Recombinant Bacteroides thetaiotaomicron 10 kDa chaperonin (groS)

What is the structure and function of Bacteroides thetaiotaomicron 10 kDa chaperonin (groS)?

Bacteroides thetaiotaomicron 10 kDa chaperonin (groS), also known as GroES or Protein Cpn10, is a small molecular chaperone that functions as a co-chaperonin with the larger GroEL (60 kDa) chaperonin. The GroS protein contains approximately 98-100 amino acids and forms a heptameric ring structure that binds to GroEL during protein folding processes .

Functionally, GroS serves as a "lid" for the GroEL cavity during protein folding, creating an enclosed environment where substrate proteins can fold without interference from the cellular environment. The GroEL-GroES complex utilizes ATP hydrolysis to drive conformational changes necessary for protein folding assistance . In B. thetaiotaomicron, the groS gene is designated as BT_1830, and the protein plays a critical role in maintaining proteostasis, particularly under stress conditions .

What are the most effective methods for expressing and purifying recombinant B. thetaiotaomicron GroS?

Recombinant B. thetaiotaomicron GroS is typically produced using heterologous expression systems, most commonly in E. coli. The recommended methodological approach involves:

• Vector selection: The gene sequence for BT_1830 (groS) should be cloned into an expression vector containing an inducible promoter (T7 or similar) and an affinity tag (His-tag is common for chaperonins).

• Expression conditions: Optimal expression occurs in E. coli BL21(DE3) or similar strains at 30°C rather than 37°C to reduce inclusion body formation. Induction with 0.1-0.5 mM IPTG for 4-6 hours typically yields good protein expression.

• Purification protocol:

  • Lysis in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol

  • Affinity chromatography using Ni-NTA resin for His-tagged protein

  • Size exclusion chromatography to obtain the heptameric form

  • Concentration to >90% purity using ultrafiltration

• Quality control: Verify functional activity through ATPase assays in conjunction with GroEL .

Commercial preparations are typically supplied at >90% purity in a liquid form containing glycerol for stability .

What assays can be used to verify the functional activity of recombinant B. thetaiotaomicron GroS?

To verify that recombinant B. thetaiotaomicron GroS is functionally active, researchers can employ several complementary approaches:

• In vitro protein folding assays:

  • Citrate synthase refolding assay: Monitor the recovery of enzymatic activity of denatured citrate synthase in the presence of GroEL and recombinant GroS

  • Malate dehydrogenase (MDH) refolding: Measure the increase in MDH activity after thermal denaturation and refolding

  • Rhodanese refolding: A classic substrate for GroEL-GroES mediated folding

• ATP hydrolysis assays:

  • GroES modulates the ATPase activity of GroEL; measure ATP hydrolysis rates with and without GroS

  • Typical conditions: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 5 mM KCl, 2 mM ATP at 25°C

• Physical interaction assays:

  • Surface plasmon resonance (SPR) to measure binding kinetics between GroEL and GroS

  • Native gel electrophoresis to visualize GroEL-GroES complex formation

• Thermal stability analysis:

  • Differential scanning fluorimetry (DSF) to assess GroS thermal stability

  • Circular dichroism (CD) spectroscopy to monitor secondary structure integrity

For quantitative assessment, compare the activity of recombinant B. thetaiotaomicron GroS with the well-characterized E. coli GroES system as a reference standard .

How does B. thetaiotaomicron GroS differ from other bacterial GroS proteins in structure and function?

Bacteroides thetaiotaomicron GroS shares the conserved co-chaperonin fold with other bacterial GroS proteins but exhibits several important differences:

• Sequence comparison:

  • B. thetaiotaomicron GroS shares approximately 50-60% sequence identity with E. coli GroES

  • The mobile loop region (responsible for GroEL interaction) shows distinct amino acid composition

• Functional differences:

  • B. thetaiotaomicron GroS appears optimized for functioning under the anaerobic conditions of the gut

  • Unlike E. coli GroES, B. thetaiotaomicron GroS may be involved in stress responses specific to the gut environment, including oxidative stress tolerance

• Expression patterns:

  • In B. thetaiotaomicron, groS expression is modulated by environmental factors including bile and dietary components

  • Expression levels differ from those seen in model organisms like E. coli

• Co-evolution with substrate proteins:

  • B. thetaiotaomicron GroS has likely co-evolved with the specific proteome of this gut bacterium

  • May show preference for B. thetaiotaomicron-specific substrate proteins

Table 1: Comparison of GroS proteins from different bacterial species

These differences suggest that while the fundamental mechanism of GroEL-GroES assisted protein folding is conserved, B. thetaiotaomicron GroS may have unique adaptations for functioning in the human gut microbiome .

