Recombinant Bifidobacterium adolescentis ATP synthase subunit alpha (atpA), partial

What is the structure and function of ATP synthase in Bifidobacterium adolescentis?

ATP synthase (F1F0-ATPase) in B. adolescentis is a multi-subunit enzyme complex crucial for energy metabolism. The structure consists of two main components: a membrane-embedded F0 part and a cytoplasmic F1 part. The F1 component contains a hexameric structure of alternating α and β subunits (α3β3), along with γ, δ, and ε subunits. The F0 component includes subunits a, b, and c that form a proton channel across the membrane.

Unlike respiratory organisms where ATP synthase primarily functions in ATP synthesis, Bifidobacterium species lack respiratory chains, causing their ATP synthase to often function in reverse. It hydrolyzes ATP to create a proton gradient across the membrane, which is essential for maintaining intracellular pH homeostasis and nutrient transport under various environmental conditions.

How is the atp operon organized in Bifidobacterium species?

The atp operon in Bifidobacterium species follows the gene order atpBEFHAGDC, where each gene encodes a specific subunit of the F1F0-ATPase complex:

• atpB: encodes the a subunit of F0

• atpE: encodes the c subunit of F0

• atpF: encodes the b subunit of F0

• atpH: encodes the δ subunit of F1

• atpA: encodes the α subunit of F1

• atpG: encodes the γ subunit of F1

• atpD: encodes the β subunit of F1

• atpC: encodes the ε subunit of F1

This organization has been verified in Bifidobacterium lactis DSM 10140, where the genes encoding the subunits of F1F0-ATPase were cloned and sequenced. The deduced amino acid sequences showed significant homology with sequences from other organisms, confirming the high conservation of this operon among bacterial species .

What methodological approaches are used to study atpA gene expression in Bifidobacterium?

Several complementary techniques are employed to study atpA gene expression in Bifidobacterium species:

• Northern blot hybridization: This technique can detect and quantify atp operon transcripts under different environmental conditions. For example, in B. lactis, cultures grown to an optical density of 0.7 at 560 nm were exposed to different pH conditions (pH 6.0, 5.5, 4.0, or 3.5), and RNA was isolated at various time points to assess transcriptional responses .

• Quantitative RT-PCR: This provides more sensitive quantification of gene expression levels and can be used to analyze expression patterns of individual genes within the atp operon.

• Primer extension: This technique identifies transcription start sites and helps characterize promoter elements that regulate gene expression.

• RNA sequencing: This provides a comprehensive view of the transcriptome, allowing for analysis of the atp operon in the context of global gene expression patterns.

• Slot blot hybridization: This method verifies gene expression using RNA isolated from treated cultures, as demonstrated in studies with B. lactis where rapid increases in atp operon transcripts were observed upon exposure to low pH .

What role does atpA play in the ATP synthase complex?

The atpA gene encodes the alpha (α) subunit of the F1 component of ATP synthase. In the F1F0-ATPase complex, the alpha subunits alternate with beta subunits to form a hexameric ring (α3β3) that constitutes the catalytic head of the enzyme. While the beta subunits contain the catalytic sites for ATP synthesis or hydrolysis, the alpha subunits serve several critical functions:

• Nucleotide binding: Alpha subunits bind nucleotides (ATP/ADP) at noncatalytic sites, which contributes to the regulation of enzyme activity.

• Structural stabilization: They maintain the integrity of the F1 complex through interactions with adjacent subunits.

• Conformational changes: Alpha subunits undergo conformational changes during the catalytic cycle, which are essential for the rotary mechanism of the enzyme.

• Regulation: They participate in the allosteric regulation of ATP synthase activity in response to cellular energy needs and environmental conditions.

The high conservation of atpA across bacterial species reflects its fundamental importance in the structure and function of ATP synthase .

How can researchers detect and identify atpA in Bifidobacterium species?

Researchers employ several molecular techniques to detect and identify atpA in Bifidobacterium species:

• PCR amplification: Primers designed from conserved regions of the atpA gene can amplify the gene from genomic DNA. This approach has been successfully used to amplify partial atpD sequences from multiple Bifidobacterium species .

• DNA sequencing: Direct sequencing of PCR products provides the nucleotide sequence of atpA, which can be compared with reference sequences for identification and analysis of genetic variations.

• Molecular typing methods: Various techniques can be used for strain-level identification:

  • Amplified Fragment Length Polymorphism (AFLP)

  • Pulsed-Field Gel Electrophoresis (PFGE)

  • Repetitive Element PCR (rep-PCR) targeting repetitive elements such as (GTG)5, ERIC, and BOX

• Hybridization techniques: Probes specific for atpA can be used in Southern blot or dot blot hybridization to detect the gene in genomic DNA samples.

