Recombinant Brucella melitensis biotype 1 ATP synthase epsilon chain (atpC)

How does recombinant atpC differ from native atpC in Brucella melitensis?

Recombinant atpC differs from native atpC primarily in its expression system and potential modifications:

• Expression system: Recombinant atpC is typically expressed in heterologous systems like E. coli, whereas native atpC is expressed within B. melitensis

• Purification tags: Recombinant versions often contain affinity tags (His-tag, GST-tag) to facilitate purification

• Post-translational modifications: The native protein may contain species-specific modifications absent in recombinant versions

• Solubility and folding: Recombinant proteins may exhibit different folding characteristics depending on expression conditions

To ensure functional similarity, researchers typically validate recombinant proteins through structural and functional assays comparing them with native proteins extracted directly from B. melitensis cultures.

What are the typical yields when expressing recombinant Brucella melitensis atpC protein in E. coli systems?

Expression yields of recombinant B. melitensis proteins in E. coli systems vary based on expression conditions. While specific data for atpC is limited, comparable Brucella proteins show the following typical yields:

Table 1: Typical Expression Yields of Recombinant Brucella Proteins in E. coli Systems

For optimal expression of B. melitensis atpC, researchers should optimize:

• Expression temperature (typically 25-30°C to enhance solubility)

• IPTG concentration (0.1-0.5 mM)

• Post-induction time (4-16 hours)

• Media composition (enriched media like Terrific Broth often improve yields)

What are the optimal protocols for cloning and expressing recombinant B. melitensis atpC for immunological studies?

For optimal cloning and expression of B. melitensis atpC, follow these methodological steps:

Cloning Protocol:

• Gene Isolation: Amplify the atpC gene from B. melitensis biotype 1 genomic DNA using high-fidelity PCR with gene-specific primers containing appropriate restriction sites

• Vector Selection: Choose an expression vector with an appropriate tag (His-tag is commonly used) and promoter (T7 promoter systems work well for Brucella proteins)

• Restriction Digestion and Ligation: Digest both PCR product and vector with compatible restriction enzymes, ligate, and transform into E. coli DH5α for plasmid propagation

• Verification: Confirm correct insertion by colony PCR and sequencing

Expression Protocol:

• Expression Host: Transform the verified plasmid into E. coli BL21(DE3) or other expression strains

• Culture Conditions: Grow in LB or TB medium at 37°C until OD₆₀₀ reaches 0.6-0.8

• Induction: Add IPTG to a final concentration of 0.5 mM and continue growth at 30°C for 6 hours

• Cell Harvest: Centrifuge cultures at 5000×g for 15 minutes at 4°C

For immunological studies, ensure protein purity exceeds 95% using chromatography techniques similar to those employed for other Brucella proteins .

How can researchers troubleshoot low expression or insolubility issues with recombinant B. melitensis atpC?

When experiencing low expression or insolubility issues with recombinant B. melitensis atpC, implement the following troubleshooting strategies:

For Low Expression:

• Codon Optimization: Analyze the atpC gene sequence for rare codons in E. coli and consider codon optimization or using a host strain with rare tRNA genes

• Promoter Strength: Test different promoter systems (T7, trc, tac)

• Host Strain Variation: Evaluate multiple E. coli strains (BL21, Rosetta, Origami)

• Induction Parameters: Systematically test various IPTG concentrations (0.1-1.0 mM) and post-induction temperatures (16-37°C)

For Insolubility Issues:

• Lower Temperature: Reduce post-induction temperature to 16-20°C and extend expression time to 16-24 hours

• Co-expression: Co-express with chaperones (GroEL/GroES, DnaK/DnaJ)

• Fusion Tags: Test solubility-enhancing fusion partners (MBP, SUMO, TrxA)

• Refolding Strategies: If inclusion bodies persist, develop refolding protocols using step-wise dialysis against decreasing concentrations of chaotropic agents

Experimental Design Table:

Table 2: Optimization Matrix for Recombinant B. melitensis atpC Expression

This experimental design allows systematic identification of optimal conditions through analysis of both expression level and solubility percentage for each condition .