How does the GroS-GroEL system contribute to oxidative stress tolerance in B. thetaiotaomicron?

The GroS-GroEL chaperonin system plays a critical role in B. thetaiotaomicron's response to oxidative stress, which is particularly important for this anaerobic gut bacterium when exposed to oxygen:

• Protection of oxidation-sensitive proteins:

  • The GroEL-GroS complex helps refold proteins damaged by reactive oxygen species (ROS)

  • Prevents aggregation of partially oxidized proteins

• Interaction with oxidative stress pathways:

  • Research indicates that B. thetaiotaomicron grown on rhamnose shows enhanced oxidative stress tolerance

  • This effect is linked to metabolic changes that reduce ROS production

• Experimental evidence:

  • When grown on rhamnose, B. thetaiotaomicron exhibits greater resilience to H₂O₂ exposure compared to growth on glucose

  • The zone of inhibition on rhamnose-containing media (DMR) was approximately 38 mm compared to 45.3 mm on glucose-containing media (DMG) when exposed to H₂O₂

• Mechanistic insights:

  • The GroEL-GroS system may be upregulated during oxidative stress

  • The chaperonin system helps maintain activity of key enzymes involved in ROS detoxification

  • GroS may protect pyruvate:ferredoxin oxidoreductase (PFOR), which is involved in ROS production when dysregulated

This oxidative stress response mechanism may be particularly important for B. thetaiotaomicron's survival during transient exposure to oxygen in the dynamic gut environment .

What are the most effective methods for genetic manipulation of the groS gene in B. thetaiotaomicron?

Genetic manipulation of the groS gene in B. thetaiotaomicron requires specialized approaches due to the challenges of working with Bacteroides species. Based on recent methodological advances, the following approaches are recommended:

• Counterselection-based mutagenesis:

  • The dual-effector counterselection system has proven effective for genetic manipulation of wild human gut Bacteroides

  • This approach uses tightly regulated anhydrotetracycline (aTC)-inducible promoters controlling expression of antibacterial effectors

  • Protocol steps:
    a) Clone homology arms flanking the groS gene into a vector containing the dual counterselection cassette
    b) Introduce the construct via conjugation with E. coli S17λpir
    c) Select for first recombination event using antibiotic resistance
    d) Induce counterselection to identify second recombination events
    e) Confirm mutants by PCR and sequencing

• Complementation strategies:

  • For complementation, the pNBU2-bla-erm or pNBU2-bla-tet vectors are recommended

  • These vectors insert in the 5' untranslated region of the tRNA-Ser

  • Clone the groS gene under a constitutive promoter for stable expression

• Site-directed mutagenesis:

  • For subtle modifications to the groS gene (point mutations), use allelic exchange vectors

  • The pExchange-tdk vector has been successfully used in B. thetaiotaomicron

• Inducible expression systems:

  • For controlled expression studies, the anhydrotetracycline-inducible system works effectively in B. thetaiotaomicron

  • This allows for dose-dependent expression of groS variants

When working with essential genes like groS, consider creating conditional knockdowns rather than complete deletions, as the complete absence of GroS may be lethal .

How can B. thetaiotaomicron GroS-GroEL be co-expressed to enhance folding of difficult-to-express proteins?

Co-expression of B. thetaiotaomicron GroS with GroEL can significantly enhance the folding of difficult-to-express recombinant proteins. The methodological approach involves:

• Vector design strategies:

  • Dual-plasmid system: Express GroEL and GroS from separate compatible plasmids (e.g., pET and pACYC)

  • Polycistronic expression: Design a single plasmid with both genes under control of the same promoter but with independent ribosome binding sites

  • Tandem promoter system: Use separate promoters for GroEL and GroS on the same plasmid

• Optimal expression conditions:

  • Lower temperatures (16-25°C) slow protein synthesis and favor proper folding

  • Consider using auto-induction media to provide gradual induction

  • Optimize the ratio of GroEL to GroS (typically 2:1 molar ratio)

• Example protocol for co-expression:

  • Transform E. coli BL21(DE3) with the GroEL-GroS expression system and the target protein plasmid

  • Grow cultures at 37°C to OD₆₀₀ of 0.6

  • Induce with 0.1 mM IPTG and shift to 20°C for 16-18 hours

  • Harvest cells and purify the target protein using standard methods

• Documented success cases:

  • Research has shown that GroEL-GroS can assist in the folding of multiple substrate proteins simultaneously when overexpressed

  • Successful examples include co-expressed maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco)

  • Relative yields of properly folded functional forms were comparable to when these proteins were expressed individually

This approach leverages the natural ability of GroEL-GroS to assist in the folding of multiple endogenous proteins simultaneously in bacterial cells. The method has proven particularly effective for large, aggregation-prone proteins that tend to form inclusion bodies when expressed alone .

