Recombinant Bradyrhizobium japonicum ATP synthase epsilon chain (atpC)

What is the ATP synthase epsilon chain (atpC) in Bradyrhizobium japonicum and what is its primary function?

The ATP synthase epsilon chain (designated as atpC or bll0439 in B. japonicum) is a critical regulatory subunit of the F₁F₀-ATP synthase complex. This protein functions as a major regulator of ATP hydrolysis and synthesis, serving as a bidirectional control element that can inhibit ATP hydrolysis while allowing ATP synthesis under appropriate conditions .

The epsilon subunit in bacteria consists of two distinct domains:

• An N-terminal β-barrel domain that interacts with the γ subunit

• A C-terminal α-helical domain (εCTD) that can adopt different conformations ("up" or "down")

These conformational changes are central to its regulatory function, allowing epsilon to act as a molecular switch that responds to cellular energy status .

How is atpC gene expression regulated in B. japonicum under different environmental conditions?

The expression of atpC in B. japonicum is dynamically regulated in response to various environmental signals and metabolic states:

• Response to plant hormones: Under treatment with 1 mM indole-3-acetic acid (IAA), atpC (bll0439) expression is significantly down-regulated by -3.02 fold. This occurs as part of a broader transcriptional response affecting approximately 15% of the B. japonicum genome .

• Growth conditions: During chemoautotrophic growth (utilizing hydrogen as an electron donor), B. japonicum undergoes significant transcriptional reprogramming affecting 1,485 transcripts (17.5% of the genome). ATP synthase components show differential expression patterns compared to heterotrophic growth conditions .

• Oxygen levels: Microoxic and anoxic conditions trigger regulatory mechanisms that affect energy metabolism components, including ATP synthase. The FixK₂ regulatory protein, activated under low oxygen conditions, influences the expression of genes involved in energy production .

The coordinated regulation of ATP synthase components ensures efficient energy conservation under changing environmental conditions .

What mechanisms control the inhibitory function of the epsilon subunit in the ATP synthase complex?

The epsilon subunit regulates ATP synthase through complex conformational dynamics that respond to cellular energy status:

• Conformational switching: The C-terminal domain of epsilon (εCTD) can adopt "up" (extended) or "down" (compact) conformations. The extended state interacts with catalytic sites in the F₁ sector, inhibiting ATP hydrolysis .

• Nucleotide sensing: In some bacterial species, binding of ATP or changes in ATP:ADP ratio can influence the conformation of epsilon, acting as a nucleotide sensor .

• Proton motive force (pmf) response: The pmf across the membrane promotes release of the epsilon C-terminal domain from its inhibitory state, allowing ATP synthesis to proceed. This mechanism prevents wasteful ATP hydrolysis when cellular energy is low .

• Redox regulation: In some systems, disulfide bond formation can stabilize specific conformations of the epsilon subunit, providing an additional regulatory mechanism linked to cellular redox status .

The inhibitory behavior is primarily directed toward preventing wasteful ATP hydrolysis, particularly under conditions where ATP conservation is critical for cellular survival .

What are the recommended protocols for producing recombinant B. japonicum atpC for structural and functional studies?

Based on protocols used for similar bacterial epsilon subunits, a methodological approach for producing recombinant B. japonicum atpC would include:

• Cloning strategy:

  • Amplify the atpC gene from B. japonicum genomic DNA

  • Clone into an expression vector (e.g., pET system) with appropriate affinity tag

  • Transform into E. coli BL21(DE3) or similar expression strain

• Protein purification:

  • Resuspend cells in appropriate binding buffer (e.g., 20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 5 mM imidazole)

  • Disrupt cells using French pressure cell (12,000 lb/in²) or sonication

  • Centrifuge lysate (17,200 × g) to remove debris

  • Purify using affinity chromatography followed by size exclusion

• Refolding strategy:
  For epsilon subunits that form inclusion bodies, a denaturation/refolding approach can be used:

  • Solubilize in 8 M urea

  • Perform direct dilution into buffer containing ethanol and glycerol for proper folding

• Activity assessment:

  • ATPase inhibition assays using purified F₁ or F₁F₀ complex

  • Conformational analysis by circular dichroism or intrinsic fluorescence

  • Structural characterization by NMR or X-ray crystallography

The recombinant protein can then be used for structural studies, binding assays, and functional characterization in reconstituted systems .

