Recombinant Bifidobacterium adolescentis Ribosome-recycling factor (frr)

What is the primary function of ribosome-recycling factor (RRF) in bacterial translation?

Ribosome-recycling factor (RRF) plays a critical role in the final stage of protein translation by dissociating ribosomes from mRNA after termination of translation. It essentially "recycles" ribosomes, making them available for new rounds of protein synthesis. The process requires coordination with elongation factor G (EF-G), which together with RRF disassembles the posttermination complex into its constituent components: mRNA, tRNA, and the ribosome. This recycling mechanism is fundamentally important for efficient protein synthesis in bacteria .

Why is the frr gene considered essential in bacteria?

The frr gene is considered essential because it encodes the ribosome-recycling factor, which is required for bacterial viability. Studies in Escherichia coli have demonstrated that strains carrying frame-shifted frr in the chromosome cannot survive unless wild-type frr is provided on a plasmid. When such strains are challenged with conditions that eliminate the plasmid-carried wild-type frr, growth ceases. Furthermore, any thermoresistant colonies that emerge from temperature-sensitive strains invariably possess a functional frr gene, either through genetic re-exchange or plasmid modification. These observations conclusively establish that frr is an essential gene for bacterial cell growth and viability .

How does the structural interaction of RRF with the ribosome facilitate recycling?

RRF interacts primarily with segments of the large ribosomal subunit's (50S) rRNA helices that are involved in the formation of two central intersubunit bridges, B2a and B3. Cryo-electron microscopy reveals that RRF binding induces considerable conformational changes in functional domains of the ribosome. The tip of RRF domain I positions approximately 13 Å toward protein L5 within the central protuberance of the 50S subunit, while domain II orients toward the small ribosomal subunit (30S). The binding sites of RRF overlap with those of EF-G and P-site tRNA, suggesting a mechanism where EF-G binding triggers the removal of deacylated tRNA from the P site by repositioning RRF toward the ribosomal E site, followed by mRNA removal through shifts in the position of 16S rRNA helix 44 .

What makes Bifidobacterium adolescentis a suitable host for recombinant protein expression?

Bifidobacterium adolescentis possesses several characteristics that make it advantageous for recombinant protein expression. First, as a common inhabitant of the human gut microbiota, it has adapted to survive harsh gastrointestinal conditions, which provides inherent stability for in vivo applications. Second, B. adolescentis exhibits remarkable genetic diversity, with genomic analyses revealing 79 distinct genetic lineages among 113 analyzed strains, allowing researchers to select optimized strains for specific applications. Third, B. adolescentis has well-characterized carbohydrate metabolism pathways, enabling manipulation of growth conditions through carbon source selection. Notably, strains like PRL2023 demonstrate superior growth capabilities on complex carbohydrates such as inulin, pullulan, and fructooligosaccharides, providing options for optimizing recombinant protein production through metabolic engineering .

What are the key genetic considerations when working with recombinant B. adolescentis?

When developing recombinant B. adolescentis strains, researchers should consider:

• Strain selection: Different B. adolescentis strains exhibit varying prevalence in human populations (ranging from 27.9% for PRL2023 to 2.9% for DSM 20087) and possess distinct genetic features. The prototype strain PRL2023 represents the most comprehensive genetic content and functional features of the species, making it an ideal starting point for recombinant work.

• Codon optimization: The GC-rich genome of bifidobacteria necessitates codon optimization for heterologous gene expression.

• Promoter selection: Indigenous promoters from highly expressed genes in B. adolescentis often provide better expression than heterologous promoters.

• Growth conditions: Different strains show statistically significant differences in growth performance on various carbon sources, with PRL2023 demonstrating superior growth on complex carbohydrates compared to type strain ATCC 15703.

• Genetic stability: The essential nature of frr genes in bacteria warrants careful consideration of expression vector design to maintain genetic stability during recombinant production .

What are the optimal approaches for expressing recombinant RRF in B. adolescentis?

Based on current research methodologies for bifidobacteria, the optimal approach for recombinant RRF expression in B. adolescentis would involve:

• Vector selection: Use of shuttle vectors containing origins of replication functional in both E. coli (for cloning) and Bifidobacterium (for expression).

• Promoter optimization: Incorporation of strong, constitutive promoters from highly expressed B. adolescentis genes or inducible promoters responsive to specific carbon sources.

• Signal sequence incorporation: For secreted production, fusion with native B. adolescentis signal peptides from abundantly secreted proteins.