How does the B. thetaiotaomicron GroEL-GroS system differ from other bacterial species in substrate specificity and folding mechanisms?

The B. thetaiotaomicron GroEL-GroS system exhibits several unique characteristics that distinguish it from other bacterial species, particularly regarding substrate specificity and folding mechanisms:

• Substrate specificity differences:

  • B. thetaiotaomicron, as a gut commensal, has evolved to fold proteins involved in polysaccharide utilization and host interaction

  • The substrate range likely includes specialized proteins not found in model organisms like E. coli

  • May have particular affinity for proteins involved in anaerobic metabolism

• Environmental adaptations:

  • Functions optimally in the anaerobic, moderately acidic environment of the gut

  • May have specialized mechanisms to handle proteins affected by bile salts and diet-derived compounds

  • Appears to have adaptations for functioning in oxidative stress conditions that B. thetaiotaomicron occasionally encounters in the dynamic gut environment

• Co-evolution with metabolic systems:

  • Evidence suggests B. thetaiotaomicron's chaperonin system has co-evolved with its expanded carbohydrate utilization machinery

  • May preferentially assist folding of polysaccharide utilization loci (PUL) proteins that are critical for B. thetaiotaomicron's ecological niche

• Regulatory differences:

  • B. thetaiotaomicron appears to regulate groS and groEL expression through mechanisms distinct from those in E. coli

  • RNA-binding proteins like RbpA, RbpB, and RbpC may play roles in post-transcriptional regulation of chaperonin expression

  • Transcriptional regulators such as BT4338 may influence chaperonin expression in response to environmental cues

Unlike E. coli, where approximately 5% of proteins interact with GroEL-GroES for folding , the proportion of the B. thetaiotaomicron proteome dependent on chaperonin assistance remains undetermined but likely differs due to its specialized gut adaptation and expanded genome dedicated to polysaccharide utilization .

How does GroS contribute to B. thetaiotaomicron's response to bile and biofilm formation?

The GroS chaperonin plays an important role in B. thetaiotaomicron's response to bile and subsequent biofilm formation, which are critical for gut colonization:

• Bile stress response:

  • Bile represents a significant environmental stressor for gut bacteria

  • Research demonstrates that bile triggers biofilm formation in many B. thetaiotaomicron isolates

  • The GroS-GroEL system likely helps protect proteins from denaturation caused by bile acids

• Biofilm formation mechanisms:

  • B. thetaiotaomicron reference strain VPI-5482 is typically a poor biofilm former in vitro, but bile exposure changes this behavior

  • The chaperonin system appears to support the folding of proteins involved in the extracellular DNA (eDNA) processing pathway

  • Specifically, proper folding of the DNase BT3563 is crucial for degrading eDNA in biofilms formed in the presence of bile

• Regulatory connections:

  • Stress response systems, including chaperonins, are coordinated with biofilm formation pathways

  • GroS may be upregulated during bile exposure to maintain proteostasis

  • The chaperonin may assist in folding key regulatory proteins that control the transition to biofilm lifestyle

• Physiological relevance:

  • This bile-dependent biofilm formation represents a key adaptation to the gut environment

  • The GroS-mediated protein folding under bile stress likely contributes to B. thetaiotaomicron's ability to colonize the mucus layer of intestinal epithelial cells

This connection between bile response, chaperonin function, and biofilm formation highlights the sophisticated adaptation mechanisms of B. thetaiotaomicron to its intestinal niche .

How does the GroS-GroEL system integrate with B. thetaiotaomicron's unique metabolic networks?

