Recombinant Bifidobacterium longum ATP synthase subunit b (atpF)

What is the genomic organization of the ATP synthase operon in Bifidobacterium longum?

The ATP synthase operon (atp) in B. longum is organized in a similar pattern to that observed in other bifidobacteria, with the gene order atpBEFHAGDC. The operon is highly conserved among eubacteria and encodes the subunits of the F1F0-ATPase . The atpF gene, encoding subunit b, is positioned between atpE and atpH in this operon. The entire atp operon is located on the circular chromosome of B. longum, which has approximately 60% GC content and contains about 1,730 coding sequences in total . The genomic context and organization of this operon are important considerations when designing recombinant expression strategies.

What primers can be used to amplify the atpF gene from Bifidobacterium longum for recombinant expression?

While specific primers for atpF amplification weren't directly mentioned in the search results, we can extrapolate from the approaches used for other atp genes. For the related atpD gene, researchers have successfully used primers designed from consensus sequences, such as atBIF-1 (5′-CACCCTCGAGGTCGAAC-3′) and atBIF-2 (5′-CTGCATCTTGTGCCACTTC-3′) for Bifidobacterium species . For atpF amplification, researchers should:

• Align atpF sequences from multiple Bifidobacterium species to identify conserved regions

• Design primers with appropriate restriction sites for subsequent cloning

• Optimize PCR conditions considering the high GC content of Bifidobacterium DNA

• Include appropriate negative and positive controls to confirm specificity

When designing primers specifically for B. longum atpF, researchers should account for the 60% GC content of the genome and consider codon optimization if the gene will be expressed in a heterologous host .

How can I confirm successful expression of recombinant B. longum atpF protein?

Successful expression of recombinant B. longum atpF can be confirmed using multiple complementary approaches:

• Western blotting: Using antibodies specific to the atpF protein or to an added epitope tag (His-tag, FLAG-tag, etc.) to detect the protein in cell lysates or culture supernatants

• Immunofluorescence staining: Visualizing the expression within bacterial cells using fluorescently labeled antibodies, as demonstrated for other recombinant B. longum proteins

• SDS-PAGE analysis: Comparing protein profiles of recombinant versus wild-type strains to identify the expected size band (approximately 22 kDa for many recombinant proteins in B. longum)

• Mass spectrometry: For definitive identification of the expressed protein

• Functional assays: Measuring ATP synthase activity to confirm that the recombinant protein is functionally incorporated into the ATP synthase complex

The choice of detection method may depend on whether the protein is expected to be cell-associated or secreted, and whether it has been modified with epitope tags for easier detection .

What growth conditions are optimal for expression of recombinant proteins in B. longum?

Based on studies with other recombinant B. longum strains, the following growth conditions have proven effective:

• Temperature: Induction at 42°C for 8 hours has been used successfully for recombinant protein expression in B. longum

• Media: Modified MRS media supplemented with cysteine and appropriate selective antibiotics is commonly used

• Growth phase: Mid to late exponential phase typically yields optimal protein expression

• Anaerobic conditions: Strict anaerobic conditions are essential for optimal growth and protein expression in B. longum

• pH: Maintaining pH between 6.0-6.5 helps optimize growth and protein stability

Interestingly, some recombinant B. longum strains have demonstrated accelerated growth rates compared to wild-type strains. For example, recombinant B. longum expressing endostatin reached an OD value of 2.207 at 24 hours, while the wild-type strain only reached 0.823 . This enhanced growth provides potential advantages for producing sufficient biomass for protein purification and in vivo functionality.

How can I design an expression system specifically optimized for atpF in B. longum?

Designing an expression system for atpF in B. longum requires careful consideration of several factors:

• Vector selection: pBV222 has been successfully used for recombinant protein expression in B. longum . Alternative vectors like pBFS63, pBFK86, and pBFK94 have also been used for gene expression in this organism .

• Promoter selection: Consider using:

  • The native atp operon promoter for physiological expression levels

  • The cscBA promoter (PcscBA) for regulated expression, as it drives the expression of sucrose permease and β-fructofuranosidase genes in B. longum

  • Inducible promoters for controlled expression

• Transcriptional terminators: Several terminators have been validated in B. longum, including:

  • Tlas (terminator for the lactic acid synthesis operon from Lactococcus lactis)

  • Trps9 (putative terminator for the 30S ribosomal protein S9 gene of B. longum 105-A)

  • TleuB (terminator for the 3-isopropylmalate dehydrogenase gene)

  • TclpP (modified terminator for the clpP operon)

• Codon optimization: Consider the high GC content (~60%) of B. longum when designing coding sequences

• Signal peptides: If secretion is desired, appropriate signal peptides should be incorporated

• Integration strategy: For stable expression, consider chromosomal integration between appropriate genes (e.g., between BL105A_1451 and BL105A_1452) using double-crossover recombination

What are the challenges in isolating functional ATP synthase complexes containing recombinant atpF from B. longum?

