Recombinant Clavibacter michiganensis subsp. sepedonicus ATP synthase subunit c (atpE)

What is the primary structure and biochemical properties of Clavibacter michiganensis subsp. sepedonicus atpE protein?

The atpE protein from C. michiganensis subsp. sepedonicus is a 77 amino acid protein that functions as subunit c of the ATP synthase complex. The full amino acid sequence is:

MDPIITAEITGNIATVGYGLAAIGPGIGVGIVAGKTVEAMARQPEMAGSLRTTMFLGIAFSEALALIGLATYFIFTN

This protein has several key properties that make it important for research:

• It forms part of the F₀ sector of ATP synthase that is embedded in the bacterial membrane

• The protein is highly conserved within bacterial species but has specific variations between subspecies

• When expressed recombinantly with an N-terminal His tag, it can be purified to >90% purity using standard methods

• The protein is typically stored as a lyophilized powder in Tris/PBS-based buffer with 6% trehalose at pH 8.0

How does the amino acid sequence of atpE differ between Clavibacter michiganensis subspecies?

Comparing the atpE sequences between C. michiganensis subspecies reveals subtle but potentially significant differences:

The full sequences show over 95% similarity, with only a few amino acid substitutions (including T6L shown above) . These differences may contribute to subspecies-specific adaptations and could be relevant for developing diagnostic tests that distinguish between the subspecies.

What research techniques are available for expressing and purifying recombinant atpE protein?

Several methodological approaches can be employed:

• Expression system selection: The most effective system is E. coli, which allows for high-yield expression of the protein with an N-terminal His tag . This approach has been documented to yield protein with greater than 90% purity.

• Purification protocol:

  • Initial centrifugation of bacterial culture

  • Lysis using appropriate buffer systems

  • Affinity chromatography using Ni-NTA columns to capture His-tagged protein

  • Elution with imidazole gradient

  • Size exclusion chromatography for further purification if needed

  • Quality assessment using SDS-PAGE

• Storage recommendations:

  • Store at -20°C/-80°C upon receipt

  • Aliquot for multiple use to avoid repeated freeze-thaw cycles

  • For working aliquots, store at 4°C for up to one week

• Reconstitution protocol:

  • Briefly centrifuge vial before opening

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

  • Add glycerol to 5-50% final concentration for long-term storage

How can recombinant atpE be used for developing detection methods for Clavibacter michiganensis in agricultural samples?

The atpE protein plays a crucial role in molecular diagnostic test development:

• Generation of specific antibodies: Recombinant atpE can be used to generate highly specific polyclonal or monoclonal antibodies that distinguish between C. michiganensis subspecies, enabling the development of serological detection methods.

• PCR-based detection systems: The atpE gene sequence has been utilized as part of multiplex PCR detection systems. In particular, researchers have developed a multiplex real-time PCR protocol that can simultaneously detect C. michiganensis subsp. sepedonicus and Ralstonia solanacearum race 3 in potato tubers .

• Internal control development: The chloroplastic ATP synthase beta-subunit gene has been used as an internal control in PCR assays to verify the absence of false negatives due to PCR inhibition or nucleic acid extraction failure . This control is particularly important because:

  • It allows verification that the PCR reaction is working properly

  • It confirms successful nucleic acid extraction from the sample

  • It prevents false negative reports in quarantine pathogen detection

• Optimization considerations: When designing multiplex PCR assays incorporating atpE-based detection, careful optimization of primer and probe concentrations is essential to prevent competitive amplification effects between targets .

What structural and functional studies can be performed with recombinant atpE to understand ATP synthase mechanics?

Researchers can employ several approaches:

• Structural analysis:

  • X-ray crystallography of the isolated subunit c or reconstituted F₀ complex

  • Cryo-electron microscopy to visualize the protein within the ATP synthase complex

  • NMR studies to analyze dynamics and interactions with other ATP synthase components

• Functional characterization:

  • Reconstitution of the protein into liposomes to study proton translocation

  • Site-directed mutagenesis to identify critical residues for function

  • ATP synthesis/hydrolysis assays with reconstituted complexes

• Protein-protein interaction studies:

  • Cross-linking experiments to identify interaction partners within the ATP synthase complex

  • Surface plasmon resonance to measure binding kinetics with other subunits

  • Co-immunoprecipitation studies using antibodies against the recombinant protein

These approaches can reveal how the bacterial ATP synthase performs the same core functions as more complex mitochondrial equivalents, despite its simpler structure .

