Recombinant Lactobacillus plantarum ATP synthase subunit delta (atpH)

What is the ATP synthase subunit delta (atpH) in Lactobacillus plantarum, and what role does it play in cellular metabolism?

The ATP synthase subunit delta (atpH) in L. plantarum is a component of the F1F0-ATP synthase complex responsible for ATP production via oxidative phosphorylation. It forms part of the central stalk of the F1 sector, connecting the F1 and F0 sectors and participating in the rotational mechanism that couples proton translocation to ATP synthesis .

The L. plantarum genome encodes six ATP synthase components, including the delta chain . This subunit plays a critical role in maintaining the structural integrity of the ATP synthase complex and ensuring efficient energy conversion. As part of the central stalk, atpH helps transmit the energy from proton movement through F0 to the catalytic sites in F1, contributing to the crucial H+/ATP ratio that determines the efficiency of energy conversion in the cell .

What expression systems are most effective for recombinant production of L. plantarum atpH?

Based on available research, several expression systems show promise for the recombinant production of L. plantarum atpH:

• Baculovirus expression system: Successfully used for the related L. reuteri atpH protein , this system is effective for producing complex bacterial proteins that may require specific folding environments.

• L. plantarum as homologous expression host: The pSIP expression system in L. plantarum WCFS1 has been demonstrated as an effective tool for expressing recombinant proteins in this organism . This approach may offer advantages for proper folding and native-like post-translational modifications.

• E. coli expression systems: Standard for recombinant bacterial protein production, though not specifically mentioned for atpH in the search results.

For expression in L. plantarum, the following methods have shown success:

• The pSIP401 expression system demonstrated high-level expression of α-amylase

• Signal peptides like Lp_2145 can significantly improve protein secretion and yield (13.1 kU/L of fermentation for α-amylase)

• Induction with 50 ng/mL SppIP for 6-10 hours at 37°C provided optimal expression for another recombinant protein in L. plantarum

What purification strategy yields the highest purity and activity of recombinant atpH?

A successful purification strategy for recombinant L. plantarum atpH should incorporate:

• Affinity chromatography: If the recombinant atpH includes an affinity tag, this allows for highly specific purification. One-step affinity procedures have yielded high-purity recombinant L. plantarum proteins (17 mg/L for tannase) .

• Buffer optimization: For the related L. reuteri atpH, recommended reconstitution involves:

  • Using deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Adding 5-50% glycerol (final concentration) for stability

  • Brief centrifugation prior to opening the vial

• Quality assessment: SDS-PAGE analysis should confirm a protein band at the expected molecular weight with purity >85%, as reported for the related L. reuteri atpH .

• Activity preservation: Avoid repeated freeze-thaw cycles, which significantly reduce protein activity .

Table 1: Comparison of purification approaches for recombinant Lactobacillus proteins

How can I measure the ATP synthase activity and specifically evaluate the contribution of the delta subunit?

Since atpH is a structural component rather than a catalytic subunit, its activity must be assessed in the context of the assembled ATP synthase complex:

• Reconstitution into proteoliposomes: Using an approach similar to that described for yeast and chloroplast ATP synthases , purified atpH can be combined with other ATP synthase components and reconstituted into liposomes. After energization with acid-base transitions, both ATP synthesis and hydrolysis rates can be measured as functions of ΔpH.

• H+/ATP ratio determination: The thermodynamic H+/ATP ratio, which reflects the efficiency of energy conversion, can be determined at equilibrium by measuring initial rates of ATP synthesis and hydrolysis at different ΔpH values and extrapolating to find the equilibrium point .

• Functional complementation: In a system where the native atpH has been deleted or mutated, the ability of recombinant atpH variants to restore ATP synthase function can provide insights into structure-function relationships.

The methodology described in research paper provides a detailed approach for such measurements:

• Create proteoliposomes containing the ATP synthase complex

• Generate a defined ΔpH by acid-base transitions

• Measure initial rates of ATP synthesis/hydrolysis using luciferin/luciferase system

• Determine ΔpH(eq) by interpolation at the point of zero rate

• Calculate H+/ATP ratio from the relationship between ΔpH(eq) and stoichiometric ratio [ATP]/([ADP]·[Pi])

What is the role of redox regulation in L. plantarum ATP synthase function, and how does the delta subunit participate?

While the search results don't specifically address redox regulation in L. plantarum ATP synthase, insights can be drawn from studies on chloroplast ATP synthase :

Chloroplast ATP synthase is activated in light and inactivated in dark by redox-modulation through the thioredoxin system. This regulation involves the γ-subunit thiols and acts as a "redox switch," ensuring the enzyme is fully active even in low light and fully inactivated in darkness .

To investigate potential redox regulation in L. plantarum ATP synthase:

• Sequence analysis: Identify potential redox-sensitive residues (typically cysteines) in the L. plantarum atpH and other ATP synthase components.

• Site-directed mutagenesis: Create variants with modified cysteine residues and test their activity under different redox conditions.

• Functional assays: Compare ATP synthesis/hydrolysis activities under varying redox conditions using techniques similar to those described in result .

• In vivo studies: Compare wild-type L. plantarum and strains expressing modified ATP synthase components under conditions that alter cellular redox state.

