Recombinant Bacillus licheniformis ATP synthase subunit b (atpF)

What is the structural organization of ATP synthase in Bacillus licheniformis and how does subunit b (atpF) contribute to its function?

ATP synthase (F₁F₀) is a multisubunit complex responsible for ATP synthesis in bacteria, including Bacillus licheniformis. The enzyme consists of two major components: the membrane-embedded F₀ sector and the catalytic F₁ sector. The b subunit, encoded by the atpF gene, forms part of the peripheral stalk that connects F₁ to F₀, thereby playing a crucial role in maintaining the structural integrity of the complex during the rotational catalysis mechanism.

The ATP synthase in B. licheniformis utilizes both H⁺ and K⁺ gradients across the membrane to drive ATP synthesis, with the b subunit contributing to the stability of the complex during this process . Unlike other membrane proteins, the b subunit is mostly hydrophilic, with only its N-terminal region anchored in the membrane. This unique structure allows it to extend from the membrane to interact with the α and δ subunits of F₁, forming a critical component of the stator complex that prevents the α₃β₃ hexamer from rotating with the central stalk during catalysis.

How is ATP synthase regulated in B. licheniformis under different growth conditions?

ATP synthase expression and activity in B. licheniformis are tightly regulated in response to environmental conditions, particularly nutrient availability. Under nutrient-rich conditions such as growth in Luria Broth (LB) medium, B. licheniformis prioritizes amino acid degradation pathways and downregulates ATP synthase expression. Conversely, in minimal medium or during nutrient limitation, the bacterium upregulates ATP synthesis to maximize energy efficiency .

During the transition from exponential to stationary phase, B. licheniformis undergoes significant metabolic remodeling. The synthesis of many vegetative proteins, including components of ATP synthase, is altered to adapt to nutrient limitation. This adaptation involves the stringent response mediated by (p)ppGpp, similar to what has been observed in the related species B. subtilis, where changes in GTP and ppGpp concentrations affect the activity of rRNA promoters through inhibition of RNA polymerase .

The AbrB protein, a transition state regulator in Bacillus species, has been implicated in the regulation of energy metabolism genes including ATP synthase components . This regulatory network ensures that ATP synthase expression and activity are synchronized with the energy demands of the cell under different growth conditions.

What methods are recommended for cloning and expressing recombinant atpF from B. licheniformis?

For successful cloning and expression of recombinant atpF from B. licheniformis, a strategic approach combining modern molecular biology techniques is recommended:

• Gene Amplification: PCR amplification of the atpF gene from B. licheniformis genomic DNA using high-fidelity DNA polymerase and specific primers containing appropriate restriction sites.

• Vector Selection: For expression in B. licheniformis itself, the pUB'-EX1 integrative expression system has proven effective for stable chromosomal integration . This approach avoids the instability issues associated with plasmid-based expression in B. licheniformis.

• Verification: Confirm successful integration through colony PCR and evaluate expression levels through Western blotting or activity assays .

This methodology has been successfully employed for expressing recombinant proteins in B. licheniformis with genetic stability through multiple generations, making it suitable for atpF expression studies.

What challenges are commonly encountered when purifying recombinant ATP synthase subunit b?

Purification of recombinant ATP synthase subunit b presents several technical challenges:

• Membrane Association: Although largely hydrophilic, the N-terminal membrane anchor of subunit b complicates extraction from the membrane. A two-step solubilization approach is recommended:

  • Initial treatment with mild detergents (e.g., 1% digitonin)

  • Followed by stronger detergents (e.g., 0.5% dodecyl maltoside)

• Stability Issues: Isolated subunit b tends to form aggregates when separated from other ATP synthase components. To address this:

  • Include stabilizing agents such as glycerol (10-15%) in purification buffers

  • Maintain ionic strength with 100-150 mM NaCl

  • Consider purification of b subunit together with interacting partners

• Protein Folding: Recombinant expression often results in improper folding of the elongated, alpha-helical structure of subunit b. Co-expression with molecular chaperones (GroEL/GroES) can significantly improve folding efficiency in bacterial expression systems.

• Functional Verification: Confirming that the purified subunit retains its native conformation is essential. Circular dichroism spectroscopy can verify the high alpha-helical content characteristic of properly folded b subunit.

How does the proton-to-ATP ratio of B. licheniformis ATP synthase compare with other bacterial species, and how can this be experimentally determined?

The proton-to-ATP ratio (H⁺/ATP) is a critical parameter that determines the efficiency of ATP synthesis and the minimum protonmotive force (pmf) required for ATP production. In ATP synthase, this ratio is principally defined by the number of c-subunits in the c-ring of the F₀ component divided by the number of catalytic sites in F₁ (typically 3) .