• Culture conditions: Growth in carbohydrate-optimized media based on the specific strain's metabolic profile. For example, with PRL2023, media containing inulin, pullulan, or fructooligosaccharides would maximize biomass production and potentially protein yield.

• Oxygen consideration: Strict anaerobic conditions during all cultivation steps, as bifidobacteria are obligate anaerobes.

• Purification strategy: Incorporation of affinity tags compatible with the Bifidobacterium genetic system for downstream purification, with tag positioning optimized to avoid interference with RRF function .

How can researchers assess the functionality of recombinant RRF from B. adolescentis?

Functional assessment of recombinant B. adolescentis RRF requires multiple complementary approaches:

• Complementation assays: Testing whether B. adolescentis RRF can complement temperature-sensitive E. coli frr mutants like MC1061-2, which carries a frame-shifted chromosomal frr and a temperature-sensitive plasmid with wild-type frr. Successful complementation would allow growth at non-permissive temperatures.

• In vitro ribosome dissociation assays: Measuring the ability of purified recombinant RRF to dissociate post-termination ribosomal complexes in conjunction with EF-G and GTP.

• Structural integrity assessment: Using circular dichroism spectroscopy to confirm proper protein folding compared to native RRF.

• Binding studies: Employing techniques like surface plasmon resonance or microscale thermophoresis to measure binding affinity to ribosomes.

• Cryo-EM structural analysis: Determining whether recombinant B. adolescentis RRF forms the expected structural interactions with ribosomal components, particularly the rRNA helices involved in intersubunit bridges B2a and B3 .

What expression systems have proven most effective for recombinant bifidobacterial proteins?

While specific data for recombinant RRF expression in B. adolescentis is limited, research on other bifidobacterial enzymes provides valuable insights. For instance, studies on α-L-arabinofuranosidase from B. adolescentis have established effective expression systems:

• E. coli-based expression: Often used for initial characterization, but may lack proper folding or post-translational modifications found in native Bifidobacterium.

• Homologous expression: Expression within B. adolescentis or closely related Bifidobacterium species preserves native protein characteristics but typically yields lower protein quantities.

• Lactococcus/Lactobacillus systems: These Gram-positive bacteria serve as intermediate hosts, offering better secretion capabilities than E. coli while maintaining reasonable yields.

For glycosidases like the arabinofuranosidase from B. adolescentis (which releases C3-linked arabinose residues from double-substituted xylose residues), plasmid-based expression systems utilizing indigenous promoters have been successfully implemented. Similar approaches would likely benefit recombinant RRF production .

How might recombinant B. adolescentis RRF contribute to understanding microbiome-host interactions?

Recombinant B. adolescentis RRF could serve as a valuable tool for investigating microbiome-host interactions through several research applications:

• Immunomodulatory studies: Recent research demonstrates that B. adolescentis correlates with vaccine efficacy, with individuals lacking this bacterium showing suboptimal antibody responses to vaccines. Recombinant RRF could be used to investigate whether this essential bacterial protein contributes to the immunomodulatory properties observed.

• Ribosomal adaptation mechanisms: Comparing RRF structure and function across different gut microbiome species could reveal adaptation mechanisms specific to the gut environment.

• Therapeutic protein delivery: Engineered B. adolescentis expressing modified RRF fusion proteins could potentially deliver therapeutic molecules to the gut, leveraging the bacterium's natural colonization abilities.

• Cross-species ribosomal interaction studies: Investigating whether B. adolescentis RRF can function with ribosomes from other bacterial species could provide insights into translation apparatus evolution within the gut microbiome.

• Probiotic enhancement: Understanding the role of RRF in B. adolescentis growth and survival could lead to optimized probiotic formulations with enhanced colonization potential .

What challenges arise when analyzing the impact of recombinant RRF expression on B. adolescentis metabolism?

Several methodological challenges must be addressed when studying how recombinant RRF expression affects B. adolescentis metabolism:

• Distinguishing native from recombinant RRF effects: Since RRF is essential, separating the metabolic impacts of native versus recombinant RRF requires careful experimental design, potentially using tagged variants for selective monitoring.

• Accounting for strain variation: The high genetic heterogeneity among B. adolescentis strains (79 distinct genetic lineages identified) means that metabolic responses to recombinant RRF expression may vary significantly between strains.

• Metabolic burden quantification: Overexpression of recombinant proteins often imposes a metabolic burden. For RRF, which interacts with essential translation machinery, this burden may manifest uniquely and requires specialized metabolic flux analysis techniques.

• Co-metabolism considerations: B. adolescentis engages in co-metabolism with other gut commensals, particularly in processing plant-derived glycans. Recombinant RRF expression could alter these interactions in complex ways that are challenging to measure in simplified laboratory systems.

