Recombinant Bifidobacterium adolescentis Elongation factor G (fusA), partial

What is Elongation Factor G and what role does it play in Bifidobacterium adolescentis?

Elongation factor G (EF-G), encoded by the fusA gene, is a critical protein involved in bacterial protein synthesis. In B. adolescentis, as in other bacteria, EF-G promotes the translocation step of protein synthesis and works with ribosome recycling factor (frr) to dissociate ribosomes from mRNA after translation termination . The protein is essential for bacterial growth and survival, as it directly influences the efficiency of protein production.

In B. adolescentis specifically, EF-G functions may be particularly important given the bacterium's adaptation to the intestinal environment, where rapid protein synthesis is needed to respond to changing environmental conditions. When B. adolescentis interacts with intestinal epithelial cells, significant changes in membrane potential have been observed, which may indirectly affect protein synthesis machinery including EF-G function .

How does the structure of B. adolescentis EF-G compare to EF-G from other bacterial species?

While the specific crystal structure of B. adolescentis EF-G has not been fully characterized in the provided research, structural comparison can be inferred from studies of other bacterial species. EF-G typically consists of five domains (I-V), with domains III-V showing mobility relative to domains I-II.

The switch I region (residues 46-56) forms an ordered helix with a distinct conformation compared to EF-Tu structures in different nucleotide states. These structural features likely influence how EF-G interacts with ribosomes and antibiotics in different bacterial species, including B. adolescentis .

What experimental systems are suitable for studying B. adolescentis EF-G function?

Several experimental systems can be employed to study B. adolescentis EF-G function:

• Cell culture models: Co-culture systems using intestinal epithelial cells (such as CaCo-2/HT-29) provide valuable insights into how B. adolescentis behaves in environments mimicking the gut. These systems can be used to assess how environmental factors affect EF-G expression and function .

• Recombinant protein expression systems: E. coli-based expression systems can be used to produce recombinant B. adolescentis EF-G for structural and functional studies. This approach allows for site-directed mutagenesis to examine specific residues important for EF-G function.

• In vitro translation systems: Reconstituted translation systems using purified components can directly assess the role of EF-G in protein synthesis and ribosome recycling.

• Bacterial genetic models: Genetic manipulation of the fusA gene in B. adolescentis or heterologous expression in other bacterial species can provide insights into its functional importance.

When studying B. adolescentis in experimental settings, it's important to consider that this anaerobic bacterium shows significant responses to various environmental factors, including oxygen exposure and interactions with eukaryotic cells .

What methods can be used to express and purify recombinant B. adolescentis EF-G for structural studies?

Expression and purification of recombinant B. adolescentis EF-G for structural studies requires careful consideration of several methodological aspects:

Expression Systems:

• E. coli-based expression: The fusA gene from B. adolescentis can be cloned into appropriate expression vectors with affinity tags (His6, GST, etc.) for efficient purification. Codon optimization may be necessary due to differences in codon usage between E. coli and B. adolescentis.

• Cell-free expression systems: These may be advantageous for proteins that affect host cell viability or for rapid screening of expression conditions.

Purification Strategy:

• Affinity chromatography: Initial purification using affinity tags (His-tag, GST-tag)

• Ion exchange chromatography: For further purification based on protein charge characteristics

• Size exclusion chromatography: Final polishing step to obtain homogeneous protein

Critical Considerations:

• Maintaining anaerobic conditions during protein extraction may be crucial to preserve native structure, as B. adolescentis is sensitive to oxidative environments

• Buffer optimization is essential to maintain protein stability

• The addition of nucleotides (GDP/GTP) may stabilize certain conformations of EF-G for crystallization

For structural studies, crystallization screening should explore various conditions, with attention to the presence or absence of nucleotides and potential binding partners, as these can significantly influence EF-G conformation .

How do mutations in the fusA gene affect B. adolescentis survival and function in gut microbiome contexts?

Mutations in the fusA gene can significantly affect B. adolescentis survival and function in the gut microbiome through several mechanisms:

• Protein synthesis efficiency: Since EF-G is crucial for translation, mutations affecting its function can alter the rate of protein synthesis, impacting bacterial growth and adaptation to changing gut conditions.

• Stress response: B. adolescentis shows significant changes in membrane potential when exposed to eukaryotic cells and inflammatory mediators . Mutations in fusA may affect how the bacterium responds to these stressors.

• Antibiotic resistance: In other bacteria, fusA mutations can confer resistance to antibiotics that target EF-G, such as fusidic acid . Although B. adolescentis is not typically a target for antibiotic therapy, such mutations could affect its survival during antibiotic treatment for other conditions.

• Interactions with host cells: Given that B. adolescentis shows altered viability and membrane potential in the presence of eukaryotic cells , fusA mutations might influence host-bacteria interactions, potentially affecting probiotic properties.

• Metabolic capacity: By analogy with other bacteria, overexpression or mutation of fusA can affect metabolic outputs. In Corynebacterium glutamicum, fusA overexpression increases L-isoleucine production , suggesting similar modifications might alter B. adolescentis metabolism.

