Recombinant Pectobacterium carotovorum subsp. carotovorum ATP synthase subunit c (atpE)

What is the atpE gene in Pectobacterium carotovorum and what does it encode?

The atpE gene in Pectobacterium carotovorum subsp. carotovorum encodes subunit c of the F0 portion of ATP synthase. This protein is a critical component of the ATP synthase complex, which produces ATP from ADP and inorganic phosphate using energy derived from a transmembrane proton motive force. Subunit c forms a ring structure in the membrane-embedded F0 portion of ATP synthase, and plays an essential role in proton translocation across the membrane.

The c-subunit contains a conserved glutamate residue (comparable to the Glu56 identified in Bacillus PS3) that is crucial for binding and transporting protons through the membrane . This proton movement drives the rotation of the central rotor, which causes conformational changes in the F1 portion that enable ATP synthesis. The atpE gene is highly conserved across bacterial species, reflecting the fundamental importance of ATP synthase in cellular bioenergetics.

What expression systems are commonly used for recombinant production of atpE?

Several expression systems can be employed for the recombinant production of atpE, with the choice depending on research objectives:

• Escherichia coli expression systems: E. coli is the most commonly used host for recombinant protein expression due to its rapid growth, high protein yields, and genetic tractability. For ATP synthase subunit c, E. coli expression systems using vectors such as pET or pBAD with appropriate promoters (T7, arabinose) allow controlled expression. This approach was demonstrated successfully with the Bacillus PS3 ATP synthase, which was expressed in E. coli for structural studies .

• Cell-free expression systems: For membrane proteins like subunit c that may be toxic when overexpressed in living cells, cell-free expression systems offer an alternative. These systems use bacterial lysates containing the transcription and translation machinery without the constraints of cell viability.

• Homologous expression: Expression in Pectobacterium carotovorum itself or closely related species can preserve native folding and assembly, particularly important if studying the protein in its physiological context.

When choosing an expression system, considerations include required protein yield, downstream applications (structural studies, functional assays), and whether post-translational modifications or proper membrane insertion are needed.

What are the essential components of a data table for tracking atpE research experiments?

A well-designed data table for atpE research should comprehensively document all experimental variables and outcomes. Based on research data management principles, such a table should include:

This table structure ensures comprehensive documentation of research materials while facilitating effective data management throughout the project lifecycle .

What techniques are most effective for site-directed mutagenesis of atpE?

Several techniques can be employed for site-directed mutagenesis of atpE, with the choice depending on the specific experimental goals:

• Homologous recombineering: This method leverages bacteriophage recombination systems (such as the Che9c-encoded recombinase protein gp61) to introduce specific mutations into the bacterial chromosome. The technique involves designing single-stranded DNA oligonucleotides containing the desired mutation flanked by homologous sequences (typically 40-50 bp on each side). After inducing expression of the recombinase, the bacteria are transformed with these oligonucleotides, leading to recombination and incorporation of the mutation into the genome .

• Homologous recombination with selection markers: For mutations that might not confer a selectable phenotype, a two-step process involving homologous recombination with a selectable marker (like antibiotic resistance) can be used. This approach involves creating a construct with the desired mutation and a selection marker, followed by a second recombination event to remove the marker while retaining the mutation .

• CRISPR-Cas9 genome editing: This more recent technique offers precise genome editing capabilities. The system uses a guide RNA to target the Cas9 nuclease to a specific genomic location, creating a double-strand break that can be repaired using a supplied template containing the desired mutation.

For the atpE gene specifically, considerations include the essential nature of ATP synthase for cell viability and the potential effects of mutations on bacterial growth. For instance, when studying an Ile66Val mutation in the atpE gene (as seen in Mycobacterium tuberculosis), researchers successfully employed homologous recombineering with carefully designed oligonucleotides .

How does the structure of ATP synthase subunit c in Pectobacterium compare to other bacterial species?

While specific structural data for Pectobacterium carotovorum ATP synthase subunit c is limited in the provided search results, comparative analysis can be made with better-characterized bacterial ATP synthases:

The c-subunit typically consists of two transmembrane α-helices connected by a polar loop. In most bacterial species, the c-subunit contains a highly conserved acidic residue (glutamate or aspartate) in the C-terminal helix that is essential for proton binding and translocation. For example, in Bacillus PS3, this corresponds to Glu56 .

The number of c-subunits in the ring varies between species (typically 8-15), which affects the bioenergetics of ATP synthesis by changing the proton-to-ATP ratio. While the exact number in Pectobacterium carotovorum is not specified in the provided materials, this would be an important aspect to determine through structural studies.

Understanding structural similarities and differences between Pectobacterium and other bacterial species would provide insights into species-specific adaptations of this essential enzyme complex.

What is the role of subunit c in proton translocation and how can this be studied experimentally?

The c-subunit plays a central role in proton translocation across the membrane, which drives ATP synthesis. The process involves:

• Protons from the periplasmic space enter the periplasmic half-channel in subunit a and bind to the conserved glutamate residue (e.g., Glu56 in Bacillus PS3) on a c-subunit .

• Protonation of this glutamate neutralizes its negative charge, allowing the c-subunit to rotate through the hydrophobic environment of the membrane .

• When the protonated glutamate reaches the cytoplasmic half-channel, it interacts with a conserved arginine residue in subunit a (e.g., Arg169 in Bacillus PS3), causing the proton to be released to the cytoplasm .

• The deprotonated c-subunit continues to rotate, eventually returning to the periplasmic half-channel to bind another proton, thus completing the cycle.