• Whole Genome Sequencing: This provides the complete sequence of the genome, including the atpA gene and its genomic context, enabling comprehensive analysis .

How does the ATP synthase activity in B. adolescentis respond to changes in pH conditions?

ATP synthase activity in Bifidobacterium species exhibits significant responsiveness to pH changes, which is critical for their survival in acidic environments such as the gastrointestinal tract. The response involves both transcriptional regulation and enzymatic activity modulation:

• Transcriptional upregulation: In B. lactis, exposure to low pH (pH 3.5-6.0) leads to a rapid increase in atp operon transcript levels. RNA isolation from bacteria at various time points (0, 10, 20, and 100 minutes) after acid exposure showed increased expression of ATP synthase genes, suggesting that regulation occurs primarily at the transcriptional level rather than at the enzyme assembly step .

• Enzymatic activity adjustments: The F1F0-ATPase in Bifidobacterium likely functions primarily in ATP hydrolysis mode at low pH, using the energy from ATP hydrolysis to pump protons out of the cell, thereby helping to maintain intracellular pH homeostasis.

• Structural adaptations: The protein complex may undergo conformational changes or post-translational modifications in response to pH changes, affecting its catalytic efficiency or substrate specificity.

This pH-responsive behavior resembles that observed in other lactic acid bacteria like Lactobacillus acidophilus, where ATP synthase activity increases as environmental pH decreases. This adaptation is crucial for Bifidobacterium's survival in the acidic environments of the gastrointestinal tract and contributes to their probiotic functionality .

What is the role of B. adolescentis ATP synthase in antibiotic resistance mechanisms?

B. adolescentis demonstrates resistance to several anti-tubercular drugs, including pyrazinamide (PZA), isoniazid (INH), and streptomycin (SM). ATP synthase may contribute to these resistance mechanisms through several pathways:

ATP synthase may indirectly contribute to these resistance mechanisms by maintaining energy homeostasis under antibiotic stress and supporting energy-dependent drug efflux systems. The enzyme's role in maintaining membrane potential could also affect the activity of membrane-targeting antibiotics .

How can recombinant B. adolescentis atpA be expressed and purified for structural studies?

The expression and purification of recombinant B. adolescentis atpA involves a systematic approach to ensure high yield and purity suitable for structural studies:

• Expression system selection: E. coli is commonly used for heterologous expression of bacterial proteins. BL21(DE3) or similar strains are preferred for their reduced protease activity and compatibility with T7 promoter-based expression systems.

• Vector design: The atpA gene is amplified from B. adolescentis genomic DNA and cloned into an appropriate expression vector (e.g., pET series) with a fusion tag for purification. Common tags include His6, GST, or MBP, which can enhance solubility and facilitate purification.

• Expression optimization: Critical parameters to optimize include:

  • Induction temperature (typically lowered to 16-25°C to enhance proper folding)

  • Inducer concentration (IPTG typically at 0.1-0.5 mM)

  • Induction time (4-16 hours)

  • Media composition (rich media like LB or minimal media for isotope labeling)

• Purification strategy:

  • Initial capture using affinity chromatography based on the fusion tag

  • Intermediate purification using ion exchange chromatography

  • Polishing step using size exclusion chromatography

  • Optional tag removal using specific proteases (e.g., TEV, thrombin)

• Quality assessment:

  • SDS-PAGE and Western blotting to verify purity and identity

  • Mass spectrometry for accurate mass determination and detection of modifications

  • Circular dichroism to verify proper folding

  • Activity assays to confirm functional integrity

For structural studies, additional considerations include sample homogeneity, stability, and concentration. Protein crystallization for X-ray crystallography or sample preparation for cryo-electron microscopy may require specific buffer conditions and additives to stabilize the protein .

How can atpA be used as a molecular marker for phylogenetic studies of Bifidobacterium species?

The atpA gene serves as an excellent molecular marker for phylogenetic studies of Bifidobacterium species due to its high conservation across bacterial species combined with sufficient sequence variability to distinguish between closely related taxa:

• Primer design: Universal primers can be designed based on conserved regions of the atpA gene to amplify the gene from various Bifidobacterium species. The approach has been successfully applied to amplify partial atpD sequences from 12 Bifidobacterium species .

• Sequence analysis: The amplified sequences are analyzed to identify species-specific variations. Specific sequence signatures have been identified for the genus Bifidobacterium and for closely related taxa such as Bifidobacterium lactis and Bifidobacterium animalis .

