Recombinant Kineococcus radiotolerans ATP synthase subunit beta (atpD)

What is Kineococcus radiotolerans and why is its ATP synthase subunit beta (atpD) of research interest?

K. radiotolerans is characterized by its orange pigmentation (likely carotenoid), catalase-positive and cytochrome c oxidase-negative properties, and dramatic colony morphology changes with age - transitioning from moist, smooth colonies to rough, dry, raised formations resembling Mycobacterium tuberculosis . The organism possesses a thick extracellular polymer shell surrounding individual cells within clusters.

The ATP synthase subunit beta (atpD) is a critical component of the F1F0-ATP synthase complex essential for energy metabolism. This protein contributes to the catalytic core that synthesizes ATP from ADP and inorganic phosphate using the proton gradient generated across the membrane. Research interest stems from understanding how this energy production system functions in an organism with remarkable stress tolerance capabilities.

What methodological considerations are important when working with recombinant K. radiotolerans atpD?

When working with recombinant K. radiotolerans atpD protein, researchers should consider several critical factors to maintain protein integrity and functionality:

• Storage conditions: Store at -20°C for short-term or -80°C for extended storage

• Handling protocol: Brief centrifugation prior to opening is recommended to bring contents to the bottom of the vial

• Reconstitution: Use deionized sterile water to reconstitute the protein to a concentration of 0.1-1.0 mg/mL

• Stability considerations: Add 5-50% glycerol (final concentration) and aliquot for long-term storage; avoid repeated freeze-thaw cycles

• Working conditions: Aliquots may be stored at 4°C for up to one week

• Shelf life determinants: Depends on multiple factors including storage state, buffer ingredients, temperature, and intrinsic protein stability (liquid form: ~6 months at -20°C/-80°C; lyophilized form: ~12 months)

The protein, produced in E. coli expression systems, typically achieves >85% purity as assessed by SDS-PAGE , making it suitable for various biochemical and structural analyses.

How does K. radiotolerans' response to radiation stress affect ATP synthase expression and function?

K. radiotolerans exhibits remarkable radiation resistance, suggesting specialized mechanisms for maintaining energy metabolism during radiation stress. Research indicates that radiation exposure triggers complex cellular responses involving DNA repair and protective systems that likely depend on ATP availability:

The bacterium expresses multiple DNA repair pathways in response to stress, including proteins involved in:

• Nucleotide excision repair (NER), including DNA-dependent ATPases and exinucleases

• Base excision repair, including uracil-DNA glycosylase and exodeoxyribonuclease III

• Recombinational DNA repair, with RecA and associated proteins (RecF, RecG, RecN, RecQ, RecR)

These repair systems require substantial energy input, suggesting a critical role for ATP synthase in maintaining cellular ATP levels during stress response. Methodological approaches to investigate this relationship should include:

• Transcriptomic analysis of atpD expression before, during, and after radiation exposure

• Proteomic analysis to assess post-translational modifications of ATP synthase components

• ATP synthesis rate measurements under various radiation conditions

• Creation of atpD mutants with altered expression levels to evaluate impact on radiation resistance

What is the relationship between K. radiotolerans' copper accumulation and ATP synthase function?

K. radiotolerans demonstrates a remarkable ability to accumulate soluble copper in its cytoplasm, with this phenotype correlating with enhanced cell growth during chronic exposure to ionizing radiation . This unusual characteristic raises important questions about ATP synthase function in the presence of high copper concentrations:

Proteomics studies reveal that approximately 40% of protein-coding ORFs on the K. radiotolerans genome are differentially expressed in response to copper treatments . Copper accumulation coincides with increased abundance of proteins involved in:

• Oxidative stress and defense mechanisms

• DNA stabilization and repair

• Protein turnover pathways

Interestingly, superoxide dismutase activity was repressed by low to moderate copper concentrations during exponential growth, suggesting complex regulation of oxidative stress defense mechanisms . ATP synthase function may be modulated during copper exposure to maintain energy homeostasis, possibly through:

• Direct interactions between copper ions and ATP synthase components

• Indirect effects through altered membrane properties

• Regulatory mechanisms responding to changes in cellular redox status

How can researchers effectively design experiments to study ATP synthase function under extreme conditions?

When investigating K. radiotolerans ATP synthase function under extreme conditions like radiation, desiccation, or high copper environments, researchers should consider:

• Standardized stress application protocols:

  • Radiation: Define precise dose rates and total exposure

  • Copper exposure: Use the range 0.1-1.5 mM Cu(II) as established in previous research

  • Consider growth phase effects (samples at 16, 22, and 32 hours have shown distinct responses)

• Appropriate analytical techniques:

  • Whole-cell proteomics with ANOVA (p-value cutoff 0.01) to detect statistically significant protein changes (defined as ±2 fold change in peptide abundance)

  • ATP synthesis and hydrolysis activity assays under stress conditions

  • Membrane potential measurements to assess proton gradient maintenance

• Genetic approaches:

  • Generation of atpD mutants with altered expression or activity

  • Creation of reporter constructs to monitor ATP synthase expression in real-time

  • Complementation studies with ATP synthase components from non-extremophilic organisms

• Structural biology methods:

  • Crystallography or cryo-EM analysis of ATP synthase components under stress conditions

  • Molecular dynamics simulations to predict structural adaptations to extreme environments

What experimental challenges exist when investigating the interaction between DNA repair systems and ATP synthase function?

K. radiotolerans expresses numerous DNA repair proteins in response to stress, including RecA, RecF, RecG, RecN, RecQ, and RecR, as well as DNA-directed RNA polymerase subunits . These energy-intensive processes likely depend on ATP synthase function, but investigating this relationship presents several methodological challenges:

Research has shown that DNA repair processes involve numerous ATP-dependent steps. For example, the RuvB helicase subunit of the RuvABC resolvasome (Krad3828) and RecA (Krad1492) were detected following copper exposure , suggesting similar responses may occur during radiation stress.

How might the study of K. radiotolerans ATP synthase advance applications in biotechnology?

Understanding the unique properties of K. radiotolerans ATP synthase could lead to several biotechnological applications:

• Radiation-resistant energy production systems:

  • The exceptional resistance of K. radiotolerans to ionizing radiation suggests its ATP synthase may have adaptations that maintain function under radiation stress

  • These features could be incorporated into engineered systems for energy production in high-radiation environments

• Bioremediation technologies:

  • K. radiotolerans' ability to thrive in radioactive waste environments and accumulate copper makes it a candidate for bioremediation

  • Understanding energy production during metal accumulation could lead to optimized bioremediation strategies

• Structural insights for protein engineering:

  • Comparative analysis of K. radiotolerans atpD with other extremophiles like Rubrobacter radiotolerans and Deinococcus radiodurans could reveal structural features contributing to stress tolerance

  • These insights could guide protein engineering efforts to create stress-resistant biocatalysts

• Novel antimicrobial strategies:

  • The unique features of K. radiotolerans ATP synthase might reveal targetable differences from host ATP synthases

  • This could potentially lead to new antimicrobial approaches for related actinobacteria of medical importance

The methodological approach would involve comparative structural analysis, functional testing under extreme conditions, and targeted mutagenesis to identify and validate key features contributing to the unique properties of this enzyme complex.