The GroS-GroEL chaperonin system in B. thetaiotaomicron is closely integrated with the bacterium's specialized metabolic networks, particularly those involved in carbohydrate utilization and stress responses:

• Polysaccharide utilization pathways:

  • B. thetaiotaomicron devotes approximately 18% of its genes to carbohydrate acquisition and utilization

  • The GroS-GroEL system likely ensures proper folding of the numerous enzymes involved in these pathways

  • Evidence suggests coordination between chaperonin expression and activation of polysaccharide utilization loci (PULs)

• Metabolic adaptation to nutrient availability:

  • When B. thetaiotaomicron utilizes different carbon sources, specific metabolic pathways are activated

  • For example, rhamnose metabolism triggers a distinct metabolic profile compared to glucose metabolism

  • The chaperonin system likely supports proper folding of enzymes needed for these different metabolic modes

• Iron metabolism connections:

  • B. thetaiotaomicron is a heme auxotroph that preferentially consumes and hyperaccumulates iron in the form of heme

  • The chaperonin system may assist in folding components of the hmu operon involved in heme metabolism

  • This connection is important as B. thetaiotaomicron can accumulate an estimated 3.6 to 8.4 mg iron in a model GI tract microbiome

• Regulatory network integration:

  • The master regulator BT4338 controls both carbohydrate utilization and gut colonization

  • This regulator influences expression of numerous genes, potentially including chaperonins

  • RNA-binding proteins (RbpA, RbpB, RbpC) may post-transcriptionally regulate both metabolic enzymes and chaperonins

The integration of the GroS-GroEL system with these metabolic networks allows B. thetaiotaomicron to maintain proteostasis while adapting to the dynamic nutritional landscape of the human gut .

What are advanced experimental applications of recombinant B. thetaiotaomicron GroS in structural biology and synthetic biology?

Recombinant B. thetaiotaomicron GroS has emerging applications in advanced experimental fields:

• Cryo-electron microscopy (cryo-EM) studies:

  • The B. thetaiotaomicron GroEL-GroS complex represents an excellent target for time-resolved cryo-EM (tr-EM)

  • Similar to studies with E. coli GroEL-GroES, the B. thetaiotaomicron complex could be analyzed at different time points to visualize conformational changes during the ATP-driven folding cycle

  • The methodology involves plunge freezing the complex at defined intervals after ATP addition

• Synthetic biology applications:

  • Engineered B. thetaiotaomicron GroS variants can potentially enhance production of difficult-to-express therapeutic proteins

  • Development of chimeric GroS proteins combining features from different bacterial species could create optimized folding environments

  • GroS-based protein fusion tags that improve solubility of partner proteins

• Microbiome engineering:

  • Modified B. thetaiotaomicron with engineered chaperonin systems could serve as delivery vehicles for therapeutic proteins in the gut

  • Potential for creating stress-resistant probiotic strains with enhanced GroS-GroEL expression

  • Development of biosensors using GroS-dependent protein folding as a readout for gut conditions

• Recombinant protein production platforms:

  • Creation of specialized expression systems co-expressing B. thetaiotaomicron GroS-GroEL for production of gut-microbiome derived enzymes

  • Development of cell-free protein synthesis systems incorporating the B. thetaiotaomicron chaperonin machinery

  • Engineered host strains with B. thetaiotaomicron-derived chaperonins optimized for expression of anaerobic bacterial proteins

These advanced applications build on fundamental knowledge of the B. thetaiotaomicron chaperonin system while extending its utility to cutting-edge research areas .

How can the GroS-GroEL system be integrated into genome-scale metabolic models of B. thetaiotaomicron?

Integrating the GroS-GroEL chaperonin system into genome-scale metabolic models (GEMs) of B. thetaiotaomicron represents an advanced challenge in systems biology. The following methodological approach can be used:

• Extending traditional GEMs to include protein folding:

  • Current GEMs of Bacteroides species (like the model for B. fragilis described in search result ) focus primarily on metabolic reactions

  • To include chaperonin functions, the model must be extended to incorporate:

    • ATP consumption by the GroEL-GroES system

    • Protein synthesis costs

    • Folding capacity constraints

• Constraint-based modeling approach:

  • Define the chaperonin capacity as a new constraint in flux balance analysis (FBA)

  • Add reactions representing:

    • GroEL-GroES complex formation

    • ATP-dependent substrate protein folding

    • GroES binding and release cycles

• Integration with expression data:

  • Use RNA-seq data from different growth conditions to constrain the activity of the chaperonin system

  • For example, data indicates that RbpA, RbpB, and RbpC (RNA-binding proteins) influence expression of many genes in B. thetaiotaomicron

  • Incorporate regulatory effects of transcription factors like BT4338 on chaperonin expression

• Modeling chaperonin-dependent growth phenotypes:

  • Define growth conditions where chaperonin function becomes limiting

  • Model the impact of environmental stressors (bile, oxidative stress) on chaperonin demand

  • Predict how chaperonin capacity affects utilization of different carbon sources

Table 2: Parameters for integrating chaperonin function into B. thetaiotaomicron GEMs