Recombinant Bifidobacterium adolescentis Serine hydroxymethyltransferase (glyA)

What is Serine hydroxymethyltransferase (SHMT) and what is its significance in Bifidobacterium adolescentis?

Serine hydroxymethyltransferase (SHMT), encoded by the glyA gene, is a crucial enzyme that catalyzes the reversible conversion of serine to glycine while transferring a one-carbon unit to tetrahydrofolate. In prokaryotes like B. adolescentis, this enzyme plays a vital role in one-carbon metabolism, amino acid biosynthesis, and nucleotide synthesis. While specific research on B. adolescentis SHMT is limited, studies in other bacteria suggest that SHMT is essential for growth and cellular processes. As a major commensal bacterium in the human gut microbiota, understanding B. adolescentis metabolic pathways, including those involving SHMT, can provide insights into its beneficial health effects and ecological role in the gastrointestinal environment .

How does the structure and function of B. adolescentis glyA compare to other bacterial species?

While the search results don't provide specific structural information about B. adolescentis glyA, comparative analysis can be approached by examining homologous genes in related bacterial species. Based on research with other bacteria, the glyA gene in B. adolescentis likely maintains conserved catalytic domains while possessing species-specific features that may reflect its adaptation to the intestinal environment.

For example, the glyA gene from Campylobacter jejuni has been successfully cloned and expressed in E. coli, leading to high levels of SHMT production . Similar approaches could be applied to B. adolescentis glyA. Genomic analysis of various B. adolescentis strains, such as prototype PRL2023 and type strain ATCC 15703, reveals considerable genetic diversity within the species , suggesting that glyA variants might have evolved different functional characteristics across strains.

What methods are recommended for isolating and identifying B. adolescentis strains for glyA research?

For isolating B. adolescentis strains suitable for glyA research, a combined approach utilizing both traditional microbiological techniques and modern genomic methods is recommended:

• Sample collection: Obtain samples from adult human intestines, as B. adolescentis is commonly found in the adult gut microbiome .

• Selective cultivation: Use selective media for bifidobacteria under strictly anaerobic conditions, as B. adolescentis is among the most oxygen-sensitive Bifidobacterium species .

• Strain identification: Perform 16S rRNA gene sequencing for preliminary identification, followed by whole genome sequencing for definitive characterization.

• Strain selection: Based on ecological and phylogenomic analysis of sequenced genomes, select strains that best represent the species' genetic diversity, similar to how PRL2023 was identified as a prototype strain .

• glyA gene screening: Design specific primers to amplify and sequence the glyA gene from isolated strains to identify natural variants of interest.

This comprehensive approach ensures the selection of well-characterized strains with confirmed taxonomic identity, which is essential for reliable glyA research.

What are the optimal cloning strategies for B. adolescentis glyA gene?

Based on successful approaches with other bacterial genes, the following cloning strategy is recommended for B. adolescentis glyA:

• Genomic library construction: Create an extensive genomic library using a suitable vector system such as pBR322, which has been successfully used for similar purposes in C. jejuni glyA cloning .

• PCR amplification: Design primers that flank the complete glyA gene, including its native promoter region to facilitate expression in heterologous hosts.

• Vector selection: For initial cloning, choose a vector system compatible with both E. coli (for ease of manipulation) and Bifidobacterium (for native expression studies).

• Transformation protocol: Due to the anaerobic nature of Bifidobacterium, optimize electroporation conditions under strict anaerobic environments to achieve successful transformation.

• Screening approach: Implement functional complementation in glyA-deficient strains as a reliable screening method to identify recombinant clones containing the functional glyA gene.

This systematic approach addresses the technical challenges associated with genetic manipulation of anaerobic bacteria while maximizing the chances of successful glyA cloning.

How can heterologous expression of B. adolescentis glyA be optimized in E. coli and other hosts?

Optimizing heterologous expression of B. adolescentis glyA requires addressing several factors:

The goal is to balance high expression levels with proper protein folding and activity. The approach used for C. jejuni glyA, which achieved high levels of SHMT in E. coli through expression from its own promoter on a multicopy plasmid , provides a useful starting model.

What are the challenges in maintaining plasmid stability in recombinant B. adolescentis?