Experimental Determination of H⁺/ATP Ratio:

The H⁺/ATP ratio can be experimentally determined by measuring the thermodynamic equilibrium between pmf and ΔG' using the following protocol:

• Prepare ATP synthase-reconstituted proteoliposomes (PLs)

• Incubate the PLs in an acidic buffer to establish a pH gradient

• Inject the PLs into assay medium containing luciferin/luciferase

• Monitor ATP synthesis/hydrolysis rates under various pmf conditions

• Determine the equilibrium pmf (pmfeq) where the net reaction rate is zero

• Measure this pmfeq at different reaction quotient (Q) values

The functional H⁺/ATP ratio can be calculated from the relationship:

$n = \frac{2.3RT \times \log_{10}Q}{F \times pmf_{eq}}$

Where:

• n is the H⁺/ATP ratio

• R is the gas constant

• T is temperature in Kelvin

• F is Faraday's constant

In engineered ATP synthase variants, modifications to the δ and α subunit fusion have demonstrated a doubled H⁺/ATP ratio compared to wild-type, resulting in the ability to synthesize ATP at half the minimum pmf . This approach could be applied to B. licheniformis ATP synthase to enhance its efficiency under energy-limited conditions.

What are the most effective strategies for site-directed mutagenesis of atpF to investigate its role in K⁺ transport through ATP synthase?

Recent research has revealed that ATP synthase can utilize both H⁺ and K⁺ gradients to drive ATP synthesis under physiological conditions . The b subunit may play a role in this dual-cation transport mechanism. To investigate this function through site-directed mutagenesis:

• Key Residues for Mutagenesis:

  • Target conserved charged residues (Asp, Glu) at the interface between subunit b and the c-ring

  • Focus on residues in the transmembrane domain that may participate in cation coordination

  • Investigate the role of the C-terminal domain in stabilizing the structure during K⁺ transport

• Functional Characterization:

  • Reconstitute the mutant ATP synthase into proteoliposomes

  • Assess ATP synthesis rates under varying K⁺ and H⁺ gradients

  • Measure direct K⁺ currents using voltage clamp techniques

  • Compare oxygen consumption rates in the presence and absence of K⁺

This systematic approach can identify residues in the b subunit that are specifically involved in K⁺ transport, potentially revealing new insights into the bioenergetic flexibility of B. licheniformis ATP synthase.

How does nutrient starvation affect the expression and activity of ATP synthase in B. licheniformis, and what experimental approaches can be used to study this?

Nutrient starvation triggers significant changes in the expression and activity of ATP synthase in B. licheniformis as part of a broader metabolic remodeling response. To investigate these changes, several experimental approaches can be employed:

• Transcriptomic Analysis:

  • RNA-seq to quantify changes in transcript levels of ATP synthase genes under nutrient limitation

  • qRT-PCR validation of specific ATP operon components, including atpF

  • Promoter fusion studies to identify regulatory elements responsive to nutrient availability

• Proteomic Analysis:

  • Quantitative proteomics using stable isotope labeling

  • 2D gel electrophoresis coupled with mass spectrometry to identify post-translational modifications

  • Blue-native PAGE to assess changes in ATP synthase complex assembly

• Metabolic Measurements:

  • Real-time monitoring of intracellular ATP levels using luciferase-based reporters

  • Measurement of membrane potential using fluorescent probes

  • Oxygen consumption rates to assess respiratory chain activity

Under phosphate starvation, B. licheniformis, like B. subtilis, activates the Pho regulon through the PhoP-PhoR two-component system . This response is interconnected with the ResD-ResE system and the transition state regulator AbrB, which may influence ATP synthase expression. Additionally, the stringent response mediated by ppGpp affects GTP levels, which in turn impacts the transcription of highly expressed genes including those encoding ATP synthase components .

What methods can be used to study the interaction between subunit b and other components of the ATP synthase complex?

Understanding the interaction network of subunit b is crucial for elucidating its role in the structural integrity and function of ATP synthase. Several cutting-edge methods can be employed:

• Cross-linking Coupled with Mass Spectrometry (XL-MS):

  • Chemical cross-linking of purified ATP synthase complexes

  • Enzymatic digestion and LC-MS/MS analysis

  • Identification of cross-linked peptides to map interaction sites

  • Data analysis using specialized software (e.g., xQuest, pLink)

  This approach can identify direct contacts between subunit b and other components at the amino acid level.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Exposure of ATP synthase to D₂O buffer

  • Quenching and pepsin digestion at various time points

  • LC-MS analysis to measure deuterium incorporation

  • Analysis of protected regions indicating protein-protein interfaces

• Single-Molecule FRET:

  • Site-specific labeling of subunit b and potential interaction partners with fluorescent dyes

  • Measurement of FRET efficiency to determine distances

  • Analysis of conformational changes during ATP synthesis

• Cryo-EM Studies:

  • Preparation of ATP synthase samples in various conformational states

  • High-resolution imaging (< 3 Å)

  • 3D reconstruction and model building

  • Validation through mutagenesis of identified interaction sites

These methodologies can provide complementary information about the dynamic interactions of subunit b within the ATP synthase complex, particularly its role in maintaining the structural connection between F₁ and F₀ during the catalytic cycle.