Isolating functional ATP synthase complexes containing recombinant atpF presents several challenges:

• Membrane protein nature: ATP synthase is a membrane-bound complex, making isolation while maintaining native conformation difficult

• Complex assembly: The F1F0-ATPase consists of multiple subunits (encoded by atpBEFHAGDC) , requiring proper assembly for functionality

• Detergent selection: Critical for solubilization while preserving activity

• Stability concerns: The complex may disassemble during purification

• Yield limitations: Membrane proteins often express at lower levels

• Activity assessment: Functional assays require reconstitution into liposomes or nanodiscs

Strategies to overcome these challenges include:

• Using mild detergents like n-dodecyl-β-D-maltoside

• Employing affinity tags on atpF for co-purification of intact complexes

• Implementing stabilizing agents during purification

• Utilizing native-PAGE to assess complex integrity

• Developing specialized activity assays for the B. longum ATP synthase

How can Recombinase-Based In Vivo Expression Technology (R-IVET) be applied to study atpF regulation in B. longum?

R-IVET can be effectively applied to study atpF regulation in B. longum by adapting the approach described for other B. longum genes :

• Construction of reporter system:

  • Generate a B. longum strain containing chromosomally integrated loxP-SpR-loxP cassette

  • Create a plasmid with promoterless Cre gene downstream of appropriate transcriptional terminators

  • Clone the putative atpF promoter region upstream of the Cre gene

• Implementation methodology:

  • Insert the atpF promoter region into the BglII site of a vector like pBFK86

  • Transform the construct into the loxP-SpR B. longum strain

  • Under conditions where the atpF promoter is active, Cre will be expressed

  • Cre will excise the SpR marker, making cells spectinomycin-sensitive

• Experimental design:

  • Expose transformed B. longum to various environmental conditions

  • Screen for loss of spectinomycin resistance

  • Conditions causing loss of resistance indicate atpF promoter activation

• Data analysis:

  • Quantify the percentage of spectinomycin-sensitive colonies under each condition

  • Determine specific stimuli that induce atpF expression

  • Compare with other ATP synthase subunit genes to identify differential regulation

This approach allows for identification of environmental conditions specifically triggering atpF expression in vivo, providing insights into its regulation during colonization and adaptation to different gut environments .

What role does the atpF gene play in the adaptation of B. longum to the human gastrointestinal tract?

The atpF gene, encoding ATP synthase subunit b, plays a critical role in energy metabolism and adaptation of B. longum to the gastrointestinal environment:

• Energy harvesting: As part of the F1F0-ATPase complex, atpF enables B. longum to maintain ATP synthesis under the low-nutrient, variable-pH conditions of the colon

• pH homeostasis: The ATP synthase complex contributes to maintaining internal pH in the acidic gut environment, with atpF serving as a crucial membrane anchor for the complex

• Competitive fitness: Efficient energy production supports the competitiveness and persistence of bifidobacteria in the colon, allowing them to effectively utilize available carbon sources

• Stress response: ATP synthase activity may be regulated in response to environmental stressors, with atpF potentially playing a role in adapting to changing conditions

• Colonization efficiency: Proper energy metabolism supports the expression of adhesion factors like fimbriae, which are important for attachment to gut surfaces

The atpF gene likely works in concert with other adaptive features of B. longum, such as its specialized oligosaccharide metabolism and host-interaction factors, to enable successful colonization and survival in the human gastrointestinal tract .

What is the optimal method for cloning the atpF gene from B. longum for recombinant expression?