What techniques are most effective for studying the conformational changes of the epsilon subunit in vitro?

Multiple complementary techniques can be employed to study the dynamic conformational changes of the epsilon subunit:

• NMR spectroscopy:

  • Provides atomic-level details of structure in solution

  • Can capture different conformational states

  • Allows monitoring of domain-domain interactions

  • Example: NMR solution structure of A. baumannii epsilon subunit revealed interaction between N-terminal β-barrel and C-terminal α-hairpin domains

• Cryo-electron microscopy (cryo-EM):

  • Visualizes the architecture of the entire ATP synthase complex

  • Can capture the extended position of the epsilon C-terminal domain

  • Provides context for interactions with other subunits

  • Example: 3.0 Å cryo-EM structure of A. baumannii F₁-ATPase showed epsilon in extended position

• Site-directed spin labeling and EPR spectroscopy:

  • Monitors distances between specific residues during conformational changes

  • Provides information about dynamics in different nucleotide conditions

• FRET (Förster Resonance Energy Transfer):

  • Measures distances between fluorophores attached to different domains

  • Can monitor conformational changes in real-time

  • Useful for studying the transition between compact and extended states

• Cross-linking studies:

  • Captures transient interactions between domains or with other subunits

  • Can be combined with mass spectrometry for detailed analysis

  • Example: Disulfide cross-linking studies showed that the "up" state inhibits ATP hydrolysis but not necessarily ATP synthesis

• Single-molecule microscopy:

  • Provides direct observation of rotational dynamics

  • Can correlate conformational states with functional outcomes

  • Reveals heterogeneity not apparent in ensemble measurements

For meaningful results, researchers should combine multiple techniques to build a comprehensive understanding of the conformational dynamics .

How does atpC contribute to B. japonicum's symbiotic relationship with soybean?

The ATP synthase epsilon chain plays indirect but significant roles in the symbiotic relationship between B. japonicum and soybean:

The coordination between energy metabolism and nitrogen fixation machinery is essential for establishing effective symbiosis, with the ATP synthase complex serving as a key component in this relationship .

What roles does atpC play in B. japonicum's response to environmental stresses?

The ATP synthase epsilon chain contributes to stress adaptation through regulation of energy metabolism:

• Oxidative stress response:

  • Paraquat-induced oxidative stress triggers genome-wide transcriptional changes in B. japonicum

  • ATP synthase regulation helps maintain energy balance during oxidative damage

  • The inhibitory function of epsilon prevents wasteful ATP hydrolysis during stress recovery

• Chemical stressors:

  • Herbicides and agricultural additives can affect B. japonicum growth

  • ATP synthase components may be regulated to optimize energy conservation under chemical stress

  • For example, dicamba solutions showed effects on B. japonicum colony-forming units, suggesting metabolic adaptations

• Nutritional stress adaptation:

  • Under carbon limitation, B. japonicum can switch to chemoautotrophic growth

  • This metabolic shift involves substantial transcriptional reprogramming affecting 17.5% of the genome

  • Energy metabolism components, including ATP synthase, are adjusted to maintain ATP production with alternative electron donors

• Low oxygen adaptation:

  • Microoxic and anoxic conditions activate regulatory proteins like FixK₂

  • ATP synthase regulation ensures efficient energy conservation under oxygen limitation

  • The epsilon subunit's inhibitory function helps prevent ATP depletion when respiration is limited

The regulatory function of epsilon becomes particularly important under stress conditions, where preventing wasteful ATP hydrolysis can be critical for bacterial survival and resilience .

How do researchers reconcile contradictory findings about the role of the epsilon subunit in ATP synthesis versus ATP hydrolysis inhibition?

Researchers have developed several frameworks to reconcile apparently contradictory findings about epsilon's role:

The current integrated model suggests that epsilon functions primarily to prevent wasteful ATP hydrolysis when cellular energy is limited, while still allowing ATP synthesis when appropriate conditions (sufficient pmf) are present. This adaptive regulation optimizes energy conservation under varying environmental conditions .