Recombinant Bacteroides thetaiotaomicron Elongation factor Tu (tuf)

What is Elongation factor Tu (EF-Tu) and what is its significance in Bacteroides thetaiotaomicron research?

Elongation factor Tu (EF-Tu) is a highly conserved bacterial protein primarily known for its canonical role in protein synthesis, where it delivers aminoacyl-tRNAs to the ribosome during translation elongation. In B. thetaiotaomicron research, EF-Tu has gained significant attention as a "moonlighting protein" - a protein that performs multiple unrelated functions beyond its primary role in translation.

Recent studies have revealed that EF-Tu functions as a mucin-adhesive protein when expressed on the bacterial cell surface. This moonlighting function plays a crucial role in bacterial adhesion to the intestinal mucosa, which is fundamental for colonization and persistence in the gut environment . The dual functionality of EF-Tu makes it an important target for understanding B. thetaiotaomicron's interactions with host tissues and other microbes in the gut microbiome.

Methodologically, researchers typically study EF-Tu function through recombinant protein expression, adhesion assays, and proteomic analyses to characterize its binding properties and surface localization patterns. Understanding these properties is essential for elucidating B. thetaiotaomicron's ecological role in the intestinal microbiota.

How does recombinant EF-Tu interact with the surface of B. thetaiotaomicron cells?

Recombinant EF-Tu demonstrates a remarkable ability to bind to the cell surface of B. thetaiotaomicron through a pH-dependent mechanism. When B. thetaiotaomicron cells are incubated with recombinant His6-tagged EF-Tu proteins under specific conditions, the proteins localize to the bacterial cell surface .

The interaction appears to be highly dependent on environmental pH. Fluorescence microscopy has shown strong fluorescence indicating cell surface localization of recombinant EF-Tu at pH 5.2, whereas fluorescence is remarkably weak at pH 8.0, suggesting minimal or no localization at higher pH levels . This pH dependency provides valuable insights into the conditions under which these interactions may occur naturally in the gut environment.

Western blotting with anti-His tag antibodies can confirm the presence of recombinant EF-Tu in B. thetaiotaomicron cell fractions, providing biochemical evidence of this interaction . The binding mechanism likely involves electrostatic interactions that are favored under slightly acidic conditions that predominate in certain gut microenvironments.

This binding phenomenon represents a form of protein sharing between bacterial species that may contribute to microbial community dynamics in the gut ecosystem.

What experimental conditions are optimal for studying recombinant EF-Tu interactions with B. thetaiotaomicron?

Optimal experimental conditions for studying recombinant EF-Tu interactions with B. thetaiotaomicron include:

pH Optimization: Maintaining a pH of approximately 5.2 is critical, as research demonstrates significantly enhanced binding of recombinant EF-Tu to B. thetaiotaomicron at this pH compared to higher pH values (8.0) . This pH condition appears to create an optimal electrostatic environment for protein attachment to the bacterial surface.

Anaerobic Culture Conditions: B. thetaiotaomicron is an obligate anaerobe, so interactions should be studied under strict anaerobic conditions to ensure physiologically relevant results. Research protocols typically include incubation of B. thetaiotaomicron with recombinant proteins under anaerobic conditions for approximately 1 hour .

Growth Medium Selection: General Anaerobic Medium (GAM) broth with adjusted pH has been used successfully in experimental protocols . The medium composition may significantly impact protein-cell interactions.

Protein Concentration: Appropriate concentration of purified recombinant proteins is essential - excessive concentrations may lead to non-specific binding, while insufficient amounts may yield false negatives.

Controls: Proper controls such as bovine serum albumin (BSA) treatment or untreated cells are essential for distinguishing specific from non-specific binding effects .

These conditions collectively create an experimental environment that closely mimics the natural gut habitat where these interactions would occur, ensuring results have physiological relevance.

What methods can be used to detect the localization of recombinant EF-Tu on B. thetaiotaomicron cell surfaces?