• Multilocus sequence analysis (MLSA): For more robust phylogenetic analysis, atpA can be combined with other genes. In lactic acid bacteria, combined sequence analysis of atpA, pheS, and rpoA genes has been successfully used for species identification .

• Comparison with 16S rRNA: The atp operon has been considered a molecular marker as an alternative to the 16S rRNA gene, providing complementary information for species identification and phylogenetic analysis .

• Phylogenetic tree construction: Methods such as neighbor-joining, maximum likelihood, or Bayesian inference can be applied to construct phylogenetic trees based on atpA sequences, providing insights into evolutionary relationships among Bifidobacterium species.

This approach is particularly valuable for distinguishing between closely related species that may not be easily differentiated based on 16S rRNA sequences alone .

What computational approaches are used for structure-based analysis of B. adolescentis atpA?

Structure-based analysis of B. adolescentis atpA employs several computational approaches to understand its functional and structural properties:

• Homology modeling: Since high-resolution structures of B. adolescentis atpA may not be available, homology modeling can be used to predict its structure based on templates from related organisms with known structures. The highly conserved nature of ATP synthase subunits makes this approach particularly effective .

• Molecular dynamics simulations: These simulations can provide insights into the dynamic behavior of atpA, including conformational changes during the catalytic cycle, interactions with other subunits, and responses to environmental factors such as pH changes .

• Docking studies: Molecular docking can predict interactions between atpA and ligands, including nucleotides (ATP/ADP) and potential inhibitors. This approach is valuable for understanding the binding mode and designing targeted modulators .

• Structure-based drug design: The high-resolution structures of ATP synthase components can inform the development of specific inhibitors or modulators. This approach has been successfully applied in the case of bedaquiline, a novel anti-tuberculosis drug targeting mycobacterial ATP synthase .

• Comparative analysis: Structural comparisons of atpA across different species can highlight conserved features essential for function and species-specific variations that might contribute to different physiological properties or drug sensitivities .

These computational approaches complement experimental studies and can guide targeted experimental investigations, potentially leading to new therapeutic strategies or improved understanding of ATP synthase function .

How does B. adolescentis ATP synthase contribute to probiotic functionality?

B. adolescentis ATP synthase plays a crucial role in the probiotic functionality of this bacterium through several mechanisms:

• Acid stress tolerance: ATP synthase helps maintain intracellular pH homeostasis by pumping protons out of the cell under acidic conditions. This is essential for survival during gastric transit and colonization of the gastrointestinal tract. The rapid increase in atp operon expression under low pH conditions, as observed in related Bifidobacterium species, supports this adaptation .

• Energy metabolism: ATP synthase is central to energy production, supporting the metabolic activities that produce beneficial compounds for the host. Genomic analysis of B. adolescentis AF91-08b2A highlighted metabolic pathways related to energy and carbohydrate metabolism that likely enhance its therapeutic efficacy .

• Stress resistance: B. adolescentis shows resistance to various antibiotics, including anti-tubercular drugs. This resistance, potentially supported by ATP synthase function, may contribute to its survival during antibiotic treatments, maintaining the beneficial gut microbiota .

• Therapeutic effects: In a DSS-induced mice colitis model, B. adolescentis AF91-08b2A significantly enhanced the disease activity index (DAI), curbed weight loss, and attenuated colonic damage. These beneficial effects may be partially attributed to the metabolic activities supported by ATP synthase .

• Adaptation to the gut environment: The ability of B. adolescentis to thrive in the gut is linked to its metabolic versatility and stress tolerance, both of which depend on proper ATP synthase function .

Recent genomic studies have identified the depletion of B. adolescentis in individuals with inflammatory bowel disease (IBD), suggesting its significance for intestinal health. The probiotic attributes of B. adolescentis, including resistance to antibiotics and stress, are likely dependent on the proper functioning of key metabolic enzymes like ATP synthase .

What experimental approaches are effective for studying the regulation of atp operon expression in B. adolescentis?

