Recombinant Pediococcus pentosaceus ATP synthase subunit c (atpE)

What is the role of ATP synthase subunit c (atpE) in Pediococcus pentosaceus?

ATP synthase subunit c (atpE) is a critical component of the F0 portion of F1F0-ATP synthase in P. pentosaceus. It forms the c-ring structure that facilitates proton translocation across the membrane, which is essential for driving ATP synthesis. In P. pentosaceus, as seen in proteomic studies, the ATP synthesis pathway involving proteins like AtpD (coded by PEPE_RS06385) shows significant regulation under various stress conditions, suggesting atpE likely plays a key role in energy homeostasis . The ATP synthase complex is central to energy production, and disruptions in ATP levels have been observed in P. pentosaceus under various treatment conditions, highlighting the importance of this pathway for bacterial survival .

How do you identify and characterize the atpE gene in Pediococcus pentosaceus?

The atpE gene in P. pentosaceus can be identified through PCR amplification using degenerate primers designed based on conserved regions of ATP synthase subunit c across related bacterial species. For amplification, design primers flanking the complete atpE coding sequence (similar to methods used for mycobacterial atpE) . The general approach includes:

• Genomic DNA extraction from P. pentosaceus cultures

• PCR amplification using designed primers specific to the atpE gene region

• Cloning of the amplified product into a suitable vector (such as pMOSBlue)

• Sequence verification and comparison with known atpE sequences

The characterization can be further enhanced through comparative genomic analysis, as P. pentosaceus has a genome size of approximately 1.76 Mbp with thousands of coding sequences . Sequence analysis should focus on conserved motifs characteristic of ATP synthase c subunits.

What expression systems are most effective for producing recombinant P. pentosaceus atpE?

Based on methodologies used for similar proteins, E. coli expression systems represent the primary choice for recombinant production of P. pentosaceus atpE. The recombinant vector construction approach involves:

• Amplification of the atpE gene using primers with appropriate restriction sites

• Cloning into an expression vector (like pLYG204.zeo plasmid, similar to methods used for mycobacterial atpE)

• Transformation into a suitable E. coli strain for protein expression

• Induction of expression under optimized conditions (temperature, inducer concentration, etc.)

When considering expression systems, researchers should be aware that membrane proteins like atpE may require specialized strains or conditions to prevent toxicity or improper folding. Alternative expression systems in Gram-positive hosts may be considered if E. coli-based expression presents challenges.

How do mutations in atpE affect ATP synthesis and bacterial survival in P. pentosaceus?

Point mutations in the atpE gene can significantly alter ATP synthase function and bacterial physiology. To study these effects:

• Introduce specific mutations using site-directed mutagenesis techniques (such as QuikChange) in recombinant vectors containing the P. pentosaceus atpE gene

• Express wild-type and mutant proteins in appropriate systems

• Assess ATP synthesis rates in reconstituted systems or whole cells

• Evaluate bacterial survival under various stress conditions

Critical mutations often occur in conserved regions of the c-subunit that interact with inhibitors or participate in proton translocation. Research indicates that under stress conditions (like condensed tannin exposure), P. pentosaceus shows altered ATP levels, with intracellular ATP content decreasing significantly . This suggests that functional atpE is essential for maintaining energy homeostasis during stress responses.

What structural differences exist between P. pentosaceus atpE and homologous proteins in other bacterial species?

Comparative structural analysis of P. pentosaceus atpE with homologs from other species requires:

• Sequence alignment of atpE from P. pentosaceus with those from diverse bacterial species

• Homology modeling based on existing crystal structures of bacterial c-subunits

• Analysis of conserved and variable regions, particularly those involved in:

  • Proton binding and translocation

  • Interaction with other ATP synthase subunits

  • Binding sites for known inhibitors

While specific structural data for P. pentosaceus atpE is not directly provided in the search results, methods similar to those used for mycobacterial ATP synthase can be applied . The analysis should consider that P. pentosaceus, as a lactic acid bacterium with unique metabolic characteristics , may possess adaptations in its ATP synthase that differ from those in other bacteria.

How does the regulation of atpE expression respond to different environmental stressors in P. pentosaceus?

To investigate atpE regulation under different stress conditions:

• Expose P. pentosaceus cultures to various stressors (acid, bile salts, antimicrobials, etc.)

• Analyze gene expression changes using RT-qPCR for atpE and related genes

• Perform proteomic analysis to quantify ATP synthase subunit levels

• Correlate expression changes with physiological parameters (growth, ATP levels)

Proteomic studies of P. pentosaceus under stress conditions have revealed significant protein regulation. For example, when exposed to condensed tannins, P. pentosaceus SF11 showed 418 differentially expressed proteins, with proteins involved in ATP synthesis being significantly affected . The AtpD protein (involved in ATP synthesis) was up-regulated in response to decreased intracellular ATP levels, suggesting a compensatory mechanism to maintain energy production .

What are the optimal conditions for cloning and expressing recombinant P. pentosaceus atpE?

The optimal cloning and expression strategy for P. pentosaceus atpE includes:

Cloning Protocol:

• PCR amplification of the atpE gene using high-fidelity polymerase

• Addition of appropriate restriction sites via primer design

• Restriction digestion and ligation into a suitable expression vector

• Transformation into an initial cloning host (e.g., E. coli DH5α)

• Sequence verification before proceeding to expression

Expression Conditions:

• Transform verified construct into an expression host (e.g., E. coli BL21(DE3))

• Culture in LB medium supplemented with appropriate antibiotics

• Induce expression at mid-log phase (OD600 ~0.6-0.8)

• Optimize induction parameters:

  • IPTG concentration: 0.1-1.0 mM

  • Temperature: 16-37°C (lower temperatures often better for membrane proteins)

  • Duration: 3-18 hours

Based on methodologies used for similar proteins, recombinant vectors like pLYG204.zeo have been successfully used for expression of similar ATP synthase components .

What purification methods are most effective for isolating recombinant P. pentosaceus atpE?

Purification of recombinant atpE protein, being a membrane protein, requires specialized approaches:

Extraction Protocol:

• Cell lysis via sonication or French press in buffer containing:

  • 50 mM Tris-HCl (pH 8.0)

  • 100-300 mM NaCl

  • Protease inhibitors

• Membrane fraction isolation via ultracentrifugation

• Solubilization of membrane proteins using:

  • 1-2% detergent (DDM, LDAO, or C12E8)

  • Incubation at 4°C for 1-2 hours with gentle agitation

Purification Strategy:

• Immobilized metal affinity chromatography (IMAC) using His-tagged protein

• Size exclusion chromatography to remove aggregates and achieve higher purity

• Optional: Ion exchange chromatography for further purification

Purity Assessment:

• SDS-PAGE with Coomassie staining

• Western blot using antibodies against the tag or the protein itself

• Mass spectrometry for identity confirmation

This approach is adapted from successful membrane protein purification protocols and should be optimized specifically for atpE.

How can researchers effectively analyze the function of recombinant P. pentosaceus atpE in vitro?

Functional analysis of recombinant atpE requires assessing its incorporation into ATP synthase complexes and measuring ATP synthesis activity:

Reconstitution into Proteoliposomes:

• Mix purified atpE with synthetic phospholipids

• Remove detergent via dialysis or Bio-Beads

• Verify proper incorporation using freeze-fracture electron microscopy

ATP Synthesis Assay:

• Establish a proton gradient across proteoliposome membranes

• Add ADP and inorganic phosphate as substrates

• Measure ATP formation using luciferase-based assays

• Calculate synthesis rates under various conditions

Proton Translocation Assays:

• Load proteoliposomes with pH-sensitive fluorescent dyes

• Monitor fluorescence changes upon establishment of membrane potential

• Calculate proton flux rates

Studies on P. pentosaceus under various conditions have shown that ATP synthesis pathways respond to environmental stressors, with measurable changes in intracellular ATP content . These physiological observations can guide the development of in vitro functional assays for the recombinant protein.

How should researchers interpret proteomic data related to P. pentosaceus atpE expression under different conditions?

Proteomic data analysis for atpE expression requires rigorous statistical and biological interpretation:

Statistical Analysis Framework:

• Apply appropriate normalization to account for technical variation

• Perform statistical tests (t-test, ANOVA) with multiple test correction

• Use fold change thresholds (typically |log2FC| > 1) to identify significant changes

• Implement clustering and principal component analysis to identify expression patterns

Biological Interpretation Approach:

• Map differentially expressed proteins to metabolic pathways

• Analyze co-expression networks to identify functionally related proteins

• Correlate atpE expression with other ATP synthase subunits

• Compare expression changes across different stress conditions

In a proteomic study of P. pentosaceus SF11 under condensed tannin treatment, researchers identified 418 differentially expressed proteins (p < 0.05), with 341 down-regulated and 77 up-regulated . Among these, proteins involved in ATP synthesis showed significant regulation, indicating the importance of energy metabolism in stress response. The study identified two differentially expressed proteins with higher fold change values (|log2FC| > 2) under CT treatment, demonstrating the application of appropriate statistical thresholds .

What bioinformatic approaches best predict structure-function relationships in P. pentosaceus atpE?

