Recombinant Lactobacillus reuteri ATP synthase subunit b (atpF)

What is the ATP synthase complex in L. reuteri and why is subunit b significant?

The ATP synthase (Complex V) in L. reuteri, like in other organisms, functions as a rotary nanomotor that catalyzes ATP synthesis using the proton gradient across the membrane. The complex consists of two main domains: F₁, which contains the catalytic sites for ATP synthesis, and F₀, which forms the proton channel in the membrane .

Subunit b (atpF) is part of the peripheral stalk (also called the stator) that connects F₁ to F₀ and prevents the F₁ domain from rotating with the central rotor. The stator is critical for the stability of the ATP synthase complex, particularly in maintaining the association between the c-ring and the F₁ complex . Without a functional stator, the mechanical energy generated by proton flow cannot be effectively converted to chemical energy in the form of ATP.

How does ATP synthase assembly occur in bacteria like L. reuteri?

ATP synthase assembly in bacteria involves a coordinated process of multiple subunits coming together to form the functional complex. Based on studies of mitochondrial ATP synthase assembly (which shares similarities with bacterial ATP synthase), the process likely involves:

• Assembly of the c-ring (part of F₀)

• Binding of the F₁ domain to the c-ring

• Addition of the stator arm components, including subunit b

• Final assembly steps to form the complete complex

In bacteria, this process occurs co-translationally and involves specific chaperones for proper folding and assembly. The assembly process may involve separate pathways that converge at the final stages, as observed in yeast mitochondrial ATP synthase .

What experimental approaches are used to express recombinant L. reuteri atpF?

For expression of recombinant L. reuteri atpF, researchers typically employ the following methodological approaches:

• Gene cloning and vector selection: The atpF gene is isolated from L. reuteri genomic DNA using PCR with specific primers. The gene is then cloned into an appropriate expression vector with a suitable promoter and affinity tag (commonly His-tag) for purification.

• Expression system selection: E. coli is often used as the host organism for heterologous expression of bacterial membrane proteins. Alternative systems include cell-free expression systems or homologous expression in L. reuteri.

• Optimization of expression conditions: Parameters such as temperature, induction timing, and inducer concentration require optimization to maximize yield while ensuring proper folding.

• Membrane protein solubilization: As a membrane protein component, specialized detergents are needed to solubilize subunit b while maintaining its native conformation.

• Purification strategy: Affinity chromatography followed by size exclusion chromatography is typically employed to obtain pure protein.

How do mutations in atpF affect ATP synthase function and L. reuteri's immunomodulatory properties?

While the direct relationship between atpF mutations and L. reuteri's immunomodulatory properties has not been extensively characterized in the provided research, we can analyze potential connections based on L. reuteri's known mechanisms.

L. reuteri strains exhibit strain-specific immunomodulatory activities, particularly in suppressing TNF production . These activities are associated with specific metabolic pathways, including cyclopropane fatty acid synthesis and polyketide synthesis . ATP synthase function, as the primary ATP-generating machinery, likely affects these metabolic pathways by controlling energy availability.

Mutations in atpF could potentially:

• Alter the efficiency of ATP synthesis, affecting energy-dependent metabolic pathways

• Change the membrane composition or properties, as the stator interacts with the membrane

• Disrupt proton gradient maintenance, which could impact other membrane-associated functions

These changes might subsequently affect the production of immunomodulatory compounds. For instance, the production of lactobacillic acid, which correlates with TNF inhibitory activity, occurs during stationary phase when energy metabolism shifts . Alterations in ATP synthase function could influence this phase-dependent production.

What are the structural and functional differences between L. reuteri atpF and homologous proteins in other probiotic bacteria?

While specific structural information about L. reuteri atpF is limited in the provided research, comparative analysis of ATP synthase subunit b across bacterial species reveals several key considerations:

• Sequence conservation: Subunit b typically shows moderate sequence conservation across species, with higher conservation in the C-terminal domain that interacts with F₁ and lower conservation in the membrane-spanning N-terminal domain.

• Length variations: The length of subunit b can vary between bacterial species, affecting the distance between F₁ and F₀ domains.

• Oligomerization state: In many bacteria, subunit b forms a homodimer, but the interaction strength and specific dimerization interfaces may differ between species.

• Species-specific adaptations: Probiotic bacteria living in the gut environment may have evolved specific adaptations in their ATP synthase components to function optimally under the pH and ionic conditions of the intestinal environment.

In L. reuteri specifically, these adaptations might relate to the bacterium's ability to colonize specific niches in the gastrointestinal tract and produce strain-specific immunomodulatory compounds.

How does the integration of recombinant atpF affect the assembly and stability of the ATP synthase complex in L. reuteri?

The integration of recombinant atpF into the ATP synthase complex presents several research challenges:

• Assembly kinetics: Recombinant atpF must incorporate correctly into the assembly pathway. Studies on ATP synthase assembly suggest that the peripheral stalk plays a critical role in stabilizing the c-ring/F₁ complex . Therefore, modified atpF might affect the assembly rate or efficiency.

• Stoichiometry maintenance: ATP synthase requires precise stoichiometry of its subunits. The expression level of recombinant atpF needs to match the native expression patterns of other subunits.

• Structural integrity: The peripheral stalk provides structural stability to the ATP synthase complex. Modifications to atpF could potentially destabilize the complex, leading to reduced activity or increased degradation.

