Recombinant Frankia sp. ATP synthase subunit b (atpF)

What is ATP synthase and what role does the subunit b (atpF) play in Frankia species?

ATP synthase is a complex molecular machine that synthesizes adenosine triphosphate (ATP) in mitochondria, eubacteria, and chloroplasts. In Frankia species, as in other organisms, ATP synthase converts the energy from proton motive force (pmf) into chemical energy in the form of ATP through a rotary mechanism . The subunit b (atpF) in Frankia sp. is part of the peripheral stalk of ATP synthase, connecting the membrane-embedded F0 portion to the catalytic F1 portion, thereby preventing rotation of the catalytic subunits during ATP synthesis. This structural stability is crucial for maintaining the enzyme's efficiency, as the central rotor can turn approximately 150 times per second during ATP synthesis .

What genomic insights have been gained about ATP synthase genes in Frankia species?

Genomic analyses of Frankia strains have revealed important insights into their metabolic capabilities and symbiotic relationships. Comparative genomic studies have identified cluster-specific genes in Frankia that contribute to host specificity and symbiotic interactions . While specific information about the atpF gene organization in Frankia is limited in the available literature, general patterns of gene conservation and specialization across Frankia clusters suggest that ATP synthase components may exhibit cluster-specific adaptations. The genomic architecture of Frankia strains varies considerably, with genome sizes ranging from smaller genomes in obligate symbionts to larger genomes in saprophytic strains , potentially affecting the regulatory contexts and expression patterns of ATP synthase genes including atpF.

How do environmental factors influence ATP synthase expression in Frankia?

Environmental factors significantly impact gene expression in Frankia species, particularly under nitrogen-fixing conditions. Studies using suppression subtractive hybridization have identified genes upregulated during nitrogen fixation in Frankia, revealing novel expression patterns . While ATP synthase genes were not specifically highlighted in these studies, the energy demands of nitrogen fixation suggest that ATP production would be critical during this process. The differentiation of vesicles (specialized nitrogen-fixing cells) in Frankia under nitrogen-free conditions likely involves coordinated regulation of energy metabolism genes, including those encoding ATP synthase components.

What expression systems are most effective for recombinant production of Frankia sp. atpF?

Based on recombinant approaches used for similar proteins, Escherichia coli expression systems with T7 promoters are recommended for Frankia sp. atpF expression. The methodology should include:

• Gene construction through oligonucleotide annealing and ligation

• Phosphorylation of oligonucleotides using T4 Polynucleotide Kinase

• Vector transformation into E. coli expression strains (e.g., T7 Express lysY/Iq)

• Co-expression with chaperone proteins to enhance production

A particularly effective strategy involves using pMAL expression vectors for creating fusion proteins with maltose-binding protein (MBP), which can increase solubility and facilitate purification. Co-expression with chaperone proteins DnaK, DnaJ, and GrpE has been shown to substantially increase yields of difficult-to-produce recombinant proteins . This approach is especially valuable for membrane-associated proteins like ATP synthase components, which often present challenges in recombinant expression systems.

What purification strategies are most suitable for recombinant Frankia sp. atpF?

Purification of recombinant Frankia sp. atpF presents specific challenges due to its hydrophobic nature and potential toxicity to host cells. Based on successful approaches with similar proteins, a multi-step purification protocol is recommended:

Optimization of detergent conditions is critical during purification to maintain protein stability while minimizing aggregation. The choice of detergent and its concentration should be empirically determined based on protein stability assays and functional tests .

How can site-directed mutagenesis of recombinant Frankia atpF provide insights into ATP synthase function?

Site-directed mutagenesis of recombinant Frankia atpF offers powerful approaches for understanding structure-function relationships in ATP synthase. Strategic mutation of conserved residues can reveal:

• Essential interactions between subunit b and other ATP synthase components

• Regions involved in stability versus catalytic regulation

• Determinants of species-specific functions

Mutagenesis methodology should include:

• PCR-based mutagenesis using complementary primers containing the desired mutation

• DpnI digestion to remove template DNA

• Transformation into high-efficiency competent cells

• Sequence verification of mutant constructs

• Expression and purification as described for wild-type protein

• Functional characterization through biochemical and biophysical assays

Comparing the effects of equivalent mutations across different species (e.g., Frankia vs. E. coli) can provide insights into evolutionary adaptation of ATP synthase in different ecological niches .

What roles might ATP synthase play in Frankia symbiotic interactions?

ATP synthase likely plays critical roles in the energy metabolism supporting Frankia symbiotic interactions with actinorhizal plants. The establishment and maintenance of symbiosis involves complex signaling and metabolic processes:

• During early infection stages, ATP production may support the synthesis of signaling molecules involved in root hair deformation

• ATP synthase activity likely increases during nodule formation to meet elevated energy demands

• Nitrogen fixation, a highly energy-intensive process, requires substantial ATP production

While Frankia uses different signaling pathways than Rhizobium (lacking traditional nod genes), it still produces extracellular factors that induce root hair deformation . The production of these factors, their export, and the subsequent infection process all require energy in the form of ATP. Regulation of ATP synthase activity may therefore be integral to successful symbiotic establishment.