Several complementary methods can effectively detect the localization of recombinant EF-Tu on B. thetaiotaomicron cell surfaces:

Fluorescence Microscopy: Using His-tagged recombinant EF-Tu proteins and fluorescently labeled anti-His antibodies allows direct visualization of protein localization on bacterial cell surfaces. This technique has successfully demonstrated pH-dependent localization of EF-Tu on B. thetaiotaomicron .

Western Blotting: This technique can detect the presence of recombinant EF-Tu in bacterial cell fractions using anti-His tag antibodies. By separating cellular components (membrane versus cytoplasmic fractions), researchers can determine the subcellular localization of the protein .

Proteomic Analysis: Mass spectrometry-based proteomic approaches can identify the presence of EF-Tu on bacterial cell surfaces with high specificity. This approach has been used to detect multiple B. longum-derived cytoplasmic proteins, including EF-Tu, on the surface of B. thetaiotaomicron after co-culture .

Functional Adhesion Assays: Indirect detection through functional analysis involves measuring changes in bacterial adhesion to substrates like porcine gastric mucin (PGM) or epithelial cells after treatment with recombinant EF-Tu. Increased adhesion indicates successful surface localization of the protein .

Flow Cytometry: Although not explicitly mentioned in the provided search results, flow cytometry using fluorescently labeled antibodies is another quantitative method for detecting surface-bound proteins.

These methods provide complementary information about protein localization and can be used in combination for comprehensive analysis of EF-Tu-bacterial surface interactions.

How does B. thetaiotaomicron growth change when exposed to recombinant EF-Tu?

When designing experiments to assess potential growth effects, researchers should consider:

Growth Curve Analysis: Monitoring optical density over time in cultures with and without recombinant EF-Tu treatment.

Viable Cell Counting: Determining if EF-Tu treatment affects culturability or viability through plating and colony counting.

Metabolic Profiling: Analyzing whether EF-Tu exposure alters metabolic outputs, which could be assessed through methods similar to those used in studies examining B. thetaiotaomicron metabolism of various carbon sources .

Competition Assays: Examining how EF-Tu treatment might affect B. thetaiotaomicron's competitive fitness in mixed cultures, similar to the consecutive batch culture (CBC) system used in other B. thetaiotaomicron research .

Without specific data on growth effects, researchers should approach this question experimentally, considering that moonlighting proteins may influence bacterial physiology beyond their characterized adhesion functions.

How does pH affect the binding efficiency of recombinant EF-Tu to B. thetaiotaomicron cell surfaces?

pH plays a critical role in determining the binding efficiency of recombinant EF-Tu to B. thetaiotaomicron cell surfaces. Experimental evidence demonstrates a strong pH dependency in this interaction, with profound implications for understanding the ecological contexts in which these interactions occur.

At pH 5.2, fluorescence microscopy reveals intense fluorescence signals on B. thetaiotaomicron JCM 5827 T cells incubated with His6-tagged recombinant EF-Tu, indicating robust cell surface localization. In stark contrast, at pH 8.0, fluorescence is remarkably weak, suggesting negligible protein localization . This pH-dependent binding pattern was consistently observed across multiple experimental approaches.

The mechanism behind this pH dependency likely involves changes in protein conformation and charge distribution. At lower pH (5.2), which approximates the slightly acidic environment of certain gut regions, electrostatic interactions favoring protein-cell surface binding may be enhanced. Additionally, pH affects the binding of these proteins to substrates like porcine gastric mucin (PGM), with enhanced binding observed at pH 5.2 compared to higher pH values .

From a methodological perspective, researchers must carefully control and report pH conditions when conducting experiments involving recombinant EF-Tu and B. thetaiotaomicron. The significant difference in binding efficiency between pH 5.2 and 8.0 highlights the importance of physiologically relevant experimental conditions that mirror the gut microenvironment.

What role does recombinant EF-Tu play in B. thetaiotaomicron adhesion to mucins and epithelial cells?

Recombinant EF-Tu significantly enhances B. thetaiotaomicron's adhesion capabilities to both mucins and epithelial cells, revealing a crucial moonlighting function beyond its canonical role in protein synthesis.