Maintaining plasmid stability in recombinant B. adolescentis presents several challenges:

• Anaerobic conditions: As one of the most oxygen-sensitive Bifidobacterium species , B. adolescentis requires strict anaerobic conditions during transformation and cultivation, which can complicate standard molecular biology procedures.

• Limited selection markers: The choice of appropriate selection markers for Bifidobacterium is restricted compared to aerobic bacteria.

• Recombination events: Homologous recombination between introduced plasmid and chromosomal DNA can lead to plasmid instability.

• Metabolic burden: Overexpression of heterologous proteins like SHMT can create metabolic stress, potentially selecting for plasmid-free cells during prolonged cultivation.

• Growth conditions: Plasmid stability can be affected by pH, temperature, and media composition, requiring optimization for B. adolescentis specific physiology.

To address these challenges, strategies include using compatible selection markers, optimizing growth conditions to reduce metabolic burden, employing integrative vectors for stable expression, and regular monitoring of plasmid retention through multiple generations.

How can functional assays for recombinant B. adolescentis SHMT activity be designed?

Designing robust functional assays for recombinant B. adolescentis SHMT requires considerations for the enzyme's specific biochemical properties and the anaerobic nature of the organism:

• Spectrophotometric assays: Measure the conversion of serine to glycine by coupling the reaction to other enzymes that produce detectable signals. For example, the production of methylenetetrahydrofolate can be monitored through its subsequent reactions.

• Radioisotope-based assays: Use 14C-labeled serine to track the transfer of the one-carbon unit to tetrahydrofolate, followed by scintillation counting.

• HPLC quantification: Develop methods to directly quantify serine consumption and glycine production using reversed-phase HPLC.

• Complementation assays: Utilize glyA-deficient E. coli strains to assess functional complementation by the recombinant B. adolescentis glyA, similar to the approach used for C. jejuni glyA .

• Anaerobic considerations: Ensure all assays are performed under appropriate anaerobic conditions to maintain enzyme stability and activity.

These methods should be validated using appropriate controls, including known SHMT enzymes from other sources, and standardized to enable comparative analyses between different B. adolescentis strains or mutant variants.

What strategies can be employed to improve SHMT yield and stability in recombinant B. adolescentis systems?

To optimize SHMT yield and stability in recombinant B. adolescentis systems, consider implementing these strategies:

• Promoter optimization: Test different promoters to identify those that provide optimal expression levels without imposing excessive metabolic burden. For example, a comparative approach like that used in the GABA production study with B. adolescentis might be valuable .

• Codon optimization: Analyze and optimize codon usage to enhance translation efficiency while maintaining protein folding and stability.

• Cultivation conditions: Systematically optimize parameters such as pH, temperature, and media composition. For instance, pH 6.0 was identified as optimal for certain recombinant protein production in B. adolescentis .

• Fed-batch fermentation: Implement controlled feeding strategies to maintain optimal growth conditions and nutrient availability, similar to the approach that achieved high production levels in the GABA study .

• Protein engineering: Introduce stability-enhancing mutations based on structural knowledge of SHMT from related species.

• Co-expression of chaperones: Consider co-expressing molecular chaperones to assist with proper protein folding.

• Post-translational modifications: Investigate the role of potential post-translational modifications in SHMT stability and activity.

A systematic approach that combines these strategies can significantly improve both yield and stability of recombinant SHMT in B. adolescentis.

How can researchers effectively analyze the impact of recombinant glyA expression on B. adolescentis metabolism?

To comprehensively analyze the impact of recombinant glyA expression on B. adolescentis metabolism, researchers should employ a multi-omics approach:

• Transcriptomics: Perform RNA-seq to identify genes whose expression changes in response to glyA overexpression, revealing potential metabolic adaptations. This is similar to the metatranscriptomic approaches used to study B. adolescentis interactions with other gut microbes .

• Proteomics: Use mass spectrometry-based proteomics to quantify changes in the protein complement, focusing on pathways connected to one-carbon metabolism and amino acid biosynthesis.

• Metabolomics: Quantify changes in metabolite levels, particularly serine, glycine, and folate derivatives, to assess the functional impact of altered SHMT activity.