How can the chromosomal integration system for B. licheniformis be optimized for studying atpF mutations in vivo?

The chromosomal integration system using pUB-MazF and pUB'-EX1 plasmids provides a foundation for engineering B. licheniformis strains with modified atpF genes . To optimize this system specifically for atpF mutation studies:

• Improved Selection Strategy:

  Selection Method	Advantages	Limitations
  Temperature-sensitive replication	No antibiotic needed for curing	Requires precise temperature control
  MazF toxin induction	Highly efficient plasmid curing	Potential metabolic burden
  Counterselection with sacB	Direct selection for plasmid loss	Requires sucrose sensitivity
  CRISPR-Cas9 system	Precise targeting of integration site	More complex construct design

• Integration Site Considerations:

  • For atpF studies, integration at the native locus maintains normal regulation

  • Alternative approach: integration at a neutral site with the entire ATP operon

  • Use of inducible promoters allows controlled expression of mutant variants

• Combinatorial Mutations:
  For comprehensive structure-function studies, implement a system for sequential integration of multiple mutations to analyze synergistic effects between different atpF domains and other ATP synthase components.

This optimized system would enable systematic in vivo analysis of atpF mutations and their effects on ATP synthase structure, assembly, and function in the native cellular environment.

How can protein-protein interactions between atpF and other ATP synthase subunits be quantified?

Quantification of protein-protein interactions between atpF and other ATP synthase subunits requires specialized techniques that can capture both the strength and specificity of these interactions:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified subunit b on a sensor chip

  • Flow solutions containing other ATP synthase subunits over the surface

  • Measure real-time association and dissociation kinetics

  • Calculate binding affinities (KD values)

• Isothermal Titration Calorimetry (ITC):

  • Titrate one subunit into a solution containing subunit b

  • Measure heat changes during binding

  • Determine thermodynamic parameters (ΔH, ΔS, ΔG) and stoichiometry

  • Calculate binding constants with high precision

• Microscale Thermophoresis (MST):

  • Label subunit b with a fluorescent dye

  • Mix with varying concentrations of interaction partners

  • Measure changes in thermophoretic mobility upon binding

  • Calculate dissociation constants in near-native conditions

• Biolayer Interferometry (BLI):

  • Immobilize subunit b on biosensors

  • Dip into solutions containing other subunits

  • Monitor wavelength shifts due to binding in real-time

  • Determine association and dissociation rate constants

• Quantitative Crosslinking (QCLMS):

  • Perform crosslinking with isotope-labeled crosslinkers

  • Analyze by mass spectrometry

  • Quantify crosslinked peptides under different conditions

  • Compare relative abundances to assess interaction strengths

These techniques can be applied to study how mutations in atpF affect its interactions with other ATP synthase components, providing insights into the molecular basis of complex assembly and stability.

How should researchers interpret discrepancies in ATP synthase activity between in vivo and in vitro measurements?

Discrepancies between in vivo and in vitro measurements of ATP synthase activity are common and can provide valuable insights into the cellular context of enzyme function:

• Common Discrepancies and Their Causes:

  Discrepancy Type	Potential Causes	Investigation Approach
  Higher in vitro activity	Absence of regulatory factors	Add cell extracts to in vitro assays
  Higher in vivo activity	Synergistic effects with other cellular components	Reconstitute with respiratory chain components
  Different ion specificity	Altered membrane environment	Systematically vary lipid composition
  Altered inhibitor sensitivity	Missing interacting proteins	Identify additional binding partners by proteomics

• Methodological Considerations:

  • Ensure identical buffer conditions where possible

  • Account for membrane potential differences

  • Consider the role of molecular crowding in vivo

  • Evaluate post-translational modifications present only in vivo

• Integrated Analysis Approach:

  • Compare kinetic parameters (Km, Vmax) across systems

  • Analyze the effect of ion gradients on activity ratios

  • Test the impact of lipid composition on functional parameters

  • Develop mathematical models to reconcile differences

Recent studies on ATP synthase have shown that it can utilize both H⁺ and K⁺ to drive ATP synthesis, with a K⁺:H⁺ stoichiometry of approximately 2.7:1 . This dual-ion mechanism may contribute to discrepancies if the ionic conditions differ between in vivo and in vitro systems.