Research approaches to study these effects include:

• Site-directed mutagenesis of conserved EF-G domains

• Competition assays in mixed bacterial communities

• Transcriptomic and proteomic analyses to assess global effects of fusA mutations

• In vivo colonization studies using gnotobiotic animal models

What techniques can be used to analyze the interaction between B. adolescentis EF-G and the ribosome during protein translation?

Analyzing the interaction between B. adolescentis EF-G and the ribosome requires specialized techniques that capture the dynamics of this critical process:

• Cryo-electron microscopy (Cryo-EM): This technique has revolutionized our understanding of ribosome-EF-G interactions by capturing different conformational states during translocation. For B. adolescentis EF-G, Cryo-EM could reveal species-specific features of ribosome binding and translocation mechanics.

• Single-molecule fluorescence resonance energy transfer (smFRET): By labeling EF-G and ribosomal components with fluorophores, researchers can track the real-time dynamics of translocation events, providing insights into the kinetics of B. adolescentis EF-G function.

• Ribosome profiling: This technique can identify ribosome pause sites that might be influenced by EF-G function, potentially revealing unique aspects of B. adolescentis translation regulation.

• Biochemical approaches:

  • Toeprinting assays to monitor translocation

  • GTPase activity assays to measure EF-G function

  • Chemical crosslinking coupled with mass spectrometry to identify specific interaction sites

• Comparative structural analysis: By comparing the interaction patterns of B. adolescentis EF-G with those from well-studied bacteria like S. aureus , researchers can identify unique features that might relate to B. adolescentis' ecological niche.

When studying these interactions, it's important to account for the environmental conditions B. adolescentis typically encounters. The bacterium shows significant physiological changes under different conditions, including altered membrane potential in the presence of eukaryotic cells , which might influence EF-G function.

What are the optimal conditions for expressing recombinant B. adolescentis fusA in heterologous systems?

The optimal conditions for expressing recombinant B. adolescentis fusA in heterologous systems require careful optimization of multiple parameters:

Expression Host Selection:

• E. coli BL21(DE3): Standard for high-level expression of recombinant proteins

• E. coli Rosetta: Provides rare tRNAs that might be needed for efficient translation of B. adolescentis genes

• B. subtilis: As a Gram-positive host, might provide a more suitable cellular environment for proper folding

Expression Vector Features:

• Strong but inducible promoters (T7, tac)

• Appropriate affinity tags (His6, GST) that don't interfere with protein function

• Inclusion of B. adolescentis-specific regulatory elements if needed

Induction and Growth Conditions:

• Temperature optimization (often lower temperatures like 16-25°C improve proper folding)

• Induction timing (typically mid-log phase)

• Induction strength (IPTG concentration typically 0.1-1.0 mM)

• Growth media supplementation (amino acids, trace elements)

Optimization Table for B. adolescentis fusA Expression:

Since B. adolescentis is sensitive to oxygen and shows altered viability in different environmental conditions , maintaining microaerobic or anaerobic conditions during growth may improve expression of properly folded protein. Additionally, considering that EF-G function depends on GTP hydrolysis, supplementation with magnesium might enhance proper folding and activity.

How can researchers assess the functional activity of recombinant B. adolescentis EF-G?

Assessing the functional activity of recombinant B. adolescentis EF-G requires multiple complementary approaches:

• GTPase Activity Assay:

  • Measure GTP hydrolysis rates using colorimetric assays (malachite green assay)

  • Compare intrinsic GTPase activity versus ribosome-stimulated activity

  • Kinetic parameters (Km, Vmax) can provide insights into EF-G efficiency

• In vitro Translation Assays:

  • Reconstituted translation systems using purified components

  • Poly(U)-directed poly(Phe) synthesis assays to measure translocation efficiency

  • Ribosome recycling assays to assess post-termination activity with recycling factor

• Ribosome Binding Assays:

  • Co-sedimentation assays to measure EF-G binding to ribosomes

  • Surface plasmon resonance (SPR) or microscale thermophoresis (MST) for binding kinetics

  • Filter binding assays using radiolabeled GTP

• Conformational Analysis:

  • Circular dichroism to assess secondary structure integrity

  • Thermal shift assays to evaluate protein stability

  • Limited proteolysis to examine domain organization

• Complementation Studies:

  • Test whether B. adolescentis EF-G can functionally replace EF-G in other bacteria

  • Use temperature-sensitive EF-G mutants for complementation assays

For a comprehensive assessment, comparing recombinant B. adolescentis EF-G activity with EF-G from well-characterized species (e.g., E. coli or S. aureus ) provides valuable benchmarks. Additionally, testing activity under conditions that mimic the intestinal environment may provide insights into B. adolescentis-specific adaptations, especially considering its observed responses to intestinal epithelial cells .

What experimental approaches can determine how B. adolescentis EF-G function changes under different environmental conditions?

