Recombinant Bacillus weihenstephanensis ATP synthase subunit alpha (atpA), partial

What is ATP synthase subunit alpha (atpA) in Bacillus weihenstephanensis?

ATP synthase subunit alpha (atpA) is a critical component of the F1 catalytic sector in the F1F0-ATP synthase complex of B. weihenstephanensis. This complex plays an essential role in energy metabolism, utilizing the proton gradient across the membrane to synthesize ATP through oxidative phosphorylation. In bacteria like B. weihenstephanensis, ATP synthase generally functions with H+ as the coupling ion rather than Na+, as observed in most aerobic or facultatively aerobic alkaliphiles . The F1 sector, containing the alpha subunit, forms the catalytic head of the ATP synthase complex where ATP synthesis occurs.

How does ATP synthase function differ between B. weihenstephanensis and other bacteria?

B. weihenstephanensis, as a psychrotolerant member of the Bacillus cereus group, possesses unique adaptations that allow it to grow at temperatures as low as 7°C . While specific information about its ATP synthase is limited in the search results, evidence from related Bacillus species suggests that unlike alkaliphilic bacteria that face bioenergetic challenges at high pH values, B. weihenstephanensis likely maintains a conventional protonmotive force for ATP synthesis . The ATP synthase in B. weihenstephanensis operates using a proton gradient rather than sodium motive force, which is consistent with observations that ATP synthases of aerobic or facultatively aerobic bacteria primarily couple ATP synthesis to H+ .

What is the significance of B. weihenstephanensis in food safety research?

B. weihenstephanensis has significant importance in food safety research as it is an important food spoilage organism and potential cereulide-producing pathogen that can grow at refrigeration temperatures (7°C) . Its psychrotolerant nature makes it particularly problematic in food processing environments. While its endospores are generally less heat resistant than mesophilic relatives, research shows that B. weihenstephanensis can readily evolve to acquire enhanced endospore heat resistance, with some mutants showing a >4-fold increase in D-value at 91°C compared to parental strains . Understanding the energy metabolism mechanisms, including ATP synthase function, could provide insights into its survival strategies under various environmental conditions.

How does temperature affect ATP synthase activity in psychrotolerant B. weihenstephanensis?

The psychrotolerant nature of B. weihenstephanensis suggests that its ATP synthase likely has adaptations for function at lower temperatures. Interestingly, research on B. weihenstephanensis indicates that increased endospore heat resistance did not negatively affect the vegetative growth capacities at lower (7°C) and upper (37°C) growth temperature boundaries . This suggests that adaptations for temperature tolerance in B. weihenstephanensis operate independently from the mechanisms governing endospore heat resistance. For ATP synthase research, this implies that the enzyme may maintain activity across a broader temperature range than ATP synthases from strictly mesophilic bacteria, though direct experimental verification would be necessary.

What structural adaptations might exist in the ATP synthase of B. weihenstephanensis?

While the search results don't directly address structural adaptations in B. weihenstephanensis ATP synthase, insights can be drawn from studies of alkaliphilic bacteria. Alkaliphilic bacteria have specific adaptations in their ATP synthases to overcome the challenges of low protonmotive force at high pH values . Similarly, B. weihenstephanensis likely has structural adaptations in its ATP synthase to maintain functionality at lower temperatures. These might include modified amino acid compositions that preserve flexibility and catalytic efficiency in cold environments. Research on the number of c-subunits in the synthase rotor ring, which has been investigated in alkaliphiles , might also be relevant for understanding energy coupling efficiency in B. weihenstephanensis.

How do ATP synthase genes in B. weihenstephanensis compare to those in related Bacillus species?

Comparative genomic analysis of ATP synthase genes across the Bacillus genus could provide insights into the evolutionary adaptations that enable B. weihenstephanensis to thrive in cold environments. The atpF gene product from B. weihenstephanensis (strain KBAB4) has a UniProt accession number A9VSA7 , which could serve as a starting point for such comparisons. The evolutionary conservation or divergence of ATP synthase subunits might correlate with the thermal adaptation profile of different Bacillus species. Analysis similar to what has been done for alkaliphilic bacteria could reveal whether specific residues or domains in the ATP synthase complex contribute to psychrotolerance.

What expression systems are optimal for producing recombinant B. weihenstephanensis ATP synthase subunits?

Based on the available product information, both Baculovirus and E. coli expression systems have been successfully used for producing recombinant B. weihenstephanensis ATP synthase subunit b . The choice between these systems depends on research objectives:

• Baculovirus expression system: Generally provides better post-translational modifications and protein folding for complex proteins. The product CSB-BP002358BON1 uses this system .