What are the most common technical challenges in expressing functional recombinant ATP synthase in B. licheniformis and how can they be addressed?

Expression of functional recombinant ATP synthase in B. licheniformis presents several technical challenges that require specific troubleshooting approaches:

• Complex Assembly Issues:

  • Challenge: Incomplete assembly of the multisubunit complex

  • Solution: Co-express all operon components with proper stoichiometry

  • Method: Use polycistronic constructs that maintain native gene organization

• Protein Stability Problems:

  • Challenge: Degradation of overexpressed subunits

  • Solution: Optimize growth conditions to reduce proteolytic activity

  • Method: Cultivate at lower temperatures (25-30°C) and use protease-deficient strains

• Integration Efficiency:

  • Challenge: Low efficiency of chromosomal integration

  • Solution: Enhance the integration system

  • Method: Implement the MazF-based system with optimized selection protocols

• Expression Level Imbalance:

  • Challenge: Overexpression of individual subunits disrupting complex assembly

  • Solution: Use tunable promoters to adjust expression levels

  • Method: Implement IPTG-inducible systems with concentration-dependent response

• Functional Verification Difficulties:

  • Challenge: Distinguishing recombinant from native ATP synthase activity

  • Solution: Introduce detectable tags or unique functional properties

  • Method: Incorporate mutations that alter inhibitor sensitivity without affecting function

For successful expression, the chromosomal integrative amplification strategy described for B. licheniformis offers significant advantages over plasmid-based systems, particularly in terms of genetic stability through repeated subculturing .

How might synthetic biology approaches be applied to engineer B. licheniformis ATP synthase with enhanced efficiency or novel properties?

Synthetic biology offers powerful approaches to engineer B. licheniformis ATP synthase with enhanced properties for both fundamental research and potential biotechnological applications:

• Rational Design Strategies:

  • Modification of the c-ring stoichiometry to alter the H⁺/ATP ratio

  • Engineering of the b subunit to enhance structural stability

  • Introduction of site-specific mutations to modify ion specificity

  • Creation of chimeric enzymes combining features from different species

• Directed Evolution Approaches:

  • Development of selection systems based on growth under limiting energy conditions

  • High-throughput screening for ATP synthase variants with desired properties

  • Continuous evolution systems that couple ATP synthesis efficiency to fitness

• Modular Engineering:

  • Swapping of functional domains between related ATP synthases

  • Creation of synthetic regulatory circuits controlling ATP synthase expression

  • Development of orthogonal systems for specialized energy conversion functions

• Novel Functionalities:

  • Engineering ATP synthase to utilize alternative ion gradients (Na⁺, Li⁺)

  • Development of variants with altered nucleotide specificity

  • Creation of light-responsive ATP synthase components

Recent work has demonstrated that engineering ATP synthase through the fusion of δ and α subunits can double the H⁺/ATP ratio, enabling ATP synthesis at lower protonmotive force . Similar approaches could be applied to B. licheniformis ATP synthase to enhance its efficiency in biotechnological applications or to adapt it for function under extreme conditions.

What role might differential expression of ATP synthase play in the adaptation of B. licheniformis to different environmental conditions?

B. licheniformis is remarkably adaptable to diverse environmental conditions, and differential expression of ATP synthase likely plays a key role in this adaptability:

• Nutrient Limitation Responses:

  • During phosphate starvation, B. licheniformis activates the Pho regulon through PhoP-PhoR, with crosstalk to the ResD-ResE system and AbrB regulator

  • This regulatory network may modulate ATP synthase expression to optimize energy conservation

  • The stringent response mediated by ppGpp affects transcription of highly expressed genes including those encoding ATP synthase

• Experimental Approaches to Study Adaptation:

  • Transcriptomics under various nutrient limitations

  • Proteomics to identify post-translational modifications

  • Metabolic flux analysis to track energy allocation

  • Comparative studies across different Bacillus species

• Environmental Response Pattern:

  Environmental Condition	ATP Synthase Expression	Metabolic Adaptation
  Rich medium (LB)	Downregulation	Amino acid degradation pathways activated
  Minimal medium	Upregulation	Amino acid biosynthesis enzymes increased
  Stationary phase	Differential regulation	Oxidative stress response proteins induced
  Oxygen limitation	Modified regulation	Shift toward fermentative metabolism
  Temperature stress	Complex regulation	Heat/cold shock responses activated

• Regulatory Integration:

  • The transition state regulator AbrB coordinates energy metabolism with other cellular processes

  • Integration of ATP synthase regulation with sporulation signals

  • Coordination with central carbon metabolism through global regulators

Understanding these regulatory networks could provide insights for designing B. licheniformis strains with optimized ATP synthase expression for specific industrial applications or environmental conditions.