B. adolescentis shows significant physiological changes under different environmental conditions , and these likely affect EF-G function. The following experimental approaches can determine these functional changes:

• In vitro Activity Assays under Varying Conditions:

  • pH gradient (intestinal pH ranges from 5.5-7.5)

  • Oxygen tension (anaerobic to microaerobic)

  • Bile salt concentrations (0.05-0.3%)

  • Short-chain fatty acid presence

  • Temperature variations (35-40°C)

• Co-culture Experimental Systems:

  • Intestinal epithelial cell lines (CaCo-2/HT-29) to mimic host-microbe interaction

  • Addition of inflammatory modulators (TNF-α) to simulate gut inflammation

  • Mixed bacterial communities to assess interspecies effects

• Structural and Biophysical Studies:

  • Hydrogen-deuterium exchange mass spectrometry to detect conformational changes

  • Thermal stability assays under different conditions

  • Fluorescence-based conformational probes

• Transcriptomic and Proteomic Approaches:

  • RNA-seq to monitor fusA expression under different conditions

  • Ribosome profiling to assess translation efficiency

  • Proteomics to quantify EF-G protein levels and post-translational modifications

• Membrane Potential and Metabolic Activity Correlation:

  • Since B. adolescentis shows changes in membrane potential when exposed to eukaryotic cells , correlating these changes with EF-G activity could reveal regulatory mechanisms

  • Fluorescent probes for membrane potential combined with activity assays

How can insights from B. adolescentis EF-G research contribute to developing new probiotics?

Research on B. adolescentis EF-G can significantly contribute to probiotic development through several mechanisms:

• Optimization of Protein Synthesis Machinery:

  • Understanding EF-G function could allow genetic modifications to enhance B. adolescentis protein synthesis efficiency

  • Improved protein synthesis may enhance bacterial survival in the competitive gut environment

  • Engineered strains with optimized translation could produce higher levels of beneficial proteins

• Stress Adaptation Enhancement:

  • B. adolescentis shows significant physiological changes when exposed to intestinal epithelial cells, including alterations in membrane potential

  • Modifications to EF-G could potentially improve adaptation to gut stressors

  • Targeted mutations based on structural insights might enhance stress resistance

• Metabolic Engineering Applications:

  • In other bacteria, overexpression of fusA and frr increases amino acid production

  • Similar approaches could enhance B. adolescentis production of beneficial metabolites

  • Co-expression of optimized fusA with other metabolic genes could create designer probiotic strains

• Enhanced Host-Microbe Interactions:

  • Understanding how B. adolescentis protein synthesis responds to host cell contact could inform selection of strains with optimal host interaction profiles

  • Engineered EF-G variants could potentially modify how B. adolescentis interacts with immune components

• Improved Manufacturing and Stability:

  • Insights into EF-G function under stress could improve production processes

  • Strains with enhanced translation efficiency might show better survival during freeze-drying and storage

The translational machinery, including EF-G, represents an underexplored target for probiotic optimization. By understanding the unique features of B. adolescentis EF-G and its response to environmental conditions , researchers can develop next-generation probiotics with enhanced functional properties and stability.

What potential biotechnological applications exist for recombinant B. adolescentis EF-G?

Recombinant B. adolescentis EF-G offers several promising biotechnological applications beyond basic research:

• Enhanced Protein Production Systems:

  • Co-expression of optimized B. adolescentis EF-G could improve translation efficiency in industrial protein production

  • By analogy with Corynebacterium glutamicum, where overexpression of fusA and frr increased L-isoleucine production by 76.5% , similar approaches could enhance production of various metabolites

• Novel Antimicrobial Development:

  • The structure of bacterial EF-G has been explored as a target for antibiotics like fusidic acid

  • Understanding B. adolescentis EF-G structure could enable development of narrow-spectrum antimicrobials that spare beneficial bifidobacteria while targeting pathogens

• Diagnostic Tools:

  • Species-specific EF-G antibodies could be developed for detection and quantification of B. adolescentis in complex microbiome samples

  • Molecular probes targeting fusA could enable real-time monitoring of B. adolescentis in microbial communities

• Synthetic Biology Chassis Development:

  • Engineered B. adolescentis with optimized translation machinery could serve as specialized chassis for gut-targeted synthetic biology applications

  • Controlled expression of modified EF-G could create tunable protein synthesis systems

• Protein Engineering Platform:

  • Understanding the structural and functional properties of B. adolescentis EF-G could inform the design of novel proteins with customized translational properties

  • Chimeric EF-G proteins combining domains from different species could create translation factors with novel properties

These applications leverage the specific properties of B. adolescentis as a beneficial gut commensal with unique environmental responses and the critical role of EF-G in bacterial physiology. The development of these technologies requires detailed understanding of EF-G structure-function relationships and how they differ between bacterial species .

How can computational modeling enhance our understanding of B. adolescentis EF-G function?

Computational modeling provides powerful tools to enhance our understanding of B. adolescentis EF-G function across multiple scales:

These computational approaches are particularly valuable for understanding B. adolescentis EF-G function given the bacterium's sensitivity to environmental conditions and the complex conformational changes EF-G undergoes during its functional cycle . By integrating computational predictions with experimental validation, researchers can develop a comprehensive understanding of how this key translational factor contributes to B. adolescentis adaptation to the gut environment.