• Flux analysis: Implement 13C-labeling experiments to trace carbon flow through central metabolic pathways before and after glyA overexpression.

• Growth phenotyping: Compare growth characteristics under various conditions to identify phenotypic changes resulting from altered SHMT levels.

• Interaction studies: Assess how glyA overexpression affects interactions with other gut microbes, potentially using co-cultivation approaches similar to those described for B. adolescentis PRL2023 .

Integration of these datasets can provide a systems-level understanding of how recombinant glyA expression reprograms cellular metabolism in B. adolescentis.

How can CRISPR-Cas systems be adapted for precise glyA editing in B. adolescentis?

Adapting CRISPR-Cas systems for B. adolescentis glyA editing requires addressing several challenges specific to this anaerobic bacterium:

• Selection of appropriate Cas variants: Test various Cas9 or Cas12 orthologs for activity in the B. adolescentis cellular environment, focusing on those previously successful in related Gram-positive bacteria.

• Delivery method optimization: Develop efficient transformation protocols under strict anaerobic conditions, potentially using electroporation methods optimized for Bifidobacterium.

• Guide RNA design: Design sgRNAs with high specificity for the glyA target region, accounting for the high GC content typical of Bifidobacterium genomes.

• Repair template optimization: Create repair templates with sufficient homology arms (>1 kb) to facilitate homology-directed repair, which may require adjustments based on B. adolescentis recombination efficiency.

• Inducible expression systems: Develop tightly controlled inducible promoters for Cas9 expression to minimize toxicity, similar to approaches used in other recombinant Bifidobacterium systems .

• Screening strategy: Implement efficient screening methods to identify successful editing events, possibly using phenotypic selection or PCR-based approaches.

This adapted CRISPR-Cas system would enable precise genetic modifications of glyA, facilitating structure-function studies and metabolic engineering of B. adolescentis.

What insights can protein crystallography provide about B. adolescentis SHMT structure-function relationships?

Protein crystallography of B. adolescentis SHMT can provide crucial insights into:

• Catalytic mechanism: Identify the precise arrangement of active site residues responsible for pyridoxal phosphate binding and serine/glycine interconversion.

• Substrate specificity determinants: Reveal structural features that determine substrate preference and catalytic efficiency compared to SHMTs from other species.

• Allosteric regulation: Uncover potential allosteric sites that might regulate enzyme activity in response to metabolic changes.

• Oligomeric state: Determine whether B. adolescentis SHMT functions as a monomer, dimer, or higher-order oligomer, which can impact its catalytic properties.

• Species-specific features: Identify unique structural elements that might reflect adaptation to the gut environment where B. adolescentis naturally resides.

To facilitate crystallography studies, researchers should:

• Express the recombinant enzyme with affinity tags for purification

• Screen multiple buffer conditions to identify those that maintain protein stability

• Test both apo-enzyme and enzyme-substrate/cofactor complexes for crystallization

• Consider surface entropy reduction mutations if crystallization proves challenging

The resulting structural information would guide rational protein engineering efforts and provide evolutionary insights into this key metabolic enzyme.

How might recombinant B. adolescentis expressing modified glyA variants impact the gut microbiome?

The introduction of recombinant B. adolescentis expressing modified glyA variants into the gut microbiome could have several ecological and functional impacts:

• Metabolic interactions: Modified SHMT activity could alter one-carbon metabolism, potentially changing the availability of metabolites like glycine and serine that are involved in cross-feeding relationships with other gut bacteria. This is particularly relevant given the observed co-metabolism between B. adolescentis and other gut commensals .

• Competitive fitness: Enhanced or reduced SHMT activity might affect the competitive fitness of B. adolescentis in the complex gut ecosystem, potentially altering community structure similar to the interactions observed in co-cultivation experiments .

• Colonization efficiency: Changes in central metabolism due to modified glyA could impact the strain's ability to colonize the gut and persist in this environment, particularly under the dynamic nutrient conditions of the intestinal tract.

• Host-microbe interactions: Altered metabolite production might influence interactions with the host epithelium, potentially affecting immune responses or barrier function.