This process can be studied experimentally through several approaches:

• Site-directed mutagenesis: Introducing specific mutations to the conserved glutamate residue or other key amino acids can provide insights into their roles in proton binding and translocation. For example, mutations that preserve the negative charge (e.g., Glu to Asp) might maintain function, while those that eliminate it (e.g., Glu to Gln) would likely abolish proton translocation.

• pH-dependent studies: Examining the effects of pH on ATP synthase activity can reveal insights about proton binding and release.

• Structural studies: Cryo-electron microscopy, as demonstrated with the Bacillus PS3 ATP synthase, can provide high-resolution structures of the complex in different rotational states, revealing the conformational changes associated with proton translocation .

• Water accessibility studies: Similar to experiments with E. coli ATP synthase, residues can be mutated to cysteines and tested for accessibility by Ag+ ions to map the proton channels .

• Proton pumping assays: Reconstituting the ATP synthase into liposomes and measuring proton translocation directly can provide functional data on the efficiency of the process.

How can homologous recombineering be applied to study atpE function in vivo?

Homologous recombineering is a powerful technique for introducing precise genetic modifications without the need for restriction enzymes or DNA ligase. It can be particularly valuable for studying atpE function in vivo:

• Experimental setup: The process begins with introducing a plasmid expressing a recombinase protein (such as gp61 from bacteriophage Che9c) into the target bacteria. For example, the episomal plasmid vector pJV75amber can be used, as described for Mycobacterium tuberculosis .

• Inducing recombinase expression: Expression of the recombinase is induced, typically with a compound like acetamide (0.2%) for 24 hours .

• Designing recombineering oligonucleotides: Single-stranded DNA oligonucleotides (approximately 70-90 nucleotides) are designed to carry the desired mutation centrally, with approximately 35-45 nucleotides of homology on each side. These oligonucleotides should be purified at a high standard (e.g., by PAGE) to ensure quality .

• Transformation and selection: The recombinase-expressing cells are transformed with the recombineering oligonucleotides. If the mutation does not confer a selectable phenotype, a co-selection strategy can be employed by simultaneously transforming with a selectable marker (like an antibiotic resistance gene) .

• Verification of mutations: Potential recombinants are screened by PCR amplification of the target region followed by Sanger sequencing to confirm the presence of the desired mutation .

For atpE specifically, this approach could be used to:

• Introduce point mutations to study the role of specific residues in proton binding and translocation

• Create mutations that alter the efficiency of ATP synthesis to understand energetic constraints

• Engineer variants with modified properties for biotechnological applications

The methodology allows for precise genomic modifications without leaving selection markers or scars, making it ideal for studying the effects of subtle mutations on ATP synthase function in the native context.

What are the challenges in expressing and purifying functional recombinant ATP synthase subunit c?

Expression and purification of functional ATP synthase subunit c present several challenges:

• Membrane protein expression: As an integral membrane protein with hydrophobic domains, subunit c can be difficult to express in heterologous systems. Overexpression often leads to protein misfolding, aggregation, or toxicity to the host cell.

• Proper membrane insertion: Ensuring correct insertion into the membrane is crucial for functional studies. This may require specific membrane-targeting sequences or expression systems designed for membrane proteins.

• Oligomeric assembly: The functional unit of subunit c is a ring comprising multiple c-subunits. Ensuring proper assembly of this oligomeric structure during recombinant expression is challenging.

• Protein extraction: Extracting membrane proteins requires detergents or other solubilizing agents that can sometimes affect protein structure or function. Finding the optimal extraction conditions that maintain native conformation is critical.

• Purification without denaturation: Maintaining the native structure during purification requires careful selection of detergents and buffer conditions. Traditional purification methods may need modification for membrane proteins.

• Functional assessment: Verifying that the purified protein retains its native function is essential but challenging, as the function of subunit c is intimately linked to the complete ATP synthase complex.

Researchers have overcome these challenges through various approaches:

• Expression of the entire ATP synthase complex rather than individual subunits, as demonstrated with the Bacillus PS3 ATP synthase expressed in E. coli

• Use of specialized membrane protein expression systems with regulated expression levels

• Employing mild detergents and native-like environments during purification

• Reconstitution into liposomes or nanodiscs for functional studies

• Structural verification by techniques like cryo-EM that can handle membrane proteins in detergent micelles or lipid environments

What are the future research directions for Pectobacterium carotovorum atpE studies?

Future research on Pectobacterium carotovorum ATP synthase subunit c (atpE) could explore several promising directions:

• Comparative structural analysis: Obtaining high-resolution structures of the P. carotovorum ATP synthase would allow comparison with other bacterial species, potentially revealing adaptations specific to this plant pathogen. Cryo-EM approaches similar to those used for the Bacillus PS3 enzyme could be applied .

• Energy coupling in pathogenesis: Investigating how ATP synthesis efficiency relates to virulence could provide insights into the bioenergetics of pathogenesis. This could be studied by creating atpE mutants with altered proton translocation properties and assessing their effects on virulence.

• Integration with quorum sensing systems: Given the importance of quorum sensing in Pectobacterium virulence, as demonstrated by studies on the expI gene , exploring potential connections between energy metabolism and quorum sensing would be valuable.

• Development of specific inhibitors: Understanding the structure and function of P. carotovorum ATP synthase could guide the development of specific inhibitors as potential control agents for this plant pathogen.

• Protein-protein interactions: Investigating how subunit c interacts with other components of the ATP synthase complex and potentially with other cellular proteins could reveal new aspects of ATP synthase regulation in this species.

• Environmental adaptations: Studying how P. carotovorum ATP synthase function responds to environmental conditions relevant to plant infection (pH, temperature, osmolarity) could provide insights into its adaptive strategies.