When B. thetaiotaomicron JCM 5827 T cells are treated with recombinant His6-EF-Tu at pH 5.2, their adhesion to porcine gastric mucin (PGM) increases compared to bovine serum albumin (BSA)-treated or untreated control cells . Similarly, adhesion to human epithelial Caco-2 cells is enhanced following His6-EF-Tu treatment at pH 5.2 . These findings demonstrate that exogenously supplied recombinant EF-Tu can functionally modify the adhesive properties of B. thetaiotaomicron.

The mechanism involves localization of EF-Tu to the bacterial cell surface, where it acts as an adhesin. EF-Tu has been identified as a "mucin-adhesive moonlighting protein," suggesting it has specific affinity for mucin glycoproteins that line the intestinal epithelium . This property enables it to serve as a molecular bridge between bacterial cells and host surfaces.

Experimentally, this phenomenon has significant implications for understanding bacterial colonization dynamics. The adhesion-promoting effect appears to be protein-specific, as different moonlighting proteins (like GroEL) show varying degrees of enhancement in adhesion assays . The pH dependency of this effect (occurring at pH 5.2 but not at pH 8.0) suggests that these interactions may be particularly relevant in specific microenvironments within the gastrointestinal tract.

How can CRISPR-Cas systems be adapted for studying protein functions in B. thetaiotaomicron?

CRISPR-Cas systems offer powerful tools for genetic manipulation in B. thetaiotaomicron, enabling researchers to investigate protein functions through targeted gene modifications. Recent advancements have specifically optimized these systems for use in Bacteroides species.

When selecting CRISPR systems for B. thetaiotaomicron, the Prevotella bryantii B14 Cas12a (Pb2Cas12a) has been identified as particularly suitable due to its protospacer-adjacent motif (PAM) sequence (5′-TTV-3′), which is well-represented throughout the B. thetaiotaomicron genome . This enables comprehensive genome-wide screening approaches.

For effective guide RNA design and delivery, researchers have determined that:

• Using the Francisella Cas12a repeats, rather than Prevotella repeats, results in efficient guide RNA processing and maturation in B. thetaiotaomicron

• Northern blotting confirms accumulation of mature gRNAs when processed from Francisella arrays, while Prevotella arrays show inefficient processing

An inducible Cas12a expression system has been developed specifically for B. thetaiotaomicron, allowing temporal control of gene targeting . This system can be combined with reporter strains (such as luciferase) to infer guide design rules and optimize targeting efficiency.

For studying specific protein functions like those of EF-Tu (tuf), researchers can employ CRISPRi (CRISPR interference) to achieve knockdown of gene expression without complete deletion. This approach is particularly valuable for studying essential genes like tuf or for investigating dosage-dependent effects on cellular functions.

Computational pipelines have been developed for automated guide RNA design specifically optimized for B. thetaiotaomicron, facilitating efficient library construction for genome-wide screens .

What methodological approaches are effective for investigating moonlighting functions of proteins in B. thetaiotaomicron?

Investigating the moonlighting functions of proteins like EF-Tu in B. thetaiotaomicron requires multifaceted methodological approaches that encompass both molecular and functional analyses:

Co-culture Systems with Membrane Filters: These systems can be employed to study protein sharing between bacterial species without direct cell-to-cell contact. For example, B. longum and B. thetaiotaomicron co-cultured using a membrane-filter system demonstrated increased adhesion of B. thetaiotaomicron to mucins compared to monoculture cells .

Comprehensive Proteomic Analysis: This approach can identify proteins that translocate from one bacterial species to another or identify unexpected proteins on bacterial cell surfaces. Mass spectrometry-based proteomics has successfully detected B. longum-derived cytoplasmic proteins, including EF-Tu, on the surface of B. thetaiotaomicron after co-culture .

Recombinant Protein Studies: Expressing purified recombinant proteins with appropriate tags (such as His6-tags) and incubating them with bacterial cells allows researchers to test moonlighting functions directly. This approach confirmed that recombinant EF-Tu can bind to B. thetaiotaomicron surfaces and enhance adhesion to mucins .