What purification strategy yields the highest purity for recombinant B. melitensis atpC?

A multi-step purification strategy is recommended for obtaining high-purity recombinant B. melitensis atpC:

Primary Purification (Affinity Chromatography):

• Cell Lysis: Resuspend bacterial pellet in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF) and lyse using sonication or French press

• Clarification: Centrifuge at 15,000×g for 30 minutes at 4°C

• IMAC Purification: Load supernatant onto Ni-NTA column equilibrated with binding buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole)

• Washing: Wash with binding buffer containing 50 mM imidazole to remove weakly bound contaminants

• Elution: Elute target protein with elution buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole)

Secondary Purification:

• Ion Exchange Chromatography: Apply dialyzed protein to Q-Sepharose column (if theoretical pI < 7) or SP-Sepharose (if pI > 7)

• Size Exclusion Chromatography: Perform final polishing step using Superdex 75 or Superdex 200 column

Purification Yield and Purity Assessment Table:

Table 3: Expected Purification Parameters for Recombinant B. melitensis atpC

For highest purity (>98%), consider adding an endotoxin removal step using Triton X-114 phase separation if the protein will be used for immunological studies .

How can the enzymatic activity of recombinant B. melitensis atpC be assessed in vitro?

The enzymatic activity of recombinant B. melitensis atpC can be assessed through multiple complementary approaches:

ATP Synthase Complex Reconstitution:

• Purify all components of the F₁-ATPase complex (α, β, γ, δ, and ε[atpC] subunits)

• Reconstitute the complex in vitro under controlled conditions

• Measure ATP synthesis/hydrolysis activity using the following assays:

ATP Hydrolysis Assay:

• Colorimetric Phosphate Release Assay:

  • Incubate reconstituted complex with ATP at 37°C

  • Measure inorganic phosphate release using malachite green or molybdate reagents

  • Calculate specific activity (μmol Pi released/min/mg protein)

• Coupled Enzyme Assay:

  • Link ATP hydrolysis to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Monitor decrease in absorbance at 340 nm

  • Calculate ATP hydrolysis rate based on NADH consumption

Regulatory Function Assessment:

• Inhibition Studies: Test atpC's regulatory role by measuring ATP hydrolysis rates with varying ATP/ADP ratios

• Binding Assays: Use isothermal titration calorimetry (ITC) to measure binding affinity between atpC and other F₁ components

Expected Activities Table:

Table 4: ATP Hydrolysis Activities of Reconstituted ATP Synthase Complexes

These assays will help determine whether the recombinant atpC retains its native regulatory functions within the ATP synthase complex .

How can researchers design knockdown/knockout experiments to study atpC function in B. melitensis?

Designing effective knockdown/knockout experiments for studying atpC function in B. melitensis requires careful consideration of this gene's potential essentiality. Below is a methodological approach:

Preliminary Essentiality Assessment:

• Bioinformatic Analysis: Compare atpC to homologs in related species where essentiality has been determined

• Growth Curve Analysis: Test growth under different energy conditions to assess potential essentiality

Conditional Knockout Strategy:

• Plasmid Construction:

  • Create a complementation plasmid containing atpC under an inducible promoter (tetracycline-responsive)

  • Construct a knockout plasmid with homologous regions flanking atpC and a selectable marker

• Two-Step Process:

  • First, introduce the complementation plasmid

  • Second, perform gene replacement to delete chromosomal atpC

Experimental Protocol:

• Transform B. melitensis with pTet-atpC (tetracycline-inducible atpC)

• Maintain expression with tetracycline

• Transform with knockout plasmid (containing ~1kb homologous regions flanking atpC)

• Select transformants on appropriate antibiotics

• Confirm deletion by PCR and Southern blot

Alternative Approaches for Partial Function Studies:

