Recombinant Brucella ovis ATP synthase subunit b 1 (atpF1)

What is Brucella ovis ATP synthase subunit b 1 (atpF1) and what is its biological role?

Brucella ovis ATP synthase subunit b 1 (atpF1) is a component of the F₀ sector of the bacterial ATP synthase complex. This complex is essential for energy production through ATP synthesis via oxidative phosphorylation. The atpF1 gene encodes a 208-amino acid protein that forms part of the membrane-embedded proton channel . The protein has several synonyms including BOV_0396, ATP synthase F(0) sector subunit b 1, ATPase subunit I 1, F-type ATPase subunit b 1, and F-ATPase subunit b 1 .

The b subunit serves as a structural stator connecting the F₀ and F₁ sectors of ATP synthase. This connection is critical for maintaining the structural integrity necessary for the rotational catalysis mechanism that drives ATP synthesis. In Brucella species, which are facultative intracellular pathogens, ATP synthase components like atpF1 are essential for energy metabolism during different stages of infection.

How does the atpF1 gene fit into the genomic context of Brucella ovis?

The atpF1 gene (BOV_0396) is part of the ATP synthase operon in the Brucella ovis genome . While specific genomic context information for atpF1 is limited in the search results, research on the Brucella ovis genome reveals interesting features:

• The B. ovis genome shows evidence of degradation compared to other Brucella species, which correlates with its narrower host range .

• B. ovis has species-specific genetic islands, including a region of approximately 28 kb (BOV_A0482-BOV_A0515) with a structure suggestive of a composite transposon .

• The genome includes several deletions, such as a 7745 bp deletion on Chromosome I (BR1078-BR1083 region in B. suis) and a smaller 3954 bp deletion leading to loss of genes encoding a transcriptional regulator and a branched chain amino acid permease .

While atpF1 is conserved in B. ovis, understanding its genomic context helps researchers interpret its role within the metabolic and pathogenic capabilities of this host-restricted pathogen.

How can Recombinant Brucella ovis ATP synthase subunit b 1 be utilized in immunological research?

Recombinant B. ovis ATP synthase subunit b 1 has significant potential in immunological research:

• Antibody production: The purified recombinant protein can be used to generate specific antibodies for diagnostic assays, immunohistochemistry, and Western blotting. These antibodies enable researchers to track the expression and localization of native atpF1 during infection.

• Vaccine development: As a conserved bacterial protein, recombinant atpF1 could be evaluated as a potential subunit vaccine candidate. Researchers can investigate its immunogenicity and protective efficacy in animal models, similar to studies conducted with the B. ovis ΔabcBA vaccine strain that has shown protection against field isolates in mice .

• Host-pathogen interaction studies: The protein can be used to investigate interactions with host components, particularly how the ATP synthase complex may contribute to bacterial survival within host cells. This is especially relevant given B. ovis' adaptation to a narrower host range .

• Diagnostic marker evaluation: Given the conservation of atpF1 across B. ovis isolates, researchers can explore its utility as a species-specific diagnostic marker to differentiate B. ovis from other Brucella species in serological assays.

What is known about the evolutionary conservation of ATP synthase subunit b 1 across Brucella species?

While the search results don't provide direct comparative data for atpF1 across Brucella species, we can infer several important points:

A comparative analysis would potentially reveal:

• Amino acid substitutions that might affect protein-protein interactions

• Conservation of functional domains across species

• Evidence of selective pressure on different regions of the protein

Researchers investigating this area should consider conducting multiple sequence alignments of atpF1 orthologs across Brucella species to identify conserved and variable regions.

What role might ATP synthase components play in the pathogenicity and host adaptation of Brucella ovis?

ATP synthase components, including subunit b 1, may contribute to B. ovis pathogenicity and host adaptation in several ways:

• Energy provision during infection: ATP synthase is crucial for generating energy under various environmental conditions encountered during infection. B. ovis field isolates have demonstrated the ability to colonize and cause lesions in mice livers and spleens, processes that require metabolic adaptation .

• Adaptation to intracellular lifestyle: The kinetics of intracellular growth of B. ovis field isolates in RAW 264.7 murine macrophage cells shows strain-specific patterns, suggesting metabolic adaptation to the intracellular environment . ATP synthase function could be modulated to optimize energy production under these conditions.

• Contribution to pH homeostasis: ATP synthase can function in reverse to maintain pH homeostasis, potentially helping B. ovis survive in acidic compartments within host cells.

• Potential role in host range restriction: The narrowed host range of B. ovis compared to other Brucella species correlates with genome degradation . While ATP synthase components are likely conserved, subtle modifications in these proteins could contribute to metabolic adaptations that influence host specificity.

Experimental evidence from B. ovis field isolates shows varying patterns of infection in mice. For example, in one study, bacterial loads in the spleen and liver at 1 day post-infection were significantly higher in mice infected with the reference strain B. ovis ATCC 25840 compared to field isolates (94 AV and 266 L), with nearly a 2-log difference .