Several experimental approaches can effectively investigate the regulation of atp operon expression in B. adolescentis:

• Transcriptional analysis:

  • Northern blot hybridization to detect and quantify atp operon transcripts under different conditions, as demonstrated in B. lactis where RNA was isolated at various time points after exposure to different pH values

  • Quantitative RT-PCR for more sensitive quantification of expression levels

  • RNA-seq for genome-wide transcriptional profiling and identification of co-regulated genes

• Promoter analysis:

  • Primer extension experiments to identify transcription start sites

  • Reporter gene assays using promoter-reporter fusions to monitor promoter activity under different conditions

  • Deletion and mutation analysis of promoter elements to identify regulatory regions

• Environmental condition testing:

  • Exposure to different pH values (e.g., pH 3.5-6.0) to assess acid-induced regulation

  • Nutrient limitation studies to understand metabolic regulation

  • Oxygen stress experiments, given the microaerophilic nature of B. adolescentis

  • Temperature variation to assess heat or cold stress responses

• Genetic manipulation:

  • Construction of deletion mutants in potential regulatory genes

  • Overexpression of regulatory elements to assess their impact on atp operon expression

  • Site-directed mutagenesis of regulatory elements in the promoter region

In B. lactis, slot blot hybridization using RNA isolated from acid-treated cultures showed that exposure to low pH led to a rapid increase in atp operon transcripts, suggesting regulation at the transcriptional level rather than at the enzyme assembly step. Similar approaches could be applied to B. adolescentis to understand species-specific regulatory mechanisms .

What are the potential applications of recombinant B. adolescentis atpA in biotechnology and medicine?

Recombinant B. adolescentis atpA holds several promising applications in biotechnology and medicine:

• Drug target development: The unique features of bacterial ATP synthases make them attractive targets for antimicrobial development. Structural studies of recombinant atpA could inform the design of specific inhibitors targeting pathogenic bacteria while sparing beneficial Bifidobacteria. The success of bedaquiline, which targets mycobacterial ATP synthase, demonstrates the potential of this approach .

• Probiotic enhancement: Understanding the structure and function of atpA could guide genetic modifications to enhance specific probiotic properties of B. adolescentis, such as acid tolerance or metabolic capabilities. This could lead to improved therapeutic strains for conditions like inflammatory bowel disease .

• Biomarker development: The specific sequence signatures identified in Bifidobacterium atpA could be used to develop diagnostic tools for monitoring gut microbiota composition, potentially helping to assess gut health and the efficacy of probiotic interventions .

• Protein engineering: The ATP synthase complex is a remarkable molecular motor. Insights from recombinant atpA studies could inform the design of novel nanomotors or energy-conversion devices for biotechnological applications .

• Antibiotic resistance studies: Given B. adolescentis's resistance to certain antibiotics, studying its ATP synthase could provide insights into natural resistance mechanisms that might inform strategies to combat antibiotic resistance in pathogens .

• Therapeutic applications: B. adolescentis has shown promise in treating inflammatory bowel disease. Understanding how ATP synthase contributes to its beneficial effects could lead to optimized therapeutic strains or novel treatment approaches for gastrointestinal disorders .

The genomic findings highlighting B. adolescentis's probiotic attributes, including resistance to antibiotics and stress, underscore the potential biotechnological value of its ATP synthase components .

How can researchers optimize expression systems for producing recombinant B. adolescentis atpA?

Optimizing expression systems for producing recombinant B. adolescentis atpA requires careful consideration of several factors:

• Host selection:

  • E. coli BL21(DE3) and its derivatives are commonly used for heterologous protein expression due to reduced protease activity

  • Lactococcus lactis or other lactic acid bacteria might provide a more suitable cellular environment for proper folding of Bifidobacterium proteins

  • Bifidobacterium expression systems could ensure native processing but typically yield lower protein amounts

• Vector design:

  • Codon optimization for the chosen host organism to address potential codon bias issues

  • Selection of appropriate promoters (constitutive vs. inducible) based on protein toxicity

  • Inclusion of fusion tags (His6, GST, MBP) to enhance solubility and facilitate purification

  • Consideration of secretion signals for extracellular production when appropriate

• Culture conditions:

  • Temperature optimization (typically lowered to 16-25°C during induction to promote proper folding)

  • Media composition (rich vs. minimal, supplementation with specific nutrients)

  • Induction parameters (inducer concentration, induction timing, duration)

  • Scale-up considerations for larger-volume production

• Protein solubility enhancement:

  • Co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Addition of solubility-enhancing compounds to the culture medium

  • Expression as part of fusion proteins with highly soluble partners

  • Optimization of cell lysis conditions to prevent aggregation

• Purification strategy refinement:

  • Development of multi-step purification protocols to achieve high purity

  • Selection of appropriate buffer conditions to maintain protein stability

  • Inclusion of stabilizing agents (glycerol, reducing agents, specific ions)

  • Implementation of quality control measures at each purification step

These optimization strategies should be approached systematically, with careful documentation of conditions and results to identify the optimal expression and purification protocol for recombinant B. adolescentis atpA .