Recombinant Serratia proteamaculans ATP synthase subunit beta (atpD)

What is the ATP synthase subunit beta (atpD) in Serratia proteamaculans?

ATP synthase subunit beta (atpD) is a critical component of the ATP synthase complex in S. proteamaculans, identified with Uniprot accession number A8G7M8. This protein functions as part of the F1 sector of F-ATPase (EC 3.6.3.14) and plays a central role in ATP production through oxidative phosphorylation. The protein consists of 460 amino acids and is essential for the catalytic function of ATP synthesis in this bacterial species .

How does atpD contribute to bacterial energy metabolism?

The beta subunit of ATP synthase contains the catalytic sites responsible for ATP synthesis. During oxidative phosphorylation, proton flow through the membrane-embedded Fo sector drives rotation of the central stalk, inducing conformational changes in the beta subunits. These conformational changes cycle the catalytic sites through different states (open, loose, and tight binding), facilitating the synthesis of ATP from ADP and inorganic phosphate. This process is fundamental to bacterial energy metabolism and survival.

What are the optimal storage conditions for recombinant S. proteamaculans atpD?

For optimal stability and activity preservation of recombinant S. proteamaculans atpD, the following storage protocols are recommended:

• Short-term storage: Store at -20°C

• Extended storage: Conserve at -20°C or -80°C

• Working aliquots: Can be stored at 4°C for up to one week

• Important note: Repeated freezing and thawing is not recommended as it can compromise protein integrity and activity

What is the recommended reconstitution protocol for experimental use?

For optimal reconstitution of lyophilized recombinant S. proteamaculans atpD:

• Briefly centrifuge the vial prior to opening to bring 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% (standard recommendation is 50%)

• Prepare multiple small aliquots for long-term storage at -20°C/-80°C

• Use reconstituted protein promptly for best results in enzymatic assays

What methods can researchers use to verify protein purity and integrity?

The commercially available recombinant S. proteamaculans atpD has a purity of >85% as determined by SDS-PAGE. Researchers should implement the following validation methods before experimental use:

• SDS-PAGE analysis with Coomassie or silver staining to confirm size and purity

• Western blotting with antibodies specific to ATP synthase beta subunit or attached tags

• Mass spectrometry to verify molecular weight and sequence integrity

• Functional assays to confirm ATP hydrolysis activity

• Circular dichroism to assess secondary structure integrity

How can researchers design effective mutagenesis studies for atpD functional analysis?

When designing mutagenesis studies for S. proteamaculans atpD:

• Target conserved residues in catalytic sites (typically involving the GXGXGKT/S sequence motif)

• Consider mutations at the interface with other ATP synthase subunits

• Design complementation systems using plasmid-expressed wild-type atpD as controls

• Include positive controls (known functional mutations) and negative controls (known disruptive mutations)

• Validate mutant protein expression levels and stability before attributing phenotypes to functional changes

• Assess effects on both ATP synthesis and hydrolysis activities separately

How is atpD expression regulated in response to environmental conditions?

While specific information for S. proteamaculans is not provided in the search results, ATP synthase expression in bacteria typically responds to:

• Energy status of the cell (ATP/ADP ratio)

• Oxygen availability (aerobic vs. anaerobic conditions)

• Growth phase (exponential vs. stationary)

• Nutrient availability

• pH and osmotic stress

Researchers investigating atpD regulation should consider these factors when designing experiments to study expression patterns under different environmental conditions.

Does atpD play a role in S. proteamaculans virulence and infection?

S. proteamaculans is a facultative pathogen with invasive activity regulated by a Quorum Sensing (QS) system consisting of the regulatory protein SprR and AHL synthase SprI . While no direct evidence links atpD to virulence mechanisms in the search results, ATP synthase activity is critical for providing energy for virulence-associated processes.

Research approaches to investigate potential connections include:

• Creating atpD knockdown mutants and assessing virulence

• Studying atpD expression during different stages of infection

• Investigating correlations between ATP synthase activity and expression of known virulence factors

• Examining atpD expression in response to host defense mechanisms

Is there evidence for horizontal gene transfer of atpD among bacterial species?

Sequence analysis of atpD is commonly used in phylogenetic studies of bacterial species. Researchers investigating potential horizontal gene transfer should:

• Perform comparative genomic analyses of atpD sequences across related bacterial species

• Look for incongruence between atpD-based phylogenies and those based on other housekeeping genes

• Analyze GC content and codon usage patterns that might indicate foreign origin

• Examine flanking regions for evidence of mobile genetic elements

How might the study of S. proteamaculans atpD inform antimicrobial development?