• E. coli expression system: Typically yields higher protein quantities and is less expensive and time-consuming. The product CSB-EP002358BON1-B uses this system .

For functional studies of ATP synthase subunits, the expression system that best preserves native protein structure and activity should be selected. Researchers should consider conducting pilot expressions in both systems and evaluating protein yield, purity, and functional activity.

What are the optimal storage conditions for recombinant B. weihenstephanensis ATP synthase subunits?

The shelf life and stability of recombinant ATP synthase subunits depend on several factors, including storage state, buffer ingredients, and storage temperature. Based on the product information, the following guidelines are recommended:

Repeated freezing and thawing is not recommended as it can lead to protein degradation and loss of activity . For long-term storage, adding glycerol to a final concentration of 5-50% is recommended, with 50% being the default in commercial preparations .

How should recombinant B. weihenstephanensis ATP synthase subunits be reconstituted for experimental use?

For optimal reconstitution of lyophilized ATP synthase subunits, the following protocol is recommended:

• Briefly centrifuge the vial prior to opening to bring the contents to the bottom.

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• Add glycerol to a final concentration of 5-50% (with 50% being typical) for preparations intended for long-term storage.

• Aliquot the reconstituted protein to minimize freeze-thaw cycles.

• Store aliquots at -20°C/-80°C for long-term storage or at 4°C for up to one week for immediate use .

This procedure helps maintain protein stability and activity for subsequent experimental applications.

What methods can be used to verify the purity and functionality of recombinant ATP synthase subunits?

Several analytical methods can be employed to assess the quality of recombinant ATP synthase subunits:

• SDS-PAGE: The commercial preparations of B. weihenstephanensis ATP synthase subunit b have a reported purity of >85% as determined by SDS-PAGE . This method allows for assessment of protein purity and molecular weight.

• Western blotting: Using specific antibodies against ATP synthase subunits can confirm protein identity. Though not specifically mentioned in the search results for B. weihenstephanensis, antibodies against ATP synthase subunits from other species exist .

• Enzymatic activity assays: For functional ATP synthase subunits, ATP synthesis or hydrolysis assays can be performed to verify catalytic activity.

• Circular dichroism spectroscopy: This technique can provide information about the secondary structure of the protein, helping to verify proper folding.

• Mass spectrometry: For detailed characterization of the protein sequence and post-translational modifications.

How can researchers study the role of ATP synthase in B. weihenstephanensis adaptation to different environments?

To investigate ATP synthase's role in environmental adaptation, researchers could design experiments incorporating the following approaches:

• Gene expression analysis: Quantify ATP synthase subunit expression levels under various environmental conditions (different temperatures, pH values, nutrient limitations) using qRT-PCR or RNA-seq.

• Mutational analysis: Create point mutations or deletions in ATP synthase genes and assess the impact on growth under different conditions. This approach could reveal which residues or domains are critical for function in specific environments.

• Comparative studies: Compare ATP synthase activity from B. weihenstephanensis grown under optimal versus stress conditions (particularly temperature stress) to identify functional adaptations.

• Protein structure analysis: Conduct structural studies (X-ray crystallography, cryo-EM) of ATP synthase from B. weihenstephanensis to identify unique features that might contribute to its psychrotolerance.

• Bioenergetic measurements: Assess proton pumping efficiency and ATP synthesis rates at different temperatures to understand how B. weihenstephanensis maintains energy homeostasis across its growth temperature range.

What approaches can be used to investigate the relationship between ATP synthase function and endospore formation in B. weihenstephanensis?

The ability of B. weihenstephanensis to readily evolve increased endospore heat resistance raises interesting questions about energy metabolism during sporulation. Researchers could investigate this relationship through:

• Temporal gene expression analysis: Monitor ATP synthase gene expression throughout the sporulation process to identify potential regulatory patterns.

• Metabolic flux analysis: Trace energy flow during vegetative growth versus sporulation to understand how ATP production and utilization change.

• Comparative proteomics: Compare ATP synthase subunit abundance and modifications between vegetative cells and sporulating cells.

• Inhibitor studies: Use specific ATP synthase inhibitors to determine how energy limitation affects sporulation efficiency and endospore heat resistance.

• Evolution experiments: Analyze ATP synthase sequences and activity in B. weihenstephanensis strains that have evolved increased endospore heat resistance to identify potential correlations with energy metabolism adaptations.