What are optimal expression systems for producing Recombinant Brucella ovis ATP synthase subunit b 1?

Based on the available information, the following expression systems and considerations are recommended:

• E. coli expression system: The search results indicate successful expression of recombinant B. ovis atpF1 in E. coli with an N-terminal His tag . E. coli remains the most common and cost-effective system for bacterial protein expression.

• Optimal vector selection:

  • pET vector systems offer tight control of expression under T7 promoter

  • pBAD vectors provide tunable expression through arabinose induction

  • Cold-shock expression vectors can improve solubility for challenging proteins

• Expression conditions optimization:

  • Temperature: Lower temperatures (16-25°C) often improve solubility

  • Induction: IPTG concentration and induction timing significantly impact yield

  • Media: Enriched media like Terrific Broth can increase biomass and protein yield

• Fusion tags consideration:

  • His-tag (as used in the reference protein) facilitates purification

  • GST or MBP tags may improve solubility if protein aggregation occurs

  • SUMO fusion can enhance expression and solubility while offering tag removal

For membrane-associated proteins like ATP synthase subunit b 1, which has a hydrophobic domain, expression conditions that prevent aggregation are particularly important. Starting with the validated E. coli system with His-tag fusion provides a solid foundation for optimization.

What purification strategies are most effective for Recombinant Brucella ovis ATP synthase subunit b 1?

Based on the properties of the recombinant protein described in the search results, the following purification strategy is recommended:

• Initial capture:

  • Immobilized Metal Affinity Chromatography (IMAC) using Ni-NTA resin for His-tagged protein

  • Lysis buffer optimization is critical: consider including mild detergents if membrane association causes solubility issues

• Secondary purification:

  • Size Exclusion Chromatography (SEC) to remove aggregates and further purify monomeric protein

  • Ion Exchange Chromatography based on the protein's theoretical pI for additional purity

• Buffer optimization:

  • Final buffer composition: Tris/PBS-based buffer with 6% Trehalose, pH 8.0 (as used for the reference protein)

  • For long-term storage, addition of 50% glycerol is recommended

• Quality control:

  • SDS-PAGE analysis to confirm >90% purity

  • Western blot to confirm identity

  • Dynamic Light Scattering to assess homogeneity

The published product achieves greater than 90% purity as determined by SDS-PAGE , suggesting that the combination of IMAC and potentially a secondary purification step is sufficient to obtain high-quality protein for research applications.

What are the recommended storage and handling procedures for maintaining Recombinant Brucella ovis ATP synthase subunit b 1 stability?

Based on the information provided in the search results, the following storage and handling recommendations should be followed:

• Storage conditions:

  • Store at -20°C/-80°C upon receipt

  • Aliquoting is necessary for multiple use to avoid freeze-thaw cycles

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

• Reconstitution protocol:

  • Centrifuge vial briefly before opening to bring contents to the bottom

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

  • Add glycerol to a final concentration of 5-50% (preferably 50%) for long-term storage

• Stability considerations:

  • Avoid repeated freeze-thaw cycles

  • The presence of 6% Trehalose in the storage buffer aids protein stability

  • pH 8.0 has been determined optimal for this specific protein

• Working solution preparation:

  • Thaw aliquots slowly on ice

  • Gentle mixing rather than vortexing is recommended to prevent protein denaturation

  • Filter sterilization through 0.22 μm filters if sterility is required for cell-based assays

These guidelines will help maintain the structural integrity and activity of the recombinant protein for experimental applications.

What are common challenges in working with Recombinant Brucella ovis ATP synthase subunit b 1 and how can they be addressed?

Researchers working with Recombinant Brucella ovis ATP synthase subunit b 1 may encounter several challenges:

• Protein solubility issues:

  • Challenge: As a membrane-associated protein, atpF1 may have hydrophobic regions that lead to aggregation.

  • Solution: Use mild detergents (0.1% Triton X-100 or 0.5% CHAPS) during extraction; employ fusion partners like MBP or SUMO; optimize buffer conditions with stabilizing agents like trehalose (as used in the reference product) .

• Protein degradation:

  • Challenge: ATP synthase components may be susceptible to proteolysis.

  • Solution: Include protease inhibitors during purification; work at 4°C; minimize handling time; add stabilizers like trehalose to storage buffer .

• Activity assessment:

  • Challenge: As a structural component of ATP synthase, isolating atpF1's individual function can be difficult.

  • Solution: Consider biochemical assays that measure binding to other ATP synthase components; use structural studies like circular dichroism to confirm proper folding.

• Freeze-thaw stability:

  • Challenge: Repeated freeze-thaw cycles can lead to protein degradation or aggregation.

  • Solution: Store in small aliquots with 50% glycerol as recommended ; maintain working aliquots at 4°C for up to one week.

• Specificity in immunological applications:

  • Challenge: Cross-reactivity with homologous proteins from other bacteria.