Adhesion Assays with Multiple Substrates: Testing adhesion to various biologically relevant substrates (porcine gastric mucin, human epithelial Caco-2 cells) under different conditions helps characterize the functional consequences of protein moonlighting .

pH-Controlled Experiments: Since pH significantly affects protein-cell surface interactions, conducting experiments at different pH values (e.g., pH 5.2 and 8.0) can reveal condition-dependent moonlighting functions .

Fluorescence Microscopy and Western Blotting: These complementary techniques provide visual and biochemical evidence for protein localization on bacterial surfaces .

These approaches collectively provide a comprehensive framework for investigating the complex, multifunctional nature of proteins like EF-Tu in B. thetaiotaomicron.

How do B. thetaiotaomicron and other bacteria exchange moonlighting proteins like EF-Tu in co-culture conditions?

The exchange of moonlighting proteins between B. thetaiotaomicron and other bacteria represents a fascinating example of interspecies molecular sharing that contributes to symbiotic relationships. This process has been particularly well-characterized between B. thetaiotaomicron and Bifidobacterium longum.

In co-culture experiments using membrane-filter systems that prevent direct cell-cell contact, B. longum-derived cytoplasmic proteins, including EF-Tu, have been detected on the surface of B. thetaiotaomicron . This indicates that protein exchange occurs through secretion and subsequent binding rather than requiring direct cellular contact.

The exchange mechanism appears to be highly selective, as proteomic analysis identified only 13 specific B. longum-derived cytoplasmic proteins on the surface of co-cultured B. thetaiotaomicron . This selectivity suggests a structured system of interspecies molecular sharing rather than random protein transfer.

The binding of these shared proteins to bacterial surfaces demonstrates species dependency, meaning that not all bacterial species can exchange proteins equally well . This specificity may reflect evolutionary adaptation to particular microbial community relationships.

Environmental factors significantly influence this exchange process. For instance, pH plays a crucial role, with protein binding occurring efficiently at pH 5.2 but poorly at pH 8.0 . This pH dependency suggests that protein exchange may be compartmentalized to specific microenvironments within the gut ecosystem.

Functionally, this protein exchange contributes to altered bacterial adhesion properties. When B. thetaiotaomicron acquires B. longum-derived EF-Tu, its adhesion to mucins increases significantly . This functional consequence suggests that protein sharing may enhance collective community functions or provide adaptive advantages in certain ecological contexts.

What experimental designs best demonstrate the impact of recombinant proteins on B. thetaiotaomicron adhesion?

Optimal experimental designs for demonstrating the impact of recombinant proteins on B. thetaiotaomicron adhesion incorporate multiple complementary approaches that control for variables while maximizing physiological relevance:

Comparative Protein Treatment Assays: Exposing B. thetaiotaomicron to different recombinant proteins (e.g., EF-Tu vs. GroEL) alongside appropriate controls (BSA-treated and untreated cells) allows for comparative analysis of protein-specific effects. This approach has revealed different degrees of adhesion enhancement by various moonlighting proteins .

Multiple Substrate Testing: Evaluating adhesion to diverse substrates provides comprehensive functional assessment. Parallel testing with porcine gastric mucin (PGM) and human epithelial Caco-2 cells has demonstrated that recombinant proteins can enhance B. thetaiotaomicron adhesion to both mucins and epithelial surfaces .

pH Variation Studies: Conducting parallel experiments at different pH values (e.g., pH 5.2 vs. pH 8.0) reveals condition-dependent effects. This approach has shown that EF-Tu enhances B. thetaiotaomicron adhesion at pH 5.2 but not at pH 8.0, highlighting the importance of physiologically relevant conditions .

Dose-Response Relationship: Testing a range of recombinant protein concentrations can establish dose-dependent effects and determine optimal concentrations for adhesion enhancement.

Time-Course Analysis: Examining adhesion at different time points after protein treatment can reveal the temporal dynamics of adhesion enhancement and determine the stability of the effect.

Correlative Microscopy and Adhesion Measurements: Combining fluorescence microscopy to visualize protein localization with quantitative adhesion assays enables researchers to correlate protein binding with functional outcomes .

Competitive Binding Assays: Including multiple bacterial strains in adhesion assays can demonstrate competitive effects and species specificity in adhesion enhancement.