CRISPR Interference (CRISPRi):

• Express catalytically inactive Cas9 (dCas9) in B. melitensis

• Design sgRNAs targeting atpC promoter region

• Induce dCas9 and sgRNA expression to repress atpC transcription

• Monitor growth defects and phenotypic changes

Site-Directed Mutagenesis:

• Identify critical residues in atpC through sequence alignment and structural modeling

• Create point mutations in these residues

• Replace wild-type atpC with mutant versions

• Assess phenotypic effects

Phenotypic Characterization Table:

Table 5: Experimental Design for atpC Functional Characterization

These approaches allow comprehensive functional characterization while accounting for potential essentiality of the atpC gene in B. melitensis .

How can researchers analyze the immune response to recombinant B. melitensis atpC in animal models?

Comprehensive analysis of immune responses to recombinant B. melitensis atpC in animal models requires evaluation of both humoral and cell-mediated immunity through a systematic approach:

Experimental Design for Immune Response Analysis:

Animal Model Selection:

• Primary models: BALB/c mice (for initial screening), C57BL/6 mice (for mechanistic studies)

• Secondary models: Guinea pigs (for hypersensitivity), Large animals (sheep, goats) for translational studies

Immunization Protocol:

• Groups:

  • Experimental (atpC + adjuvant)

  • Adjuvant-only control

  • Positive control (whole-cell vaccine)

  • Negative control (saline)

• Schedule: Prime (Day 0) and boost (Days 21 and 42)

• Route: Subcutaneous or intramuscular

• Sampling: Pre-immune (Day 0), post-prime (Day 21), post-boost (Days 42 and 56)

Comprehensive Immune Response Analysis:

1. Humoral Immunity Assessment:

• ELISA: Measure specific IgG, IgG1, IgG2a titers

• Western Blot: Confirm antibody specificity

• Avidity ELISA: Determine antibody maturation using chaotropic agents

2. Cell-Mediated Immunity Assessment:

• Lymphocyte Proliferation: Measure T-cell proliferation in response to atpC restimulation using thymidine incorporation or CFSE dilution

• ELISpot: Enumerate IFN-γ, IL-2, IL-4-secreting cells

• Flow Cytometry: Analyze T-cell subsets (CD4+, CD8+) and activation markers

• Cytokine Profiling: Measure cytokine production (IFN-γ, TNF-α, IL-2, IL-4, IL-10) in culture supernatants by ELISA

3. Functional Assays:

• Macrophage Activation: Assess activation of macrophages by immune serum

• Opsonophagocytosis: Evaluate antibody-mediated uptake of Brucella by phagocytes

• Protection Studies: Challenge immunized animals with virulent B. melitensis

Expected Results Template:

Table 8: Expected Immune Response Parameters to Recombinant atpC Immunization

This comprehensive approach allows detailed characterization of both humoral and cell-mediated immune responses to recombinant atpC, facilitating comparison with other Brucella antigens .

What structural and functional differences exist between ATP synthase epsilon chains across Brucella species and how do they impact virulence?

Structural and functional comparison of ATP synthase epsilon chains across Brucella species reveals subtle differences that may impact bacterial physiology and virulence:

Sequence and Structural Analysis:

• Primary Sequence Comparison: ATP synthase epsilon chains show >95% sequence identity across Brucella species, with key differences primarily in non-catalytic regions

• Domain Organization: All Brucella epsilon chains contain an N-terminal beta-barrel domain and a C-terminal helix-turn-helix motif

• Species-Specific Variations: Most variations occur in surface-exposed loops that may interact with other F₁ subunits

Functional Implications of Structural Differences:

• Regulatory Efficiency: Subtle sequence variations may affect the regulatory efficiency of ATP synthesis/hydrolysis

• Protein-Protein Interactions: Species-specific residues may alter interactions with other ATP synthase components