ATP synthase is an essential enzyme for bacterial survival, making it a potential target for antimicrobial development. Research strategies include:

• Screening for compounds that specifically inhibit S. proteamaculans atpD

• Structure-based drug design targeting unique features of bacterial ATP synthases

• Investigating synergistic effects between ATP synthase inhibitors and established antibiotics

• Developing delivery systems that can target ATP synthase inhibitors to bacterial cells

• Examining resistance mechanisms that might emerge in response to ATP synthase inhibition

What is the relationship between atpD function and bacterial adaptation to diverse environments?

S. proteamaculans has been isolated from soil environments, suggesting adaptation to various ecological niches . Research questions to explore include:

• How does atpD sequence and activity vary among S. proteamaculans strains from different environments?

• Are there specific adaptations in the ATP synthase complex that enhance survival under particular stress conditions?

• How does the efficiency of ATP synthesis change under different environmental conditions?

• What regulatory mechanisms control atpD expression during environmental transitions?

How does the QS system in S. proteamaculans potentially influence atpD expression and function?

The Quorum Sensing system in S. proteamaculans involving SprI and SprR regulates bacterial invasion capabilities . Research approaches to investigate potential QS-ATP synthase connections include:

• Comparing atpD expression patterns between wild-type and SprI/SprR mutants

• Examining ATP production in QS system mutants

• Investigating whether ATP synthase activity influences the production of QS signaling molecules

• Analyzing potential binding sites for QS regulators in the atpD promoter region

What are common challenges in expressing and purifying functional recombinant atpD?

Researchers frequently encounter these challenges when working with recombinant ATP synthase components:

• Solubility issues: ATP synthase subunits can form inclusion bodies in heterologous expression systems

  • Solution: Optimize expression conditions (temperature, inducer concentration) or use solubility tags

• Proper folding: Ensuring correct tertiary structure

  • Solution: Co-express with chaperones or use specialized expression strains

• Activity preservation: Maintaining native-like function

  • Solution: Include appropriate cofactors during purification; avoid harsh purification conditions

• Tag interference: Affinity tags may affect function

  • Solution: Use cleavable tags or confirm activity with and without tags

How can researchers distinguish between the ATP synthesis and hydrolysis activities of atpD?

ATP synthase can catalyze both ATP synthesis and hydrolysis. Experimental approaches to distinguish these activities include:

• Directional assays:

  • For synthesis: Measure ATP production using luciferase-based assays

  • For hydrolysis: Measure inorganic phosphate release from ATP

• Condition manipulation:

  • Synthesis requires a proton gradient (typically created using liposomes)

  • Hydrolysis can be measured in simple buffer systems with ATP

• Inhibitor profiling:

  • Certain inhibitors affect synthesis and hydrolysis differently

  • Use of specific inhibitors can help distinguish the predominant activity

What controls are essential when evaluating potential atpD inhibitors?

When screening for compounds that inhibit S. proteamaculans atpD:

• Include positive controls (known ATP synthase inhibitors like oligomycin or DCCD)

• Include negative controls (compounds with similar structures that don't inhibit ATP synthase)

• Test for direct binding to atpD using techniques like isothermal titration calorimetry

• Verify specificity by testing activity against other ATPases

• Confirm the mechanism of inhibition (competitive, non-competitive, uncompetitive)

• Evaluate effects on whole bacterial cells to confirm target engagement in vivo

How might structural studies of S. proteamaculans atpD advance our understanding of bacterial ATP synthases?

Future research directions for structural studies include:

• High-resolution structure determination using X-ray crystallography or cryo-EM

• Comparative structural analysis with ATP synthases from other bacterial species

• Investigation of conformational changes during the catalytic cycle

• Structure-based identification of species-specific features that could be targeted for antimicrobial development

• Elucidation of protein-protein interactions within the ATP synthase complex

What role might atpD play in bacterial stress responses and antibiotic resistance?

Energy metabolism is intimately connected to stress responses in bacteria. Research questions to explore include:

• How does atpD expression change during exposure to antibiotics or other stressors?

• Does ATP synthase activity influence the expression of efflux pumps or other resistance mechanisms?

• Can modulation of ATP synthase activity enhance or reduce antibiotic efficacy?

• Is there a connection between the energy state of the cell and the activation of stress response pathways?

How can systems biology approaches enhance our understanding of atpD in the context of bacterial metabolism?

Integrative approaches to study atpD function include: