Recombinant Bifidobacterium animalis subsp. lactis ATP synthase subunit c (atpE)

What is the genomic context of the atpE gene in Bifidobacterium animalis subsp. lactis?

The atpE gene is part of the highly conserved atp operon in B. animalis subsp. lactis. The complete operon structure is atpBEFHAGDC, where atpE encodes subunit c of the F1F0-ATPase complex . This gene organization is consistent across various bifidobacterial species, though some genetic variations may exist between strains. In B. animalis subsp. lactis DSM 10140, the entire atp operon has been cloned and sequenced, revealing significant homology with ATP synthase subunits from other organisms . The operon produces transcripts of approximately 7.3 kb (corresponding to the complete operon) and 4.5 kb (corresponding to atpC, atpD, atpG, and atpA genes) .

What are the key structural characteristics of ATP synthase subunit c in B. animalis subsp. lactis?

ATP synthase subunit c forms the c-ring structure in the membrane-intrinsic F0 portion of F1F0-ATPase. This protein contains highly conserved residues essential for proton translocation and interaction with other ATP synthase subunits. In bifidobacteria lacking a respiratory chain, F1F0-ATPase primarily functions to create a proton gradient driven by ATP hydrolysis rather than ATP synthesis . The specific structural details of B. animalis subsp. lactis subunit c would include transmembrane domains characteristic of this highly hydrophobic protein, though the search results don't provide the exact amino acid sequence or detailed structural information for this specific subspecies.

How is atpE gene expression regulated in response to environmental pH changes?

The atp operon in B. animalis subsp. lactis shows acid inducibility, with transcription levels increasing significantly upon exposure to low pH environments . Research with B. lactis DSM 10140 demonstrated a rapid increase in atp operon transcripts when cultures were exposed to acidic conditions (pH 3.5-6.0), suggesting regulation occurs primarily at the transcriptional level rather than during enzyme assembly . This pH-responsive regulation likely represents an adaptation mechanism allowing the organism to maintain pH homeostasis in acidic environments, which is particularly important for probiotic strains that must survive passage through the gastrointestinal tract.

What promoter elements control atpE transcription in bifidobacteria?

Transcription initiation sites for the atp operon in B. lactis DSM 10140 have been mapped using primer extension techniques. Interestingly, analysis revealed no consensus promoter sequences at these sites . This finding suggests that the atp operon in bifidobacteria may utilize unique or non-canonical promoter elements for transcriptional regulation. The transcription of the atp operon produces two main mRNA transcripts: one of approximately 7.3 kb corresponding to the complete operon, and another of 4.5 kb corresponding to a subset of genes (atpC, atpD, atpG, and atpA) .

What experimental approaches are most effective for quantifying atpE expression levels?

Based on the methodologies described in the search results, several approaches can be effective for quantifying atpE expression:

• Northern blot hybridization - Used successfully to detect and quantify atp operon transcripts in B. lactis under different pH conditions

• Primer extension - Applied to map transcription initiation sites

• Slot blot hybridization - Used to verify acid inducibility of the atp operon using RNA isolated from acid-treated cultures

• Quantitative PCR - While not explicitly mentioned for atpE, qPCR could provide sensitive quantification of transcript levels

When designing primers for these approaches, researchers should target unique regions of atpE to avoid cross-reaction with other genes in the operon or elsewhere in the genome.

What are the optimal expression systems for producing recombinant B. animalis subsp. lactis atpE protein?

While the search results don't specifically address recombinant expression of atpE from B. animalis subsp. lactis, several considerations are important based on general principles and the characteristics of this gene:

• Expression Host Selection:

  • E. coli systems (BL21, Rosetta) may be suitable for initial expression attempts

  • Lactococcus lactis or other Gram-positive hosts might better accommodate the codon usage and protein folding requirements

  • Homologous expression in Bifidobacterium could preserve native folding but offers lower yields

• Vector Design Considerations:

  • Inclusion of a purification tag (His6, GST) while accounting for the hydrophobic nature of subunit c

  • Codon optimization based on the host (especially important given the high G+C content of B. animalis genomes - 61.55%)

  • Inducible promoters to control expression timing

The hydrophobic nature of subunit c likely presents challenges for recombinant expression and may require specialized approaches for membrane protein expression.

What purification strategies effectively isolate recombinant subunit c while maintaining its native structure?

Purification of recombinant ATP synthase subunit c requires specialized approaches due to its highly hydrophobic nature and tendency to form oligomeric structures:

• Detergent-Based Extraction:

  • Mild detergents (DDM, CHAPS) to solubilize membrane fractions

  • Selective extraction protocols that maintain the integrity of the c-ring structure

• Chromatography Approaches:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Size exclusion chromatography to separate monomeric from oligomeric forms

  • Ion exchange chromatography exploiting the charged residues in subunit c

• Verification of Structure:

  • Circular dichroism to confirm secondary structure content

  • Blue native PAGE to analyze oligomeric state

  • Mass spectrometry to verify protein integrity

The purification strategy should account for the specific research goals - whether functional studies require the intact c-ring or if monomeric subunit c is sufficient.

How can researchers verify the proper folding and functionality of recombinantly expressed atpE protein?

Verification of proper folding and functionality for recombinant ATP synthase subunit c can be approached through multiple complementary methods:

• Structural Assessment:

  • Circular dichroism spectroscopy to evaluate secondary structure content

  • Fluorescence spectroscopy if tryptophan residues are present

  • Limited proteolysis to assess accessibility of cleavage sites

• Functional Assays:

  • Reconstitution into liposomes and measurement of proton translocation

  • Assembly assays with other ATP synthase subunits

  • ATP hydrolysis/synthesis assays with reconstituted complexes

• Binding Studies:

  • Interaction analysis with natural inhibitors (e.g., oligomycin)

  • Co-immunoprecipitation with other ATP synthase subunits

  • Surface plasmon resonance with antibodies against conformational epitopes

Each approach provides different and complementary information about the structural integrity and functional capacity of the recombinant protein.

How can genetic engineering approaches modify atpE to enhance acid tolerance in B. animalis subsp. lactis?

Given the acid inducibility of the atp operon in B. animalis subsp. lactis and its role in pH homeostasis , targeted modifications of atpE could potentially enhance acid tolerance:

• Site-Directed Mutagenesis Strategies:

  • Modification of key residues involved in proton binding and translocation

  • Alterations to enhance interaction with other subunits under acidic conditions

  • Introduction of stabilizing mutations from acid-tolerant bacterial species

• Promoter Engineering:

  • Enhanced or constitutive expression of atpE through promoter modifications

  • Introduction of additional pH-responsive elements to increase expression at lower pH

• Experimental Validation:

  • Growth curve analysis at various pH values

  • Measurement of intracellular pH under acid stress

  • ATP synthesis/hydrolysis assays at different pH values

Such modifications could enhance the probiotic potential of B. animalis subsp. lactis by improving survival during gastrointestinal transit and in fermented food environments.

What approaches can be used to study protein-protein interactions between subunit c and other components of the ATP synthase complex?

Several methodologies can elucidate the interactions between ATP synthase subunit c and other components of the complex:

• In Vitro Approaches:

  • Cross-linking studies followed by mass spectrometry

  • Surface plasmon resonance with purified components

  • Co-immunoprecipitation with antibodies against specific subunits

• Genetic Approaches:

  • Bacterial two-hybrid systems

  • Suppressor mutation analysis

  • Site-specific mutagenesis of interaction interfaces

• Structural Biology Methods:

  • Cryo-electron microscopy of the assembled complex

  • X-ray crystallography of subcomplexes

  • NMR studies of specific domain interactions

Understanding these interactions is crucial for elucidating the assembly and function of the ATP synthase complex in B. animalis subsp. lactis, which has adapted to function primarily in the direction of proton extrusion rather than ATP synthesis in this non-respiratory organism .

How can researchers develop antibodies specific to B. animalis subsp. lactis subunit c for detection and localization studies?

Development of specific antibodies against ATP synthase subunit c requires careful consideration of several factors:

• Antigen Selection and Preparation:

  • Identification of immunogenic regions unique to B. animalis subsp. lactis subunit c

  • Use of recombinant full-length protein or synthetic peptides corresponding to exposed epitopes

  • Conjugation to carrier proteins for small peptide antigens

• Antibody Production Strategies:

  • Polyclonal antibodies for broad epitope recognition

  • Monoclonal antibodies for specific epitope targeting

  • Recombinant antibody fragments for improved specificity

• Validation Protocol:

  • Western blotting against purified protein and cell extracts

  • Immunoprecipitation to verify native protein recognition

  • Immunofluorescence microscopy to confirm specificity in intact cells

  • Cross-reactivity testing against related bacterial species

• Application Considerations:

  • For membrane proteins like subunit c, optimization of fixation and permeabilization protocols is critical

  • Considering the high conservation of ATP synthase components, careful epitope selection is essential to ensure specificity

How does the atpE gene and subunit c from B. animalis subsp. lactis compare with those from other probiotic bacteria?

Comparative analysis of ATP synthase components across probiotic bacteria reveals both conservation and adaptation:

The high G+C content of Bifidobacterium genomes (61.55% for B. animalis subsp. lactis) versus the typically lower G+C content of Lactobacillus species suggests different evolutionary pressures and potentially different codon usage patterns in these genes . While the core function of ATP synthase subunit c is conserved, specific adaptations may exist in probiotic bacteria to optimize function in the gastrointestinal environment.

What genetic variations exist in the atpE gene among different strains of B. animalis subsp. lactis, and how might they impact protein function?

While B. animalis subsp. lactis is generally considered genetically monomorphic , strain ATCC 27673 shows significant genomic differences from other strains, including 96 unique genes and six distinct genomic islands . The search results don't specifically address atpE variations, but several patterns can be inferred:

• Core Metabolic Genes:

  • Essential genes like those in the atp operon typically show higher conservation

  • Mutations that significantly alter ATP synthase function would likely be deleterious

• Strain-Specific Adaptations:

  • Subtle variations might exist that optimize function for specific ecological niches

  • Non-synonymous substitutions could affect proton binding, c-ring assembly, or interaction with other subunits

• Regulatory Variations:

  • Differences in promoter regions might affect expression levels or pH-responsiveness

  • Variations in untranslated regions could impact mRNA stability or translation efficiency

Future genomic and proteomic studies comparing atpE across multiple B. animalis subsp. lactis strains could reveal the extent and functional significance of any variations that exist.

How has the ATP synthase evolved in Bifidobacterium compared to other bacterial genera?

Evolutionary analysis of the ATP synthase complex reveals interesting patterns across bacterial lineages:

• Phylogenetic Relationships:

  • The atpD genes (encoding the β subunit) of Lactobacillus species cluster with those of Listeria, Lactococcus, Streptococcus, and Enterococcus

  • Bifidobacterium forms a distinct cluster reflecting its taxonomic position

  • The higher G+C content and highly biased codon usage of Lactobacillus atpD compared to its genome average suggests potential horizontal gene transfer events

• Functional Adaptations:

  • In respiratory bacteria, ATP synthase primarily functions in ATP synthesis

  • In fermentative bacteria like Bifidobacterium, the complex functions primarily to create a proton gradient through ATP hydrolysis

  • These different functional roles have likely driven different selective pressures

• Regulatory Evolution:

  • The acid inducibility of the atp operon in B. lactis represents an adaptation to acidic environments

  • Different mechanisms of pH regulation have evolved across bacterial genera

These evolutionary patterns reflect both the conservation of this essential enzyme complex and its adaptation to diverse ecological niches and metabolic strategies.

What approaches can be used to study the assembly of the c-ring from individual subunit c proteins in B. animalis subsp. lactis?

Investigating c-ring assembly requires specialized approaches that can capture this complex process:

• In Vitro Reconstitution Studies:

  • Purified recombinant subunit c monitored for oligomerization under controlled conditions

  • Effects of lipid composition, pH, and ionic strength on assembly kinetics

  • Analysis of intermediate structures during assembly process

• Real-time Monitoring Techniques:

  • Fluorescence resonance energy transfer (FRET) between labeled subunits

  • Single-molecule microscopy to observe assembly events

  • Hydrogen-deuterium exchange mass spectrometry to track conformational changes

• Computational Approaches:

  • Molecular dynamics simulations of subunit interactions

  • Prediction of key residues involved in oligomerization

  • Modeling of assembly pathways and energy landscapes

These studies would provide valuable insights into the biogenesis of the ATP synthase complex in B. animalis subsp. lactis and potentially identify strain-specific features of this process.

How do mutations in atpE affect the acid tolerance and probiotic efficacy of B. animalis subsp. lactis?

The acid inducibility of the atp operon in B. lactis suggests its importance for acid tolerance, making atpE mutations potentially significant for probiotic functionality:

• Systematic Mutagenesis Approaches:

  • Site-directed mutagenesis of key functional residues

  • Random mutagenesis followed by selection under acid stress

  • CRISPR-based genome editing for precise chromosomal modifications

• Phenotypic Assessment Framework:

  • Survival curves at various pH values

  • Intracellular pH measurement during acid challenge

  • ATP synthesis/hydrolysis rates under acidic conditions

• Probiotic Functionality Testing:

  • Simulated gastrointestinal transit assays

  • Adhesion to intestinal cell lines

  • Competitive growth with intestinal microbiota

  • Animal models of probiotic colonization

This research direction could lead to engineered strains with enhanced acid tolerance for improved survival during gastrointestinal transit and in fermented food products.

What is the role of ATP synthase subunit c in maintaining cellular pH homeostasis in B. animalis subsp. lactis?

ATP synthase plays a critical role in pH homeostasis in Bifidobacterium and other fermentative bacteria:

Understanding this function is particularly relevant for probiotic applications, as it contributes to the ability of B. animalis subsp. lactis to survive transit through the acidic stomach environment.