• Stability Under Stress: Differences in thermodynamic stability may affect function under various stress conditions

Impact on Virulence:
Analysis of transcriptional profiles and virulence studies suggests:

• Metabolic Adaptation: Different Brucella species show distinct transcriptional regulation of ATP synthase components during host cell infection

• Growth Rate Correlation: Expression levels of ATP synthase genes correlate with growth rates in different intracellular environments

• Stress Response Integration: The epsilon chain likely interfaces with stress response systems that are crucial for intracellular survival

Comparative Analysis Table:

Table 9: Comparative Analysis of ATP Synthase Epsilon Chain Across Brucella Species

These differences, though subtle, may contribute to host specificity and virulence differences among Brucella species by affecting metabolic adaptation to various intracellular environments .

How does the interaction between atpC and other ATP synthase subunits change under different environmental stresses relevant to Brucella infection?

The interactions between atpC (epsilon chain) and other ATP synthase subunits in Brucella exhibit dynamic changes under different environmental stresses, which likely play crucial roles during infection:

Key Environmental Stresses During Infection:

• Acidic pH: Encountered in phagolysosomes (pH 4.0-5.5)

• Nutrient Limitation: Restricted carbon sources and micronutrients

• Oxidative Stress: Reactive oxygen species produced by host cells

• Iron Restriction: Host sequestration of iron as antimicrobial defense

Changes in Protein-Protein Interactions:

1. Under Acidic Conditions:

• The epsilon chain undergoes conformational changes that modify its interaction with the gamma and beta subunits

• This conformational change likely protects against wasteful ATP hydrolysis under acidic conditions

• Studies of bacterial ATP synthases show that acidic pH promotes a more compact conformation of the epsilon chain

2. During Nutrient Limitation:

• ADP/ATP ratio changes affect epsilon chain conformation

• High ADP levels promote an extended conformation that inhibits ATP hydrolysis

• This mechanism conserves ATP during nutrient-limited conditions of intracellular life

3. Under Oxidative Stress:

Experimental Approaches to Study These Interactions:

Table 10: Methods to Analyze atpC Interactions Under Stress Conditions

These dynamic interactions likely contribute to Brucella's ability to maintain energy homeostasis during infection, which is critical for intracellular survival and virulence .

What role does the ATP synthase epsilon chain play in the antibiotic resistance mechanisms of B. melitensis?

The ATP synthase epsilon chain (atpC) may contribute to antibiotic resistance in B. melitensis through several mechanisms, though research in this specific area is emerging:

Potential Roles in Antibiotic Resistance:

1. Energy-Dependent Efflux Systems:

• ATP synthase provides energy for ATP-binding cassette (ABC) transporters

• Efficient ATP generation is critical for maintaining efflux pump activity

• Studies of B. melitensis and related species show that expression of ATP synthase components correlates with resistance to certain antibiotics

2. Persister Cell Formation:

• ATP synthase regulation affects bacterial energy state

• Low ATP levels are associated with persister cell formation

• Studies on Brucella persister cells demonstrate altered expression of energy metabolism genes

• Toxin-antitoxin systems involved in persister formation may interact with ATP synthase function

3. Metabolic Adaptation and Resistance:

Evidence from Related Research:

Studies on B. canis demonstrated that deletion of regulatory factors affecting metabolism (such as MucR) resulted in altered sensitivity to various antibiotics including ciprofloxacin, doxycycline, and rifampin . While not directly focused on atpC, these findings suggest metabolic regulators impact antibiotic resistance in Brucella species.

Experimental Data on Antibiotic Sensitivity:

What are the most sensitive detection methods for analyzing atpC expression in Brucella under different infection conditions?