These experimental approaches collectively provide robust evidence for the impact of recombinant proteins on B. thetaiotaomicron adhesion properties while controlling for confounding variables.

How can researchers distinguish between different functions of proteins like EF-Tu in B. thetaiotaomicron?

Distinguishing between canonical and moonlighting functions of proteins like EF-Tu in B. thetaiotaomicron presents a significant methodological challenge that requires sophisticated experimental approaches:

Subcellular Localization Analysis: Different functions of a protein are often associated with distinct subcellular localizations. Using fractionation techniques followed by Western blotting or proteomic analysis can reveal whether EF-Tu is present in expected locations (cytoplasm for translation) versus unexpected locations (cell surface for adhesion) .

Targeted Mutagenesis Studies: Creating mutations in specific domains of EF-Tu can help distinguish which regions are essential for different functions. If a mutation disrupts adhesion but not translation (or vice versa), this suggests independent functional domains.

Inducible Expression Systems: Using CRISPR-based or other inducible expression systems that allow controlled modulation of protein levels can help determine dose-dependent effects on different functions .

Complementation Assays: Complementing EF-Tu deficiency with either wild-type or function-specific mutant variants can reveal which functions are essential under different conditions.

Competitive Inhibition Experiments: Using antibodies or peptides that specifically block certain protein domains can selectively inhibit moonlighting functions while leaving canonical functions intact.

In vitro Functional Assays: Purified recombinant EF-Tu can be tested separately in translation assays versus adhesion assays to characterize each function independently.

Correlation with Environmental Conditions: As demonstrated by pH-dependent binding studies, certain functions may predominate under specific environmental conditions . Systematic testing across various conditions can reveal function-specific regulation.

Temporal Analysis During Growth Phases: Different functions may predominate during different growth phases or physiological states of the bacterium.

These approaches collectively provide a framework for dissecting the multifunctional nature of proteins like EF-Tu in B. thetaiotaomicron, allowing researchers to characterize each function independently while understanding their integration in bacterial physiology.

What are the implications of protein moonlighting for B. thetaiotaomicron's role in microbial community interactions?

The moonlighting capabilities of proteins like EF-Tu have profound implications for B. thetaiotaomicron's ecological role within complex microbial communities:

Facilitation of Symbiotic Relationships: The discovery that B. thetaiotaomicron can utilize moonlighting proteins from other bacteria (like B. longum) reveals a previously unrecognized mechanism for symbiotic interactions. This protein sharing enhances adhesion properties and potentially other functions, suggesting that bacteria can functionally complement each other through molecular exchange .

Microenvironment-Specific Adaptations: The pH-dependent nature of moonlighting protein interactions suggests that B. thetaiotaomicron may leverage these functions specifically in certain gut microenvironments. This adaptive flexibility could enhance its ecological fitness across the heterogeneous gastrointestinal tract .

Contribution to Biofilm Formation: Enhanced adhesion to mucins and epithelial cells through moonlighting proteins likely influences biofilm formation and spatial organization of microbial communities. B. thetaiotaomicron's ability to utilize both endogenous and exogenous moonlighting proteins may determine its positioning within multispecies biofilms .

Immune System Interactions: Surface-exposed moonlighting proteins like EF-Tu may mediate interactions with the host immune system, potentially influencing recognition patterns and immune responses to B. thetaiotaomicron.

Colonization Dynamics: The adhesion-enhancing properties of moonlighting proteins likely affect B. thetaiotaomicron's ability to initially colonize and persist within the gut ecosystem. This has implications for community succession and stability .

Metabolic Networking: Beyond adhesion, protein moonlighting may facilitate metabolic interactions between B. thetaiotaomicron and other community members, potentially contributing to the efficient utilization of complex substrates like dietary polysaccharides.

Evolutionary Considerations: The species-specific nature of moonlighting protein sharing suggests evolutionary adaptation to particular microbial community structures. This raises questions about the co-evolution of B. thetaiotaomicron with specific partner species.

These implications collectively suggest that protein moonlighting represents an important and previously underappreciated mechanism through which B. thetaiotaomicron participates in the complex ecological dynamics of the gut microbiome.