• Genetic transfer: There's potential for horizontal gene transfer of the modified glyA to other gut microbes, though this would require detailed risk assessment.

To study these effects, researchers could employ gnotobiotic animal models with defined microbial communities, followed by multi-omics analyses to track community dynamics and metabolic changes after introduction of the recombinant strain.

What are the best approaches for analyzing SHMT enzyme kinetics in recombinant B. adolescentis?

For rigorous analysis of SHMT enzyme kinetics in recombinant B. adolescentis, researchers should consider the following methodological approaches:

• Enzyme preparation: Extract SHMT under anaerobic conditions to preserve native structure and activity. Purification should be rapid and include stabilizing agents like pyridoxal phosphate.

• Reaction conditions optimization: Systematically test buffers, pH ranges (5.5-7.5), temperatures (30-40°C), and ionic strengths to determine optimal conditions that reflect the intestinal environment.

• Substrate concentration range: Design experiments with serine concentrations spanning at least 0.1-10x the expected Km value to enable accurate determination of kinetic parameters.

• Initial velocity measurements: Ensure measurements are made within the linear range of the reaction, typically by limiting the reaction time or enzyme concentration.

• Data analysis: Apply appropriate kinetic models (Michaelis-Menten, Hill equation, etc.) to determine key parameters:

• Comparative analysis: Compare kinetic parameters with SHMT from other species to identify unique features of the B. adolescentis enzyme.

Following these approaches will yield reliable kinetic data that can inform metabolic modeling and enzyme engineering efforts.

How can researchers overcome the challenges of anaerobic techniques when working with recombinant B. adolescentis?

Working with B. adolescentis, one of the most oxygen-sensitive Bifidobacterium species , presents unique challenges that require specialized anaerobic techniques:

• Anaerobic chamber setup: Invest in a high-quality anaerobic chamber with oxygen monitoring capabilities. Maintain O2 levels below 5 ppm and supplement with catalysts to remove trace oxygen.

• Media preparation: Pre-reduce all media by boiling under nitrogen gas flow followed by addition of reducing agents like cysteine-HCl (0.05%) and resazurin as an oxygen indicator.

• Buffer degassing: Systematically remove dissolved oxygen from all buffers using vacuum degassing combined with nitrogen or argon purging.

• Anaerobic transfer techniques: Develop proficiency in techniques for transferring cultures without oxygen exposure, such as using syringes with stoppered vessels.

• Enzyme work: Perform all enzyme extractions and assays under strictly anaerobic conditions to preserve SHMT activity, potentially using oxygen-scavenging enzyme systems as additional protection.

• Molecular biology adaptations: Modify standard molecular biology protocols for anaerobic conditions, including DNA extraction, transformation, and protein purification.

• Real-time oxygen monitoring: Implement continuous oxygen monitoring during critical procedures to ensure anaerobic integrity.

• Storage considerations: Develop protocols for sample storage that maintain anaerobic conditions, particularly for long-term preservation of strains and enzyme preparations.

These methodological adaptations are essential for obtaining reliable results when working with recombinant B. adolescentis and its oxygen-sensitive enzymes like SHMT.

What experimental controls are essential for validating recombinant B. adolescentis glyA expression studies?

A comprehensive set of controls is crucial for rigorously validating recombinant B. adolescentis glyA expression studies:

• Negative controls:

  • Empty vector transformants to assess background activity and plasmid effects

  • Heat-inactivated enzyme preparations to confirm enzymatic nature of observed activity

  • glyA knockout strains to establish baseline metabolism without SHMT

• Positive controls:

  • Wild-type B. adolescentis strain expressing native glyA levels

  • Well-characterized SHMT from model organisms (e.g., E. coli) as reference for activity assays

  • Purified commercial SHMT for standardization of enzyme assays

• Expression controls:

  • qRT-PCR to quantify glyA transcript levels

  • Western blotting with anti-SHMT antibodies to confirm protein expression

  • Activity assays with varying substrate concentrations to verify functional enzyme production

• Environmental controls:

  • Strict monitoring of anaerobic conditions throughout experiments

  • Consistent media composition across experiments

  • Temperature monitoring during growth and assays

• Growth controls:

  • Growth curves comparing recombinant and wild-type strains

  • Monitoring of plasmid stability over multiple generations

  • Assessment of cell morphology and viability

• Batch controls:

  • Experimental replicates from independent transformations

  • Technical replicates for all measurements

  • Time-course experiments to ensure reproducibility

Implementing these controls ensures that observed phenotypes and biochemical characteristics can be confidently attributed to the recombinant glyA expression rather than experimental artifacts or secondary effects.