The optimal cloning method for B. longum atpF should address the unique challenges of this organism:

• DNA extraction:

  • Use specialized protocols accounting for the thick cell wall of Gram-positive bacteria

  • Include enzymatic treatment (lysozyme, mutanolysin) to improve DNA yield

  • Consider the high GC content (~60%) when optimizing extraction buffers

• PCR amplification:

  • Design primers with appropriate restriction sites or homology regions

  • Use high-fidelity polymerases suitable for GC-rich templates

  • Include DMSO or other GC-enhancing additives in PCR reactions

  • Optimize annealing temperatures carefully due to GC-rich nature

• Cloning strategy options:

  Strategy	Advantages	Considerations
  Restriction enzyme cloning	Well-established, simple	Requires convenient restriction sites
  Gibson assembly	Seamless, multiple fragments	Requires homology regions
  TOPO cloning	Rapid, high efficiency	Limited to specific vectors
  In-Fusion cloning	Flexibility with multiple fragments	More expensive reagents

• Vector selection:

  • pBV222 has been validated for B. longum expression

  • Consider shuttle vectors that replicate in both E. coli and B. longum

  • Selection marker compatibility (e.g., chloramphenicol resistance)

• Transformation considerations:

  • Electroporation is preferred for B. longum (typical settings: 25 μF, 200 Ω, 2.5 kV)

  • Recovery in 1/2MRSCS medium under anaerobic conditions

  • Verification by colony PCR and sequencing

For chromosomal integration, double-crossover recombination has been successful, using homologous regions like those between BL105A_1451 and BL105A_1452 in the B. longum genome .

How can I assess the impact of atpF mutations on ATP synthase function in B. longum?

Assessing atpF mutations requires a multi-faceted approach:

• Site-directed mutagenesis strategies:

  • Design mutations based on structure-function relationships

  • Create a complementation system using the native atpF promoter

  • Consider using the Cre/loxP system for markerless mutations

• Phenotypic characterization methods:

  • Growth rate analysis under different energy sources

  • pH tolerance assessment

  • Membrane potential measurements using fluorescent dyes

  • Oxygen sensitivity testing

  • Competitive growth assays with wild-type strains

• Biochemical measurements:

  • ATP production quantification in wild-type vs. mutant strains

  • Membrane-bound ATPase activity assays

  • Proton pumping efficiency measurements

  • Protein complex assembly analysis by blue native PAGE

• Structural analysis:

  • Membrane protein extraction and purification

  • Protein-protein interaction studies focusing on atpF

  • Stability assessment of the F1F0 complex in mutants

• In vivo assessment:

  • Colonization ability in gastrointestinal models

  • Competitive index determination in mixed cultures

  • Persistence under simulated gut conditions

When analyzing results, researchers should consider that the atpF mutations may have pleiotropic effects due to ATP synthase's central role in energy metabolism, potentially affecting various cellular processes beyond direct ATP production.

What techniques can be used to study the expression pattern of atpF in different environmental conditions?

Several complementary techniques can be employed to study atpF expression patterns:

• Transcriptional analysis:

  • RT-qPCR: Quantify atpF mRNA levels under different conditions

  • RNA-seq: Profile whole-transcriptome changes including atpF

  • Northern blotting: Visualize atpF transcript size and abundance

  • Primer extension: Map transcription start sites precisely

• Promoter activity assessment:

  • R-IVET system using Cre/loxP as described for B. longum

  • Reporter gene fusions (e.g., luciferase, GFP) with the atpF promoter

  • Beta-galactosidase assays for promoter strength quantification

• Protein-level analysis:

  • Western blotting with atpF-specific antibodies

  • Proteomics to quantify relative abundance under different conditions

  • Immunofluorescence microscopy for localization studies

• Environmental conditions to test:

  Condition	Relevance	Measurement parameters
  pH variation	Gut transit simulation	pH 4.0-7.5 in 0.5 increments
  Carbon source	Nutrient adaptation	Growth on different oligosaccharides
  Oxygen levels	Microaerobic adaptation	0-5% oxygen exposure
  Bile concentration	Intestinal stress	0.1-1.0% bile salt exposure
  Temperature	Fever response	37°C vs. 39-42°C

• Data integration:

  • Correlate atpF expression with growth parameters

  • Compare with other ATP synthase subunits

  • Analyze in context of global stress responses

The R-IVET system is particularly valuable as it allows for in vivo monitoring of gene expression, potentially revealing condition-specific regulation patterns that might be missed in vitro .

How can recombinant B. longum expressing modified atpF be used to study microbiota-host interactions?