Detecting atpC expression in Brucella under various infection conditions requires highly sensitive methods due to the relatively low abundance of this transcript. The following techniques offer complementary approaches:

RNA-Based Detection Methods:

1. Quantitative RT-PCR (RT-qPCR):

• Sensitivity: Can detect 10-100 copies of target transcript

• Protocol Optimization:

  • Use pathogen-specific primers to avoid host RNA interference

  • Implement Brucella genome-directed primers (BmGDP) for specific amplification

  • Include multiple reference genes (16S rRNA, GAPDH) for normalization

• Sample Processing:

  • Rapid RNA stabilization immediately upon sample collection

  • Host-pathogen RNA separation to enrich Brucella transcripts

2. RNA-Seq with Pathogen Enrichment:

• Sensitivity: Genome-wide coverage with detection of low-abundance transcripts

• Advantages: Unbiased detection of novel transcripts, splice variants

• Protocol Considerations:

  • Selective depletion of host rRNA and mRNA

  • Linear amplification of bacterial transcripts

  • Deep sequencing (>30 million reads) to ensure coverage of low-abundance transcripts

Primer Design for atpC Detection:

Table 12: Recommended Primers for B. melitensis atpC Detection

Protein-Based Detection Methods:

Immunoblotting:

• Generate specific antibodies against B. melitensis atpC

• Use highly sensitive detection systems (chemiluminescence, fluorescence)

• Compare expression across infection time points

Mass Spectrometry:

• Selected Reaction Monitoring (SRM) for targeted detection

• Use isotopically labeled peptide standards for absolute quantification

Infection Model Considerations:
For meaningful results, standardize infection parameters including:

• MOI (multiplicity of infection): 50-100 bacteria per cell

• Cell type: Macrophages (J774.A1, RAW264.7) or non-phagocytic cells (HeLa)

• Time points: Early (15min, 1h, 4h) and late infection (24h, 48h)

These methods enable precise quantification of atpC expression during different stages of infection, providing insights into its role in Brucella pathogenesis .

What bioinformatic tools and databases are most useful for studying the structure and evolution of Brucella ATP synthase components?

Researchers studying the structure and evolution of Brucella ATP synthase components can utilize a comprehensive set of bioinformatic tools and databases:

Sequence Analysis Tools:

1. Primary Sequence Analysis:

• NCBI BLAST/PSI-BLAST: Identify homologs across bacterial species

• Clustal Omega/MUSCLE: Generate multiple sequence alignments of atpC and related proteins

• MEGA X: Perform phylogenetic analysis of ATP synthase components

2. Evolutionary Analysis:

• PAML: Detect positive selection in ATP synthase genes

• ConSurf: Map conservation onto protein structures

• FunDi/MISTIC: Identify co-evolving residues in ATP synthase complexes

Structural Analysis Tools:

3. Protein Structure Prediction:

• AlphaFold2/RoseTTAFold: Generate accurate structural models of atpC

• SWISS-MODEL: Homology modeling using existing ATP synthase structures

• I-TASSER: Integrate threading with ab initio modeling

4. Molecular Dynamics:

• GROMACS/NAMD: Simulate atpC dynamics under different conditions

• AMBER: Analyze protein-protein interactions within ATP synthase complex

• Normal Mode Analysis: Identify functional motions in atpC

Key Databases:

Table 13: Essential Databases for Brucella ATP Synthase Research

Specialized Resources for ATP Synthase Research:

• AtpBD: Database of ATP synthase sequences and structures

• BRENDA: Enzyme-specific information on ATP synthases

• BrucellaBase/VFDB: Virulence factor databases with ATP synthase information

Workflow for Comprehensive Analysis:

• Retrieve atpC sequences from multiple Brucella species using PATRIC/NCBI

• Perform multiple sequence alignment and phylogenetic analysis

• Identify conserved and variable regions across species

• Generate structural models and analyze species-specific variations

• Simulate protein dynamics under conditions relevant to infection

• Map results onto metabolic networks and virulence pathways

This integrated bioinformatic approach provides insights into the structural basis of atpC function and its evolutionary adaptations across Brucella species .

What are the best approaches for integrating transcriptomic, proteomic, and functional data to understand atpC's role in Brucella pathogenesis?