Structure-function prediction for P. pentosaceus atpE involves:

Sequence-Based Analysis:

• Multiple sequence alignment with homologous proteins

• Identification of conserved motifs and functional residues

• Prediction of transmembrane segments using algorithms like TMHMM

• Conservation analysis to identify functionally important residues

3D Structure Prediction:

• Homology modeling using templates from related species

• Molecular dynamics simulations to assess stability

• Docking studies with known inhibitors or interacting molecules

• Validation of models using Ramachandran plots and other quality metrics

Functional Prediction:

• Identification of proton-binding sites through conservation and structural analysis

• Prediction of protein-protein interaction interfaces with other ATP synthase subunits

• Virtual mutagenesis to assess the impact of amino acid substitutions

While specific structural data for P. pentosaceus atpE is not provided in the search results, the general approach can be adapted from studies on similar proteins, integrating genomic and proteomic data available for P. pentosaceus, which has a genome size of 1.76 Mbp with 1754 coding sequences .

How can comparative genomics enhance our understanding of P. pentosaceus atpE evolution and function?

Comparative genomic approaches provide valuable insights into atpE evolution and function:

Evolutionary Analysis Protocol:

• Collect atpE sequences from diverse bacterial species

• Construct phylogenetic trees using maximum likelihood methods

• Calculate selection pressures (dN/dS ratios) across different lineages

• Identify signatures of positive or purifying selection

Functional Domain Analysis:

• Map conserved domains across species

• Identify lineage-specific adaptations in P. pentosaceus

• Correlate sequence conservation with known functional regions

• Examine synteny of the ATP synthase operon across species

Horizontal Gene Transfer Assessment:

• Analyze GC content and codon usage bias in the atpE gene

• Compare gene trees with species trees to identify inconsistencies

• Assess the presence of mobile genetic elements near the ATP synthase operon

This approach can help understand how P. pentosaceus atpE has evolved within the context of the organism's adaptation to various ecological niches, including fermented foods and the human gastrointestinal tract .

What are the major technical challenges in working with recombinant P. pentosaceus atpE?

Researchers face several technical challenges when working with recombinant atpE:

Expression Challenges:

• Membrane protein toxicity in heterologous expression systems

• Protein misfolding and aggregation

• Low expression yields

• Improper insertion into membranes

Purification Challenges:

• Detergent selection for optimal solubilization without denaturation

• Maintaining protein stability during purification

• Achieving sufficient purity for structural studies

• Preserving native conformation and activity

Functional Analysis Challenges:

• Reconstituting functional ATP synthase complexes in vitro

• Distinguishing specific atpE effects from those of other subunits

• Developing reliable activity assays that mimic physiological conditions

Strategies to overcome these challenges include using specialized expression hosts, membrane protein-specific tags, and optimized detergent screens based on successful approaches with similar proteins .

How might research on P. pentosaceus atpE contribute to understanding antimicrobial resistance mechanisms?

Research on P. pentosaceus atpE has significant implications for antimicrobial resistance:

Research Approaches:

• Screen for natural compounds that specifically target P. pentosaceus atpE

• Analyze resistance mutations that emerge under selective pressure

• Perform comparative studies with atpE from pathogenic bacteria

• Develop in vitro selection systems to identify novel resistance mechanisms

Potential Applications:

• Design of new antimicrobials targeting ATP synthase

• Development of combination therapies to prevent resistance

• Identification of resistance markers for diagnostic purposes

• Understanding cross-resistance mechanisms between different antimicrobial classes

P. pentosaceus has shown resistance to various environmental stressors, including acidity (87% logarithmic survival rate at pH 2) and bile salts (99% logarithmic survival rate at 0.5% w/v) . Proteomic analysis has identified 120 proteins involved in acid and bile salt resistance mechanisms, suggesting complex adaptive responses that may involve energy metabolism pathways .

What emerging technologies could advance research on P. pentosaceus atpE structure and function?

Several emerging technologies hold promise for advancing atpE research:

Structural Biology Advances:

• Cryo-electron microscopy for membrane protein structures

• Solid-state NMR for dynamics studies in lipid environments

• X-ray free-electron laser (XFEL) crystallography for time-resolved structures

• Hydrogen-deuterium exchange mass spectrometry for conformational analysis

Functional Analysis Technologies:

• Single-molecule FRET to study conformational changes during catalysis

• High-throughput mutagenesis coupled with deep sequencing

• Nanodiscs for stabilization of membrane proteins in near-native environments

• Microfluidic platforms for rapid functional screening

Systems Biology Approaches:

• Multi-omics integration (genomics, transcriptomics, proteomics, metabolomics)

• Machine learning for predicting protein-protein interactions

• CRISPR-Cas9 genome editing in P. pentosaceus for in vivo functional studies

These technologies could help overcome current limitations in understanding the structure-function relationships of P. pentosaceus atpE and potentially lead to novel applications in biotechnology and antimicrobial development.