What analytical methods are most appropriate for studying recombinant Frankia sp. atpF structure and function?

Multiple complementary analytical approaches are recommended for comprehensive characterization of recombinant Frankia sp. atpF:

Advanced cryo-electron microscopy (cryo-EM) has revolutionized structural biology of membrane protein complexes and would be particularly valuable for studying how atpF integrates into the complete ATP synthase complex . This method allows visualization of the protein in a near-native environment without the need for crystallization.

What are the main challenges in working with recombinant Frankia sp. atpF?

Researchers face several significant challenges when working with recombinant Frankia sp. atpF:

• Expression toxicity in host systems, potentially requiring tightly regulated expression systems

• Protein instability outside the native membrane environment

• Difficulty in assessing proper folding and function in isolation from other ATP synthase components

• Limited availability of Frankia-specific genetic tools compared to model organisms

These challenges can be addressed through careful optimization of expression conditions, including induction timing and temperature, co-expression with chaperones, and development of fusion constructs that enhance stability . Additionally, using bacterial strains specifically designed for membrane protein expression, such as C41(DE3) or C43(DE3), may improve yields.

How can metagenome-assembled genomes (MAGs) advance research on Frankia ATP synthase?

Metagenome-assembled genomes (MAGs) offer promising avenues for studying uncultured Frankia strains, including their ATP synthase components:

• MAGs provide access to genetic information from uncultivated Frankia strains

• Comparative genomics using MAGs can reveal evolutionary patterns in ATP synthase genes

• MAGs with high completeness (>90%) and low contamination (<5%) provide reliable data for gene analysis

The analysis of atpF genes from diverse Frankia MAGs could reveal adaptations to different host plants or environmental conditions. This approach is particularly valuable for studying obligate symbionts that cannot be cultured axenically, potentially revealing novel structural or functional adaptations in their ATP synthase components .

What potential applications might emerge from research on recombinant Frankia sp. atpF?

Research on recombinant Frankia sp. atpF could lead to several promising applications:

• Development of novel antimicrobials targeting bacterial ATP synthases

• Engineering of Frankia strains with enhanced symbiotic capabilities

• Creation of biosensors for detecting environmental contaminants

• Design of bioenergetic systems inspired by natural ATP synthases

The significant structural and regulatory differences between human and bacterial ATP synthases make bacterial ATP synthase a promising target for new antibiotics to combat multiple drug-resistant organisms . Understanding the unique features of Frankia ATP synthase could contribute to this effort, particularly against actinobacterial pathogens with similar ATP synthase structures.

How should researchers approach functional reconstitution of recombinant Frankia sp. atpF?

Functional reconstitution of recombinant Frankia sp. atpF requires careful consideration of lipid environments and interaction partners:

• Selection of appropriate lipid composition for proteoliposome preparation

• Co-reconstitution with other ATP synthase components to form functional subcomplexes

• Development of assays to measure specific atpF-dependent functions

The methodology should include:

• Preparation of liposomes with defined lipid composition

• Incorporation of purified atpF using detergent-mediated reconstitution

• Removal of detergent through dialysis or adsorbent beads

• Verification of incorporation through density gradient centrifugation

• Functional assessment through proton translocation or ATP synthesis assays

Successful reconstitution approaches for ATP synthase subunits from other organisms provide valuable templates that can be adapted for Frankia proteins .

What comparative genomic approaches are most informative for studying Frankia atpF evolution?

Comparative genomic analysis of atpF genes across Frankia strains can reveal evolutionary patterns and functional adaptations. Recommended approaches include:

• Multiple sequence alignment of atpF sequences from diverse Frankia clusters

• Phylogenetic analysis to determine evolutionary relationships

• Selection pressure analysis to identify sites under positive or purifying selection

• Synteny analysis to examine conservation of gene neighborhood

• Comparative analysis with atpF from other bacterial genera

Special attention should be given to comparing atpF sequences between different Frankia clusters, particularly between strains with different host specificities (e.g., Sp+ vs. Sp- strains) . Such analysis could reveal whether ATP synthase components contribute to the specificity of Frankia-plant interactions or have adapted to the particular metabolic demands of different symbiotic relationships.

What protocols are recommended for analyzing the expression of atpF in different Frankia growth conditions?

Analysis of atpF expression under different growth conditions requires sensitive and specific methods:

For studying differential expression, suppression subtractive hybridization (SSH) has proven effective in identifying genes upregulated during nitrogen fixation in Frankia . This approach could be adapted to identify conditions that specifically affect atpF expression. The methodology involves:

• RNA isolation from Frankia grown under different conditions

• cDNA synthesis and adapter ligation

• Hybridization to remove common sequences

• PCR amplification of differentially expressed sequences

• Cloning and sequencing of enriched fragments

• Verification by Northern blot or RT-qPCR

This approach would be particularly valuable for understanding how atpF expression changes during the transition from free-living to symbiotic states in Frankia.