Integrating multi-omics data to understand atpC's role in Brucella pathogenesis requires sophisticated methodological approaches:

Multi-Omics Data Integration Strategy:

1. Coordinated Experimental Design:

• Matched Sampling: Collect transcriptomic, proteomic, and functional data from the same experimental conditions

• Temporal Resolution: Include multiple time points (0, 1, 4, 12, 24, 48h post-infection)

• Consistent Models: Use identical infection models across all omics platforms

2. Data Generation Methods:

Transcriptomics:

• RNA-Seq with specific enrichment for Brucella transcripts

• Targeted RT-qPCR validation of ATP synthase components

• Single-cell RNA-Seq to capture heterogeneity in bacterial populations

Proteomics:

• TMT-based quantitative proteomics for relative protein abundance

• Phosphoproteomics to detect post-translational modifications

• Protein-protein interaction studies via co-immunoprecipitation

Functional Assays:

• ATP synthesis/hydrolysis activity measurements

• Bacterial fitness assessments in various conditions

• Virulence assays in cellular and animal models

3. Computational Integration Methods:

Table 14: Computational Methods for Multi-Omics Integration

Case Study Approach:
Based on existing Brucella research, an ideal integration strategy would follow this workflow:

• Generate Baseline Data:

  • Transcriptome and proteome profiles of wild-type Brucella

  • ATP synthase activity measurements under standard conditions

• Create Perturbations:

  • Generate atpC mutants (point mutations in key residues)

  • Subject bacteria to relevant stresses (pH, oxidative, nutrient limitation)

• Measure Multi-omics Responses:

  • Transcriptome changes in response to perturbations

  • Corresponding proteomic alterations

  • Metabolic shifts using metabolomics

  • Functional outcomes (survival, virulence)

• Integrate Data:

  • Apply network analysis to identify modules connecting atpC to virulence factors

  • Use time-course data to establish causality

  • Validate key connections through targeted experiments

This integrated approach has successfully revealed complex regulatory networks in Brucella, as demonstrated in previous studies examining host-pathogen interactions during infection .

By implementing these methodologies, researchers can establish a comprehensive understanding of how atpC contributes to Brucella's pathogenic mechanisms, potentially identifying novel therapeutic targets.

What are the most promising therapeutic applications targeting ATP synthase in Brucella infections?

ATP synthase in Brucella represents a promising therapeutic target due to its essential role in bacterial energy metabolism. Several therapeutic strategies show particular promise:

ATP Synthase Inhibitor Development:

• Small Molecule Inhibitors: Design of specific inhibitors targeting unique features of Brucella ATP synthase

• Diarylquinolines: Adaptation of compounds similar to bedaquiline (TB drug targeting ATP synthase)

• Natural Product Derivatives: Screening of compounds like resveratrol and oligomycin for Brucella-specific activity

Combination Therapy Approaches:

• Metabolic Sensitization: ATP synthase inhibitors may sensitize Brucella to conventional antibiotics

• Persister Elimination: Targeting ATP synthase to prevent or reverse persister formation

• Host-Directed Therapy: Combining ATP synthase inhibitors with immunomodulators

Therapeutic Potential Assessment:

Table 15: Evaluation of ATP Synthase-Targeting Therapeutic Strategies

Current Progress and Promising Leads:
While specific inhibitors for Brucella ATP synthase are still under development, research on related bacterial ATP synthases has identified several promising pharmacophores that could be adapted for Brucella treatment.

Future Research Priorities:

• Structural characterization of Brucella ATP synthase to enable structure-based drug design

• High-throughput screening of compound libraries against purified Brucella ATP synthase

• Development of cellular assays to measure ATP synthase inhibition in live Brucella

• In vivo validation of lead compounds in animal models of brucellosis

These therapeutic approaches could help address the limitations of current brucellosis treatments, particularly persistent infections and antibiotic resistance .