The study in result challenges the conventional model that ATP synthase down-regulation prevents wasteful ATP hydrolysis in the dark. Instead, it suggests this regulation affects protein transport across thylakoid membranes. Similar unexpected functions might exist in bacterial systems.

How can recombinant atpH be used as a tool for studying bacterial metabolism and adaptation to environmental stress?

Recombinant atpH can provide valuable insights into bacterial metabolism and stress adaptation through several research approaches:

• Structure-function analysis: By creating site-directed mutants of atpH, researchers can investigate how specific residues contribute to ATP synthase assembly, stability, and function under different environmental conditions.

• Strain comparison studies: Comparing atpH sequences and expression levels across different L. plantarum strains (e.g., LRCC5310 vs. BRD3A ) can reveal adaptations in energy metabolism related to different ecological niches.

• Global gene expression analysis: As demonstrated in the study of L. plantarum gene expression in the human gastrointestinal tract , ATP synthase regulation can be examined as part of the broader metabolic response to environmental challenges.

• Metabolic flux analysis: Manipulating atpH expression or function can help elucidate how energy generation impacts other metabolic pathways during stress adaptation.

The genomic approach described in search result revealed that in L. plantarum, ATP metabolism genes (including "a copper transporting ATPase gene") are among those differentially expressed during passage through the human intestinal tract, highlighting the connection between energy metabolism and environmental adaptation.

What role might the ATP synthase delta subunit play in probiotic functionality of L. plantarum strains?

The ATP synthase delta subunit may contribute to probiotic functionality of L. plantarum in several ways, though this connection is not directly addressed in the search results:

• Energy efficiency for gut colonization: Optimized ATP synthesis efficiency could provide competitive advantages during colonization of the gastrointestinal environment.

• Acid stress tolerance: The ability to maintain PMF (proton motive force) and ATP synthesis under acidic conditions is crucial for survival in the gastrointestinal tract and could be influenced by ATP synthase regulation.

• Integration with immunomodulatory functions: As seen in recombinant L. plantarum expressing immune-relevant proteins , energy metabolism may indirectly support the expression and presentation of proteins that interact with the host immune system.

• Support for bacteriocin production: ATP generation is essential for the production of bacteriocins, which contribute to competitive exclusion of pathogens. L. plantarum BRD3A, for example, produces several bacteriocins that confer antimicrobial properties .

• Adaptation to different intestinal regions: L. plantarum gene expression varies between ileum and colon , suggesting region-specific adaptations in energy metabolism that may involve ATP synthase regulation.

Research on L. plantarum LRCC5310 shows that its genome encodes a nearly complete vitamin B₆ biosynthetic pathway in addition to ATP synthase components , illustrating how energy metabolism interconnects with other probiotic-relevant functions.

What are the most common challenges in expressing recombinant L. plantarum atpH, and how can they be addressed?

Based on experiences with similar recombinant proteins in L. plantarum, common challenges include:

• Low expression levels: This can be addressed by:

  • Optimizing codon usage for the expression host, as done for recombinant spike protein expression in L. plantarum

  • Selecting appropriate signal peptides - Lp_2145 demonstrated 6.2-fold higher expression compared to native signal peptides for α-amylase expression

  • Optimizing induction conditions - 50 ng/mL SppIP at 37°C for 6-10h yielded highest expression for another recombinant protein

• Protein solubility issues: Strategies to improve solubility include:

  • Lowering induction temperature

  • Using fusion tags that enhance solubility

  • Including stabilizing additives like glycerol in extraction buffers

• Variable secretion efficiency: The choice of signal peptide significantly impacts secretion:

Table 2: Effect of signal peptides on α-amylase expression in L. plantarum

• Protein stability: To maintain stability:

  • Store purified protein at -20°C (short-term) or -80°C (long-term)

  • Add 5-50% glycerol as cryoprotectant

  • Prepare single-use aliquots to avoid freeze-thaw cycles

How can I validate the structural integrity and functional activity of purified recombinant atpH?

A comprehensive validation strategy should include:

• Structural integrity assessment:

  • SDS-PAGE to confirm the expected molecular weight (similar to analysis in )

  • Western blotting with specific antibodies

  • Circular dichroism spectroscopy to evaluate secondary structure

  • Limited proteolysis to assess proper folding

• Functional validation:

  • Binding assays with other ATP synthase components

  • Reconstitution experiments incorporating atpH into partial or complete ATP synthase complexes

  • ATP synthesis/hydrolysis assays of reconstituted complexes using methods similar to those described in result

• Stability evaluation:

  • Thermal shift assays to determine melting temperature

  • Time-course activity measurements under various storage conditions

  • Testing stability at different pH values and salt concentrations (the recombinant spike protein expressed in L. plantarum was stable at 50°C, pH 1.5, and high salt concentration )

• In vivo complementation:

  • Expression of recombinant atpH in strains with deleted or mutated native atpH to assess functional replacement

  • Growth rate and ATP production measurements in complemented strains

Real-time RT-qPCR can be used to quantify expression levels, as demonstrated for recombinant α-amylase in L. plantarum, where transcript levels correlated with protein expression and activity .