• Regulatory impacts: In some organisms, the expression of ATP synthase subunits is coordinated through feedback mechanisms. Introducing recombinant atpF might disrupt this regulation, affecting the balance between nuclear-encoded and endogenously produced components.

To experimentally assess these effects, researchers typically employ techniques such as Blue Native PAGE to analyze complex assembly, activity assays to measure ATP synthesis rates, and structural approaches such as cryo-EM to examine the integrated complex.

What are the optimal conditions for functional characterization of recombinant L. reuteri atpF?

Functional characterization of recombinant L. reuteri atpF requires careful consideration of experimental conditions:

Table 1: Optimal Conditions for Functional Characterization of Recombinant atpF

For functional assays, researchers can measure:

• ATP synthesis activity: Using luciferin/luciferase assays to detect ATP production

• Proton translocation: Using pH-sensitive fluorescent dyes to monitor proton movement

• Protein-protein interactions: Using crosslinking or pull-down assays to assess integration into the ATP synthase complex

• Thermal stability: Using differential scanning fluorimetry to assess protein stability

How can researchers address challenges in the purification of recombinant L. reuteri atpF?

Purification of membrane proteins like atpF presents several challenges requiring specific methodological approaches:

• Solubilization optimization:

  • Screen multiple detergents (DDM, LMNG, LDAO) at various concentrations

  • Evaluate solubilization efficiency by Western blotting

  • Consider using detergent mixtures for improved extraction

• Protein aggregation prevention:

  • Include glycerol (10-15%) in all buffers

  • Maintain samples at low temperatures (4°C)

  • Add stabilizing agents such as arginine (50-100 mM)

• Purification strategy refinement:

  • Implement a two-step affinity chromatography approach

  • Include an ion-exchange step to remove contaminants

  • Utilize size exclusion chromatography as a final polishing step

• Functional validation:

  • Assess protein folding using circular dichroism

  • Verify membrane association using liposome binding assays

  • Confirm protein-protein interactions with other ATP synthase subunits

Troubleshooting Guide:

• For low expression yields: Optimize codon usage or try expression at lower temperatures

• For protein degradation: Add protease inhibitors and reduce purification time

• For loss of function: Verify native-like secondary structure and consider lipid addition

How can researchers effectively measure the impact of atpF modifications on L. reuteri's immunomodulatory functions?

To investigate connections between atpF modifications and immunomodulatory functions, researchers should implement a multi-faceted experimental approach:

• Generation of defined atpF variants:

  • Site-directed mutagenesis of conserved residues

  • Domain swapping with homologous proteins

  • Construction of chimeric proteins

• Metabolomic analysis:

  • Monitor production of cyclopropane fatty acids, especially lactobacillic acid, which correlates with TNF inhibitory activity

  • Analyze changes in tryptophan metabolism and indole derivative production

  • Quantify polyketide synthesis, which has been linked to AhR activation

• Immunological assays:

  • Measure TNF suppression in activated human monocytes or macrophages

  • Assess AhR activation using reporter cell lines like H1L6.1c3

  • Evaluate effects on IL-22 expression and other cytokines

• Correlation analysis:

  • Relate ATP synthesis rates to immunomodulatory compound production

  • Examine temporal relationships between energy metabolism shifts and immunomodulatory activity

  • Compare strain-specific differences in both ATP synthase properties and immunomodulatory functions

The experimental design should include appropriate controls, such as comparing atpF-modified strains with other immunomodulatory mutants like cfa (cyclopropane fatty acid synthase) knockouts, which completely lose TNF inhibitory activity .

What emerging technologies could enhance studies of L. reuteri atpF structure and function?

Several cutting-edge technologies show promise for advancing our understanding of L. reuteri atpF:

• Cryo-electron microscopy (Cryo-EM): This technique can reveal the atomic structure of the entire ATP synthase complex, including the precise positioning and conformation of subunit b within the native complex.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This approach can identify dynamic regions of atpF and map interaction interfaces with other subunits.

• Single-molecule biophysics: Techniques such as magnetic tweezers or optical traps can measure the mechanical properties of the stator, including its contribution to the stiffness of the ATP synthase complex.

• Computational approaches:

  • Molecular dynamics simulations to model atpF behavior in membranes

  • Evolutionary coupling analysis to identify co-evolving residues

  • Machine learning to predict the impact of mutations

• CRISPR-Cas9 genome editing: Precise modification of the native atpF gene in L. reuteri to study functions in the natural context.

How might atpF engineering contribute to the development of next-generation probiotics?

Engineering ATP synthase subunit b could potentially enhance probiotic properties through several mechanisms:

• Enhanced colonization:

  • Modifying atpF to optimize ATP synthase efficiency under gut conditions

  • Improving acid tolerance through proton gradient management

  • Adapting to specific host intestinal environments

• Immunomodulatory enhancement:

  • Optimizing energy production to increase synthesis of immunomodulatory compounds

  • Engineering metabolic shifts to favor production of beneficial metabolites

  • Creating strains with constitutive production of immunomodulatory factors

• Strain-specific targeting:

  • Developing L. reuteri variants optimized for specific health conditions

  • Creating strains with enhanced activity in particular intestinal regions

  • Engineering bacteria for targeted release of therapeutic compounds

This approach aligns with the concept of developing next-generation probiotics with enhanced therapeutic properties, as mentioned in the research on L. reuteri PKS gene clusters and their AhR activation potential .