  • Solution: Perform extensive cross-reactivity testing; use regions of atpF1 unique to B. ovis for antibody generation if species specificity is required.

How can researchers distinguish between Brucella ovis ATP synthase subunit b 1 and homologous proteins from other bacterial species?

Distinguishing B. ovis ATP synthase subunit b 1 from homologous proteins in other species is crucial for specificity in research applications:

• Sequence-based approaches:

  • Perform multiple sequence alignments to identify regions unique to B. ovis atpF1

  • Design PCR primers or peptide antibodies targeting B. ovis-specific regions

  • Use mass spectrometry with peptide mapping to identify species-specific peptide signatures

• Immunological discrimination:

  • Develop monoclonal antibodies against unique epitopes of B. ovis atpF1

  • Perform epitope mapping to identify species-specific binding regions

  • Use competitive ELISA to distinguish between homologous proteins

• Genetic context analysis:

  • The genomic context of atpF1 in B. ovis may differ from other species

  • B. ovis has undergone genome degradation with specific deletions and genetic islands

  • PCR amplification of the gene with its flanking regions could provide species-specific patterns

• Functional differences:

  • Assess potential differences in protein-protein interactions

  • Compare binding affinities to other ATP synthase components

  • Evaluate thermal stability differences that might reflect adaptation to different host environments

This multi-faceted approach would enable researchers to ensure they are specifically studying B. ovis atpF1 rather than homologous proteins from other bacterial species, which is particularly important in diagnostic development or host-pathogen interaction studies.

How might Recombinant Brucella ovis ATP synthase subunit b 1 contribute to vaccine development strategies?

Recombinant B. ovis ATP synthase subunit b 1 holds potential for vaccine development through several approaches:

Field isolate studies have demonstrated that B. ovis strains can vary in virulence , suggesting that an effective vaccine would need to provide protection against diverse isolates. Recombinant atpF1, if sufficiently conserved across isolates, could contribute to this broad protection.

What research gaps exist in understanding the role of ATP synthase in Brucella ovis pathogenesis?

Several significant research gaps remain in understanding ATP synthase's role in B. ovis pathogenesis:

• Metabolic adaptation during infection:

  • How ATP synthase activity is regulated during different stages of infection

  • Whether ATP synthase components are differentially expressed in response to host environments

  • Comparison of ATP synthase activity between virulent field isolates and attenuated strains

• Contribution to intracellular survival:

  • The specific role of ATP synthase in adapting to nutrient limitation within host cells

  • Whether ATP synthase components interact with host factors

  • How ATP synthase activity relates to the observed differences in intracellular growth between B. ovis strains

• Structural biology insights:

  • High-resolution structures of B. ovis ATP synthase components are lacking

  • How species-specific variations in ATP synthase structure might influence function

  • Potential structural interactions between ATP synthase and other bacterial factors

• Host-specific adaptation:

  • How ATP synthase components in B. ovis might be adapted to its narrow host range

  • Comparison with ATP synthase from zoonotic Brucella species with broader host ranges

  • Correlation between genome degradation in B. ovis and potential modifications in energy metabolism

• Therapeutic targeting potential:

  • Whether ATP synthase components represent viable therapeutic targets

  • If species-specific variations could be exploited for selective inhibition

  • How disruption of ATP synthase affects bacterial virulence in vivo

Investigating these gaps would provide valuable insights into the fundamental biology of B. ovis and could lead to new strategies for diagnosis, prevention, and treatment of infections.

What emerging technologies and approaches are advancing the study of bacterial ATP synthase components?

Several cutting-edge technologies are enhancing our understanding of bacterial ATP synthase components:

• Cryo-electron microscopy (Cryo-EM):

  • Enables high-resolution structural determination of large protein complexes like ATP synthase

  • Allows visualization of different conformational states during the catalytic cycle

  • Could reveal structural adaptations specific to B. ovis ATP synthase

• Advanced mass spectrometry techniques:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for protein dynamics

  • Cross-linking mass spectrometry (XL-MS) to map protein-protein interactions

  • Protein footprinting to identify exposed surfaces and binding interfaces

• Single-molecule techniques:

  • Förster resonance energy transfer (FRET) to study conformational changes

  • Optical tweezers to measure mechanical forces during ATP synthesis

  • Single-molecule tracking in live bacteria to study ATP synthase mobility and localization

• Systems biology approaches:

  • Multi-omics integration to correlate ATP synthase expression with metabolic states

  • Flux balance analysis to model energy production during different infection stages

  • Network analysis to understand ATP synthase regulation in context of global metabolism

• CRISPR-based technologies:

  • CRISPRi for conditional knockdown of ATP synthase components

  • CRISPR-based screening to identify genetic interactions with ATP synthase genes

  • Base editing for precise modification of key residues without complete gene disruption

• In vivo imaging advances:

  • ATP sensors to visualize energy production in live bacteria during infection

  • Super-resolution microscopy to study ATP synthase distribution and clustering

  • Intravital microscopy to track bacterial metabolism in animal models