Recombinant B. longum with modified atpF offers unique opportunities for studying microbiota-host interactions:

• Tracking colonization dynamics:

  • Incorporate reporter tags (fluorescent, luminescent) fused to atpF

  • Monitor bacterial persistence and localization in the gut

  • Assess competitive fitness against wild-type strains

• Modulating energy metabolism:

  • Engineer atpF variants with altered ATP production efficiency

  • Study how bacterial energy status affects host-microbe interactions

  • Investigate impacts on colonization resistance against pathogens

• Host immune response studies:

  • Examine how energy metabolism alterations affect immunomodulatory properties

  • Investigate if ATP synthase components are recognized by host pattern recognition receptors

  • Determine if atpF modifications alter the serpin-mediated immunomodulation reported for B. longum

• Therapeutic applications:

  • Develop atpF modifications that enhance survival in specific gut regions

  • Optimize energy production for improved therapeutic protein delivery

  • Engineer strains with enhanced persistence for prolonged beneficial effects

• Biomarker development:

  • Use atpF expression patterns as indicators of gut environmental conditions

  • Develop diagnostic applications based on ATP synthase activity profiles

These approaches build on previous work with recombinant B. longum, such as strains expressing endostatin protein that demonstrated beneficial effects on gut microbiota composition and potential therapeutic applications for IBD and colitis-associated cancer .

What are the most promising approaches for improving ATP production efficiency in recombinant B. longum strains?

Several promising approaches can be pursued to enhance ATP production efficiency:

• Genetic engineering strategies:

  • Optimize atpF and other ATP synthase subunit genes for enhanced assembly

  • Upregulate expression of rate-limiting components of the ATP synthase complex

  • Engineer pH-responsive promoters to activate ATP synthase genes under specific conditions

  • Introduce heterologous ATP synthase components with higher efficiency

• Metabolic engineering approaches:

  • Optimize the bifid shunt pathway that is central to bifidobacterial energy metabolism

  • Enhance substrate utilization pathways for improved carbon flux

  • Reduce competing pathways that divert energy resources

  • Increase membrane integrity to maintain proton motive force

• Strain adaptation techniques:

  • Serial passage under energy-limited conditions

  • Directed evolution targeting improved ATP production

  • Adaptive laboratory evolution in gut-simulating environments

• Consortium engineering:

  • Co-culture with complementary microbes that provide metabolic precursors

  • Design syntrophic relationships that enhance energy harvest

• Environmental optimization:

  • Identify ideal pH, carbon source, and mineral compositions for maximum ATP production

  • Develop specialized growth media formulations for industrial applications

This optimization is particularly important given that recombinant B. longum strains often show accelerated growth rates compared to wild-type strains, suggesting altered energy metabolism that could be further enhanced .

What are the emerging applications of recombinant B. longum strains in treating inflammatory bowel diseases?

Recombinant B. longum strains show considerable promise for IBD treatment:

• Therapeutic protein delivery systems:

  • B. longum has been successfully engineered to express endostatin, demonstrating beneficial effects on gut microbiota and potential applications for IBD and colitis-associated cancer

  • The ATP synthase system can be optimized to enhance energy production for protein expression

  • Strain engineering can improve gut persistence and therapeutic efficacy

• Microbiome modulation effects:

  • Recombinant B. longum-Endo increases beneficial bacteria (Lactobacillus, Bifidobacterium, Allobaculum, Parabateroides)

  • Simultaneously decreases potentially pathogenic bacteria (Desulfovibrio, Helicobacter, Enterorhabdus)

  • These changes may contribute to reduced inflammation and improved gut barrier function

• Immunomodulatory mechanisms:

  • B. longum naturally produces a eukaryotic-type serine protease inhibitor (serpin) potentially involved in immunomodulation

  • Recombinant strains can be designed to enhance serpin production or express additional immunomodulatory factors

  • Targeted delivery of anti-inflammatory molecules to gut tissue

• Precision medicine applications:

  • Patient-specific microbiome analysis to guide recombinant B. longum design

  • Customized therapeutic protein expression based on inflammatory profiles

  • Combination therapies with conventional IBD treatments

• Clinical development considerations:

  Development Stage	Key Considerations	Timeline
  Preclinical testing	Safety, colonization efficiency, therapeutic efficacy	1-2 years
  Phase I trials	Dose optimization, safety assessment	1-2 years
  Phase II trials	Efficacy in defined IBD populations	2-3 years
  Regulatory approval	Genetically modified organism regulations	1-2 years

These applications build on the growing understanding of B. longum's natural properties and the expanding toolkit for genetic manipulation of this important probiotic species .