How reliable are atpE-based molecular diagnostic tests for detecting Clavibacter michiganensis in field samples?

Molecular diagnostic tests targeting atpE must balance several factors:

• Sensitivity considerations:

  • For quarantine pathogens like C. michiganensis subsp. sepedonicus, achieving a low detection level is crucial

  • The bacteria can survive latently in potato tubers at low concentrations

  • Optimized PCR protocols can detect very low numbers of bacterial cells

• Specificity factors:

  • The high conservation of atpE within subspecies (>99% ANI within subspecies) ensures reliable detection

  • Sufficient sequence variation between subspecies (89-95% ANI between subspecies) allows discrimination

  • Careful primer design is essential to avoid cross-reactivity with other soil bacteria

• Validation requirements:

  • Comparison with established detection methods is necessary

  • Testing across diverse field samples with varying bacterial loads

  • Assessment of potential inhibitory compounds in plant material

• Practical implementation:

  • Multiplex approaches that detect multiple targets simultaneously offer time and labor savings

  • Internal controls are essential to certify the absence of false negatives

  • Standard protocols must be followed to ensure reproducibility across testing laboratories

How can structural comparisons between C. michiganensis atpE and homologs from other bacteria inform antimicrobial development?

Structural analysis provides several research opportunities:

• Comparative structural biology:

  • ATP synthase subunit c is essential for bacterial energy metabolism

  • The protein shows sufficient structural differences between pathogenic bacteria and eukaryotic hosts

  • These differences can be exploited for selective targeting

• Drug discovery approaches:

  • Virtual screening against the structure of C. michiganensis atpE to identify potential inhibitors

  • Structure-based drug design targeting unique features of the bacterial protein

  • Fragment-based screening to identify molecules that bind to functional sites

• Validation methodologies:

  • In vitro assays using purified recombinant protein to measure binding and inhibition

  • Bacterial growth inhibition studies with identified compounds

  • Validation in plant infection models to assess efficacy in reducing disease

• Resistance mechanism studies:

  • Monitoring of natural variations in atpE sequences across bacterial populations

  • Directed evolution experiments to identify potential resistance mutations

  • Designing inhibitor combinations to address potential resistance

The ATP synthase is increasingly recognized as a promising antibiotic target due to its essential role in bacterial metabolism and the structural differences between bacterial and host proteins .

What role does atpE play in the pathogenicity and host adaptation of Clavibacter michiganensis subspecies?

This complex question requires several research approaches:

• Comparative genomics:

  • Analysis of atpE sequence variations between pathogenic and non-pathogenic Clavibacter strains

  • Examination of selection pressure on atpE across different host-adapted subspecies

  • Correlation of atpE sequence with host range and virulence

• Functional genetics studies:

  • Creation of atpE mutants with altered ATP synthase function

  • Assessment of these mutants for changes in virulence, growth rate, and stress tolerance

  • Complementation studies to confirm the role of specific atpE variants

• Host-pathogen interaction analysis:

  • Examination of ATP production during different stages of infection

  • Assessment of energy requirements during colonization of different plant tissues

  • Evaluation of ATP synthase activity under host defense response conditions

The taxonomic differences between C. michiganensis subspecies (with ANI values between subspecies of 89.18–95.01%) suggest potential adaptation to different hosts, which may be reflected in metabolic enzymes like ATP synthase.

How can protein engineering of recombinant atpE advance our understanding of proton translocation mechanisms in ATP synthases?

Protein engineering offers several research possibilities:

• Site-directed mutagenesis approaches:

  • Systematic mutation of conserved residues in the proton channel

  • Creation of chimeric proteins combining elements from different bacterial species

  • Introduction of reporter groups at specific positions to monitor conformational changes

• Functional assay development:

  • Reconstitution of mutant proteins into liposomes to measure proton translocation

  • ATP synthesis/hydrolysis assays with reconstituted complexes

  • Biophysical measurements of protein dynamics during catalysis

• Advanced structural studies:

  • Time-resolved structural analysis to capture intermediate states

  • Molecular dynamics simulations to model proton movement through the c-ring

  • Single-molecule studies to observe rotational dynamics

These approaches can help elucidate the path of transmembrane proton translocation and provide models for understanding the roles of specific residues in ATP synthase function .

