon aurantiacus compare to homologs in other bacterial species?

Comparative genomic analysis of kdpC reveals both conservation and divergence across bacterial species. While the core function remains similar, adaptations to specific ecological niches have driven evolutionary changes.

For researchers investigating evolutionary relationships, methodological approaches should include:

• Multiple sequence alignment using tools like MUSCLE or CLUSTAL

• Phylogenetic tree construction to visualize evolutionary relationships

• Selection pressure analysis to identify conserved vs. rapidly evolving regions

• Structural modeling to compare predicted three-dimensional conformations

These analyses can reveal functional constraints on kdpC evolution and identify species-specific adaptations that might relate to ecological specialization, such as the predatory lifestyle of H. aurantiacus .

What expression systems are most effective for producing recombinant Herpetosiphon aurantiacus kdpC?

Optimizing expression of recombinant kdpC requires careful consideration of several factors due to its membrane-associated nature. Based on experimental evidence with similar proteins, researchers should consider:

A recommended experimental approach includes:

• Construct multiple expression vectors with different fusion tags (His, GST, MBP)

• Test expression in various host systems under different conditions (temperature, inducer concentration)

• Conduct small-scale expression tests before scaling up

• Verify protein integrity by Western blotting and functional assays

Current recombinant preparations utilize a Tris-based buffer with 50% glycerol for storage, which helps maintain stability during freeze-thaw cycles .

What purification strategies are most effective for recombinant kdpC protein?

Purification of membrane proteins like kdpC presents unique challenges requiring specialized approaches:

• Cell lysis optimization:

  • Gentle disruption methods (sonication with cooling periods)

  • Buffer optimization (pH 7.5-8.0, 100-300 mM NaCl)

  • Addition of protease inhibitors to prevent degradation

• Membrane extraction:

  • Differential centrifugation to isolate membrane fractions

  • Careful selection of detergents (DDM, LDAO, or CHAPS) for solubilization

  • Detergent concentration optimization to maintain protein structure

• Chromatography sequence:

  • Initial capture: Affinity chromatography based on fusion tag

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography

  • Consider on-column detergent exchange if necessary

• Quality control:

  • SDS-PAGE and Western blotting to confirm identity and purity

  • Mass spectrometry for accurate molecular weight determination

  • Circular dichroism to verify secondary structure integrity

For storage, manufacturers recommend maintaining aliquots at -20°C for extended periods, while working stocks can be kept at 4°C for up to one week . Researchers should avoid repeated freeze-thaw cycles to maintain functional integrity.

What functional assays can be used to characterize recombinant kdpC activity?

To thoroughly characterize recombinant kdpC function, researchers should implement multiple complementary approaches:

• Binding assays:

  • Isothermal titration calorimetry (ITC) to measure potassium binding affinity

  • Surface plasmon resonance (SPR) to study interaction kinetics with other Kdp components

  • Fluorescence-based assays using potassium-sensitive dyes

• Transport assays:

  • Reconstitution into proteoliposomes for direct transport measurements

  • Electrophysiological approaches (patch clamp or planar lipid bilayers)

  • Radioactive tracer (86Rb+) uptake studies in reconstituted systems

• Structural characterization:

  • Circular dichroism to assess secondary structure

  • Limited proteolysis to identify domain boundaries

  • Thermal shift assays to evaluate stability under different conditions

• In vivo complementation:

  • Expression of recombinant kdpC in kdpC-deficient strains

  • Growth assays under potassium limitation

  • Measurement of intracellular potassium levels using atomic absorption spectroscopy

These methodological approaches provide complementary data that together can establish the functional integrity and specific activities of recombinant kdpC .

How can kdpC research contribute to understanding bacterial osmoregulation mechanisms?

Research on kdpC offers valuable insights into bacterial osmoregulation mechanisms through multiple experimental approaches:

• Comparative genomics:

  • Analysis of kdpC conservation across bacteria from diverse osmotic environments

  • Identification of species-specific adaptations in sequence or regulation

  • Correlation of kdpC variations with ecological niches

• Physiological studies:

  • Investigation of kdpC expression under various osmotic challenges

  • Characterization of growth phenotypes of kdpC mutants under osmotic stress

  • Real-time monitoring of cytoplasmic potassium during osmotic shifts

• Regulatory network analysis:

  • Identification of transcription factors controlling kdpC expression

  • Mapping of signaling pathways that respond to osmotic stress

  • Integration of kdpC regulation with other osmoregulatory systems

This research has broader implications for understanding bacterial adaptation to fluctuating environments, particularly in the context of the predatory lifestyle of Herpetosiphon aurantiacus, which may encounter varying osmotic conditions during interaction with different prey organisms .

What potential applications exist for studying kdpC in the context of antimicrobial development?

The study of kdpC from Herpetosiphon aurantiacus has several potential applications in antimicrobial research:

• Novel target identification:

  • The essential nature of potassium transport makes kdpC a potential antimicrobial target

  • Structure-based drug design focused on inhibiting kdpC function

  • Screening for compounds that disrupt the assembly of the KdpFABC complex

• Predation mechanisms:

  • Understanding how H. aurantiacus uses ion transport during predation

  • Identifying secreted factors that might target prey potassium homeostasis

  • Developing antimicrobial peptides based on H. aurantiacus predatory mechanisms

• Biocontrol applications:

  • Engineering enhanced predatory bacteria for targeting specific pathogens

  • Developing H. aurantiacus as a biocontrol agent against plant or animal pathogens

  • Creating synergistic combinations of predatory bacteria and conventional antibiotics

Methodologically, these applications require:

• High-throughput screening systems for identifying kdpC inhibitors

• In vitro and in vivo models to test efficacy against pathogenic bacteria

• Safety and specificity assessments for potential therapeutic applications

Given that H. aurantiacus can prey on clinically relevant organisms such as Staphylococcus aureus and Candida albicans , understanding its predatory mechanisms, including the role of potassium transport, could lead to novel antimicrobial strategies.

How might systems biology approaches enhance our understanding of kdpC in bacterial physiology?

Systems biology approaches offer powerful tools for comprehensively understanding kdpC's role within the broader context of bacterial physiology:

• Multi-omics integration:

  • Combining transcriptomics, proteomics, and metabolomics data under various conditions

  • Correlating kdpC expression with global metabolic shifts

  • Identifying unexpected connections between potassium transport and other cellular processes

• Network analysis:

  • Constructing protein-protein interaction networks centered on kdpC

  • Mapping genetic interactions through synthetic lethality screens

  • Identifying regulatory hubs that control kdpC expression

• Computational modeling:

  • Creating mathematical models of bacterial potassium homeostasis

  • Simulating the impact of environmental changes on the Kdp system

  • Predicting emergent properties of the potassium transport network

• Single-cell analyses:

  • Measuring cell-to-cell variability in kdpC expression

  • Correlating kdpC activity with individual cell behavior during predation

  • Tracking potassium dynamics in real-time at the single-cell level

These approaches can reveal how kdpC contributes to the complex predatory behavior of Herpetosiphon aurantiacus and how potassium transport integrates with other cellular systems during different physiological states .