What are common challenges in expressing recombinant proteins in B. longum and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant B. longum:

• Low transformation efficiency:

  • Solution: Optimize electroporation parameters (25 μF, 200 Ω, 2.5 kV)

  • Use glycine in growth media to weaken cell wall

  • Consider heat treatment before electroporation

  • Ensure DNA is free of salts and in appropriate buffer

• Plasmid instability:

  • Solution: Integrate genes into chromosome using double-crossover recombination

  • Maintain selective pressure throughout culture

  • Use compatible selectable markers (chloramphenicol, spectinomycin)

  • Consider codon optimization to reduce metabolic burden

• Low protein expression:

  • Solution: Test different promoters (native atp promoter, PcscBA)

  • Optimize ribosome binding sites for B. longum

  • Consider using transcriptional terminators (Tlas, Trps9, TleuB, TclpP)

  • Express protein during exponential growth phase

• Protein misfolding/inactivity:

  • Solution: Express at lower temperatures (30-37°C instead of 42°C)

  • Include molecular chaperones as co-expression partners

  • Use native signal peptides for secreted proteins

  • Consider fusion partners to enhance solubility

• Difficult protein detection:

  • Solution: Use epitope tags (His, FLAG, etc.)

  • Develop specific antibodies for Western blotting

  • Apply immunofluorescence staining techniques

  • Implement mass spectrometry-based detection

Encouragingly, some recombinant B. longum strains display accelerated growth compared to wild-type (OD 2.207 vs. 0.823 at 24 hours), which can facilitate biomass production and protein yield .

How can I optimize PCR conditions for amplifying GC-rich regions like those in B. longum atpF?

Amplifying GC-rich regions from B. longum requires specialized PCR optimization:

• Polymerase selection:

  • Use high-fidelity polymerases designed for GC-rich templates

  • Enzymes with proofreading capability (Q5, Phusion, PfuUltra)

  • Hot-start formulations to reduce non-specific amplification

• Buffer and additive optimization:

  Additive	Concentration Range	Effect
  DMSO	2-10%	Reduces secondary structure
  Betaine	0.5-2.5 M	Equalizes GC/AT stability
  Glycerol	5-15%	Stabilizes polymerase
  Formamide	1-10%	Lowers DNA melting temperature
  7-deaza-dGTP	Partial replacement of dGTP	Reduces GC hydrogen bonding

• Thermal cycling parameters:

  • Extended initial denaturation (5-10 minutes at 98°C)

  • Higher denaturation temperature (95-98°C)

  • Touchdown PCR approach (decreasing annealing temperature)

  • Longer extension times (60s/kb rather than standard 30s/kb)

• Primer design considerations:

  • Design primers with balanced GC content when possible

  • Avoid primer regions with potential secondary structures

  • Include GC clamps at 5' end but not 3' end

  • Consider using primers successfully used for similar genes (like atBIF-1/atBIF-2 for atpD)

• Template preparation:

  • Use highly purified DNA free of PCR inhibitors

  • Optimize template concentration (usually lower than standard PCR)

  • Consider linearizing plasmid templates

These optimizations have been successfully applied to amplify GC-rich genes from Bifidobacterium species, including the atpD gene (using primers atBIF-1 and atBIF-2), which is part of the same operon as atpF .

How does atpF from B. longum compare to homologous genes in other probiotic bacteria?

A comparative analysis of atpF across probiotic species reveals important insights:

• Sequence conservation patterns:

  • atpF is part of the highly conserved atp operon found across eubacteria

  • The gene order (atpBEFHAGDC) is preserved in bifidobacteria but differs from some other probiotics

  • Sequence identity is highest among Bifidobacterium species, with more divergence in other genera

• Structural and functional comparisons:

  • The b subunit serves as a peripheral stalk in F1F0-ATPase across species

  • Length and domain organization are generally conserved

  • Species-specific adaptations may relate to membrane composition differences

• Phylogenetic relationships:

  • atpF sequences align with established taxonomic relationships

  • Can complement 16S rRNA and atpD-based phylogenetic analyses

  • Useful for distinguishing closely related taxa like B. lactis/B. animalis and L. gasseri/L. johnsonii

• Expression regulation:

  • pH-responsive regulation appears common across lactic acid bacteria

  • Different promoter strengths and regulatory elements exist between species

  • Energy metabolism coordination varies with fermentation patterns

• Comparative expression systems:

  Species	Expression System	Special Features	Efficiency
  B. longum	pBV222, pBFK86	High GC content	Variable, strain-dependent
  L. lactis	NICE system	Nisin-inducible	High, well-characterized
  L. acidophilus	pTRK-based vectors	Constitutive expression	Moderate to high

This comparative understanding helps researchers select appropriate expression systems and predict functional conservation when working with ATP synthase components across probiotic species .