What are the current knowledge gaps in understanding B. melitensis atpC function and how might they be addressed?

Despite progress in understanding Brucella biology, several critical knowledge gaps remain regarding B. melitensis atpC function:

Current Knowledge Gaps:

1. Structural Characterization:

• Lack of experimentally determined structure for B. melitensis atpC

• Limited understanding of species-specific structural features

• Insufficient data on conformational changes during regulation

2. Regulatory Mechanisms:

• Incomplete understanding of transcriptional regulation of atpC

• Limited data on post-translational modifications

• Poor characterization of protein-protein interactions within the ATP synthase complex

3. Functional Significance:

• Uncertain contribution to virulence and intracellular survival

• Limited understanding of role in stress responses

• Unknown contribution to antimicrobial resistance

4. Host-Pathogen Interactions:

• Unclear whether atpC is recognized by the host immune system

• Limited data on expression changes during different infection phases

• Poor understanding of role in metabolic adaptation to host environment

Research Approaches to Address These Gaps:

Table 16: Research Strategies to Address Knowledge Gaps

Integrative Research Priorities:

• Comprehensive Structural Biology: Determine atpC structure in different conformational states

• Systems Biology Approach: Map atpC within global regulatory and metabolic networks

• In vivo Expression Studies: Track atpC expression during all stages of infection

• Comparative Analysis: Analyze atpC function across Brucella species with different host tropisms

• Translational Research: Develop atpC-based diagnostics and therapeutics

Technological Innovations Needed:

• Better genetic manipulation tools for Brucella

• Improved methods for studying host-pathogen interactions at single-cell resolution

• Enhanced computational approaches for integrating multi-omics data

Addressing these knowledge gaps would significantly advance our understanding of Brucella pathogenesis and potentially reveal new targets for diagnostic and therapeutic development .

How might ongoing technological advances in protein engineering and synthetic biology be applied to study or manipulate Brucella atpC?

Cutting-edge technologies in protein engineering and synthetic biology offer promising approaches to advance Brucella atpC research:

Advanced Protein Engineering Applications:

1. Structure-Function Manipulation:

• Site-Directed Mutagenesis: Create precise mutations in functional domains to analyze regulatory mechanisms

• Domain Swapping: Exchange domains between Brucella species to identify species-specific functions

• Protein Labeling: Introduce minimal fluorescent tags for live-cell imaging of ATP synthase dynamics

2. Protein Interaction Studies:

• Proximity Labeling: Adapt BioID or APEX2 systems for mapping atpC interactions in living Brucella

• Split Fluorescent Proteins: Develop complementation assays to visualize ATP synthase assembly

• Nanobody Development: Create specific nanobodies against atpC for intracellular tracking and perturbation

Synthetic Biology Approaches:

3. Genetic Circuit Design:

• Inducible Expression Systems: Develop tightly regulated systems for atpC expression

• Genetic Sensors: Create reporters linked to ATP synthase activity

• CRISPR Interference: Implement tunable repression of atpC expression

4. Minimal ATP Synthase Systems:

• Reconstitution Studies: Build minimal functional ATP synthase complexes

• Orthogonal Systems: Introduce non-native ATP synthase components to study compatibility

Emerging Technologies with High Potential:

Table 17: Cutting-Edge Technologies for Brucella atpC Research

Practical Implementation Strategy:

• First Phase: Develop tagged versions of atpC that maintain native function

• Second Phase: Create conditional expression/degradation systems

• Third Phase: Implement synthetic circuits to manipulate ATP synthase activity

• Fourth Phase: Engineer novel functions or regulatory mechanisms

Potential Applications:

• Attenuated Vaccine Development: Engineered atpC variants for balanced attenuation

• Diagnostic Tools: Engineered bacteria with reporters linked to ATP synthase activity

• Biosensors: Bacteria with ATP synthase-based detection systems

• Fundamental Research: Understanding minimal requirements for ATP synthase function