• Integration of multi-omics data: Comprehensive assessment requires integration of transcriptomics, proteomics, and metabolomics data, with particular attention to translation-related pathways that might be affected by altered RRF levels .

How do different carbon sources affect recombinant protein production in B. adolescentis?

The choice of carbon source significantly impacts recombinant protein production in B. adolescentis due to the bacterium's specialized carbohydrate metabolism. Comparative growth studies reveal substantial differences in growth performance across different strains and carbon sources:

*Statistical significance determined by Mann-Whitney test with Benjamini-Hochberg correction

These growth differences suggest that carbon source selection should be tailored to the specific B. adolescentis strain used for recombinant protein production. For expression systems using PRL2023, inulin and fructooligosaccharides appear particularly promising as they support robust growth. Additionally, the ability of B. adolescentis to co-metabolize plant-derived glycans such as xylan with other gut commensals suggests potential for developing complex media formulations that optimize recombinant protein production through microbial consortia approaches .

What are common pitfalls in recombinant RRF expression and how can they be addressed?

Common challenges in recombinant RRF expression include:

• Toxicity from overexpression: Since RRF interacts with essential translation machinery, overexpression can be toxic. Solution: Use tightly regulated inducible promoters and optimize induction parameters for balance between yield and cellular viability.

• Protein misfolding: RRF has a unique two-domain structure that may be challenging to fold correctly in recombinant systems. Solution: Consider expressing with chaperones, optimizing growth temperature, or using periplasmic expression strategies to enhance proper folding.

• Poor solubility: Recombinant RRF may form inclusion bodies. Solution: Express as fusion proteins with solubility-enhancing partners like thioredoxin or SUMO, optimize growth at lower temperatures, or develop effective refolding protocols.

• Low expression levels: The essential nature of RRF may lead to selection against high-level expression. Solution: Use strong ribosome binding sites, optimize codon usage for B. adolescentis, and balance promoter strength with cellular tolerance.

• Loss of function: Modifications for purification may interfere with activity. Solution: Carefully position affinity tags to avoid interfering with binding interfaces, particularly domains that interact with rRNA helices involved in intersubunit bridges B2a and B3 .

How can researchers optimize recombinant protein yield in B. adolescentis culture systems?

To optimize recombinant protein yield in B. adolescentis:

• Strain selection: Choose strains with robust growth characteristics, such as PRL2023, which has superior ability to metabolize complex carbohydrates and is widely distributed among human populations (found in 27.9% of examined microbiomes).

• Media optimization: Develop a defined media formulation based on strain-specific carbohydrate utilization profiles. For PRL2023, inulin and fructooligosaccharides support maximal growth (OD values between 0.8-1.1).

• Growth conditions: Maintain strict anaerobic conditions (typically <0.1 ppm O₂) throughout cultivation. Control pH between 6.0-6.5 for optimal bifidobacterial growth.

• Induction timing: For inducible systems, determine optimal induction point by monitoring growth curves; typically, mid-logarithmic phase balances biomass accumulation with metabolic activity.

• Harvest timing: Determine the optimal harvest time that balances protein accumulation against potential degradation, typically through time-course expression studies.

• Scale-up considerations: When transitioning from laboratory to larger-scale production, carefully control oxygen exposure during media preparation and inoculation, as even brief oxygen exposure can significantly impact bifidobacterial viability and protein production .

What analytical methods are most appropriate for verifying the structural integrity of recombinant B. adolescentis RRF?

To verify structural integrity of recombinant B. adolescentis RRF, researchers should employ multiple complementary analytical methods:

• Circular dichroism (CD) spectroscopy: Provides information about secondary structure elements. RRF typically contains both α-helices and β-sheets in a characteristic arrangement; CD can verify these structural features are preserved in the recombinant protein.

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS): Confirms proper oligomeric state and detects potential aggregation issues.

• Thermal shift assays: Measure protein stability and can identify if recombinant RRF has comparable thermal stability to native protein.

• Limited proteolysis: Pattern of proteolytic fragments can indicate whether the protein is properly folded with expected domain organization.

• Nuclear magnetic resonance (NMR) spectroscopy: For more detailed structural analysis, can provide residue-level information about protein folding.

• Cryo-electron microscopy: Most definitive for functional validation, can visualize whether recombinant RRF binds to ribosomes with the expected orientation, particularly interacting with the large ribosomal subunit's rRNA helices involved in intersubunit bridges B2a and B3, and whether it induces the expected conformational changes in ribosomal functional domains .