What are the potential applications of engineered B. adolescentis expressing modified glyA in microbiome research?

Engineered B. adolescentis strains with modified glyA offer several promising applications in microbiome research:

• Metabolic interaction studies: Modified glyA variants could serve as tools to investigate cross-feeding relationships in the gut microbiome, particularly how one-carbon metabolism links different bacterial species, similar to the co-cultivation studies that revealed interactions between B. adolescentis and other gut commensals .

• Microbiome tracers: B. adolescentis strains with tagged or modified glyA could function as traceable probes to monitor bacterial colonization and persistence in complex microbial communities.

• Synthetic ecology models: Engineered strains could help develop defined microbial communities with predictable metabolic networks, advancing our understanding of ecological principles governing the gut microbiome.

• Mechanistic studies: Strains expressing glyA variants with altered kinetic properties could reveal how SHMT activity influences bacterial fitness in the intestinal environment.

• Microbiome modulators: Strategically engineered B. adolescentis strains might influence community composition by altering the availability of one-carbon metabolites.

These applications could significantly advance our understanding of microbial ecology in the gut and potentially lead to new approaches for microbiome modulation in research contexts.

How might the study of recombinant B. adolescentis glyA contribute to our understanding of probiotic mechanisms?

Studying recombinant B. adolescentis glyA can provide valuable insights into probiotic mechanisms through several avenues:

• Metabolite production: Modified SHMT activity could alter the production of glycine and other one-carbon metabolism products that may influence host physiology or interact with the intestinal epithelium.

• Ecological fitness: Understanding how glyA expression affects B. adolescentis survival and competitive ability in the gut environment could inform the design of more effective probiotics that can successfully establish in the intestinal ecosystem.

• Host-microbe interactions: Altered metabolic profiles resulting from modified glyA expression might affect how B. adolescentis interacts with host cells, potentially impacting immune responses or epithelial barrier function.

• Strain-specific effects: Comparative studies of glyA across different B. adolescentis strains could help explain strain-specific probiotic effects, similar to how the PRL2023 prototype strain was identified as having distinctive properties .

• Next-generation probiotics: The molecular understanding gained from glyA studies could contribute to rational design of next-generation probiotics with enhanced beneficial properties, aligning with recent research identifying B. adolescentis PRL2023 as a candidate for such applications .

These contributions would advance our understanding of the molecular mechanisms underlying probiotic effects, moving beyond correlative observations toward causal mechanisms.

What integrative approaches can combine recombinant B. adolescentis glyA research with other -omics technologies?

Integrating recombinant B. adolescentis glyA research with other -omics technologies can provide comprehensive insights through these multidisciplinary approaches:

• Multi-omics integration pipeline:

  • Genomics: Identify strain-specific variants of glyA and associated genetic elements

  • Transcriptomics: Analyze expression patterns of glyA and metabolically connected genes

  • Proteomics: Quantify SHMT protein levels and post-translational modifications

  • Metabolomics: Measure changes in one-carbon metabolites and connected pathways

  • Metagenomics: Track recombinant B. adolescentis in complex communities

• Ecological systems biology:

  • Apply ecological modeling to predict how modified glyA expression affects community dynamics

  • Use network analysis to map metabolic interactions between B. adolescentis and other gut microbes

  • Employ similar approaches to those used in studying B. adolescentis PRL2023 interactions with gut commensals

• Host-microbe interaction framework:

  • Combine microbiome profiling with host transcriptomics/proteomics

  • Correlate SHMT activity with host metabolomic profiles

  • Employ gnotobiotic models with defined bacterial communities

• Computational integration approaches:

  • Develop predictive metabolic models incorporating SHMT kinetics

  • Apply machine learning to identify patterns across multi-omics datasets

  • Implement visualization tools for complex data integration