What are the main challenges in producing functionally active recombinant atpE, and how can they be addressed?

Several technical challenges must be overcome:

• Membrane protein expression issues:

  • Challenge: As a hydrophobic membrane protein, atpE can be difficult to express in soluble form

  • Solution: Use specialized E. coli strains designed for membrane protein expression, optimize growth temperature (typically lower than standard), and employ specialized media formulations

• Protein folding and stability:

  • Challenge: Maintaining native conformation outside the membrane environment

  • Solution: Addition of stabilizing agents like trehalose (6%) , careful buffer optimization, and storage as lyophilized powder

• Functional reconstitution:

  • Challenge: Ensuring the recombinant protein maintains native activity

  • Solution: Reconstitution into liposomes or nanodiscs with appropriate lipid composition, assembly with other ATP synthase components for functional studies

• Long-term storage stability:

  • Challenge: Preventing protein degradation during storage

  • Solution: Store at -20°C/-80°C with addition of 5-50% glycerol for long-term storage, aliquot to avoid repeated freeze-thaw cycles

How can researchers effectively evaluate the quality and activity of recombinant atpE preparations?

Quality control involves multiple analytical approaches:

• Purity assessment:

  • SDS-PAGE analysis with Coomassie or silver staining (should show >90% purity)

  • Western blotting using anti-His antibodies to confirm identity

  • Mass spectrometry for accurate molecular weight determination and sequence verification

• Structural integrity evaluation:

  • Circular dichroism spectroscopy to assess secondary structure

  • Fluorescence spectroscopy to evaluate tertiary structure

  • Limited proteolysis to confirm proper folding

• Functional activity testing:

  • Reconstitution into liposomes to measure proton translocation

  • Assembly with other ATP synthase components to assess complex formation

  • ATP synthesis/hydrolysis assays with reconstituted complexes

• Stability monitoring:

  • Thermal shift assays to measure protein stability

  • Long-term storage studies at different temperatures

  • Freeze-thaw stability assessment

What emerging technologies could advance the study of recombinant atpE and its applications?

Several cutting-edge approaches show promise:

• Cryo-electron microscopy advancements:

  • High-resolution structural determination of complete bacterial ATP synthases in different rotational states

  • Visualization of inhibitor binding sites for drug development

  • Analysis of conformational changes during proton translocation

• Single-molecule techniques:

  • Observing ATP synthase rotation in real-time

  • Measuring forces generated during ATP synthesis

  • Detecting conformational changes during catalysis

• Synthetic biology approaches:

  • Engineering of minimal ATP synthases with defined components

  • Creation of hybrid ATP synthases with novel properties

  • Development of ATP synthases as nanomachines for biotechnology applications

• AI and computational methods:

  • Machine learning for predicting protein-protein interactions within the ATP synthase complex

  • Molecular dynamics simulations of proton movement through the c-ring

  • In silico screening for novel inhibitors targeting bacterial ATP synthases

The application of these technologies to bacterial ATP synthases could provide insights comparable to recent studies on ATP synthases from other organisms, revealing the path of transmembrane proton translocation and providing models for understanding decades of biochemical analysis .

How might comparative studies of atpE across Clavibacter subspecies contribute to our understanding of bacterial evolution and host adaptation?

This research direction offers several insights:

• Evolutionary analysis opportunities:

  • The high conservation within subspecies (>99% ANI) versus moderate diversity between subspecies (89-95% ANI) provides a window into recent evolutionary divergence

  • Comparison of synonymous versus non-synonymous substitutions can reveal selection pressures

  • Reconstruction of ancestral sequences can illuminate the evolutionary trajectory of this protein

• Host adaptation studies:

  • Correlation of atpE sequence variations with host range and environmental niches

  • Analysis of energy metabolism requirements in different plant hosts

  • Investigation of how metabolic efficiency contributes to host specialization

• Taxonomic refinement:

  • The clear delineation between subspecies based on ANI and dDDH values supports the reclassification of Clavibacter michiganensis subspecies

  • Genomic approaches provide higher resolution than traditional taxonomic methods

  • Integration of functional data with genomic analysis can provide a more comprehensive taxonomic framework

These comparative approaches can contribute to both fundamental understanding of bacterial evolution and practical applications in plant pathology and disease management.