Recombinant Bacteroides thetaiotaomicron Protein grpE (grpE)

What is Bacteroides thetaiotaomicron and why is it significant in microbiome research?

Bacteroides thetaiotaomicron is a Gram-negative, obligate anaerobic bacterium that serves as a prototype for understanding carbohydrate metabolism by colonic bacteria . It is a universal member of the human gut microbiota, particularly within the large intestine . This organism occupies a hub position in the distal colon, influencing both host physiology and incoming enteric pathogens .

B. thetaiotaomicron's proteome consists of 4,779 proteins, including an extensive system for obtaining and breaking down dietary polysaccharides that would otherwise be difficult for humans to digest . The bacterium encodes numerous enzymes such as glycoside hydrolases and polysaccharide lyases that enable it to break down complex dietary fibers . This metabolic capacity makes B. thetaiotaomicron a key player in the symbiotic relationship between gut microbes and human hosts, particularly in nutrient acquisition and processing.

What is the grpE protein and what are its known functions in bacterial systems?

While the search results don't specifically describe a grpE protein in B. thetaiotaomicron, we can draw insights from well-characterized grpE proteins in other bacteria. In Escherichia coli, grpE was first identified when a mutation (grpE280) prevented bacteriophage lambda DNA replication in vivo . The E. coli grpE protein is classified as a heat shock protein with a molecular weight of approximately 23,000 Da under both denaturing and native conditions .

The grpE protein functions as a nucleotide exchange factor for the molecular chaperone DnaK (Hsp70). A key characteristic of this interaction is that grpE forms a stable complex with DnaK that can withstand high salt concentrations (up to 2M KCl), but is disrupted by ATP . This ATP-mediated regulation of the DnaK-grpE complex is a critical aspect of its function in protein folding and stress response mechanisms.

How should researchers approach the identification and initial characterization of B. thetaiotaomicron grpE?

For initial characterization of putative grpE proteins in B. thetaiotaomicron, researchers should employ a multi-faceted approach:

• Genomic analysis: Begin with in silico identification using sequence homology to known grpE proteins from E. coli and other related species. Utilize resources like the recently developed transcriptome atlas for B. thetaiotaomicron (available at: http://micromix.helmholtz-hiri.de/bacteroides/)[3].

• Expression profiling: Examine expression patterns across different growth conditions, particularly under heat shock and other stress conditions, as grpE is typically a stress-response protein .

• Functional conservation assessment: Test for functional complementation using E. coli grpE mutants to determine if the B. thetaiotaomicron homolog can rescue the phenotype.

• Protein interaction mapping: Investigate whether the putative B. thetaiotaomicron grpE interacts with DnaK homologs, as this interaction is fundamental to grpE function .

• Structural analysis: Compare predicted structural features with known grpE proteins to identify conserved domains associated with nucleotide exchange function.

What expression systems are most effective for producing recombinant B. thetaiotaomicron grpE protein?

The choice of expression system for recombinant B. thetaiotaomicron grpE should consider the anaerobic nature of this bacterium and potential challenges in protein folding:

Expression System Comparison Table:

When designing an expression strategy:

• Consider using inducible promoters to control expression levels, particularly if high levels of grpE are potentially toxic.

• For E. coli expression, co-expression with DnaK may enhance solubility and proper folding of recombinant grpE, given their known interaction in E. coli .

• Optimize growth conditions based on the experimental research design framework with clear protocols and procedures using appropriate variables .

What purification strategies maximize yield and activity of recombinant B. thetaiotaomicron grpE?

Drawing from the E. coli grpE purification strategies, researchers should consider:

• Affinity chromatography: Design constructs with appropriate tags (His, GST, etc.) for initial capture. Alternatively, leverage the natural affinity between grpE and DnaK by using DnaK-based affinity columns, as demonstrated for E. coli grpE purification .

• Elution strategies: If using a DnaK affinity approach, ATP can be used to specifically elute grpE, as ATP disrupts the DnaK-grpE interaction .

• Size exclusion chromatography: Use as a polishing step to separate monomeric grpE (approximately 23 kDa) from aggregates and to confirm its oligomeric state under native conditions .

• Activity validation: Confirm functional activity through DnaK binding assays and nucleotide exchange activity measurements.

How can researchers verify the structural integrity and functionality of purified recombinant B. thetaiotaomicron grpE?

A comprehensive validation approach should include:

• SDS-PAGE and Western blotting: Confirm size and purity, with expected molecular weight around 23 kDa based on E. coli grpE .

• Native gel electrophoresis and size exclusion chromatography: Assess oligomeric state under native conditions.

• Circular dichroism (CD) spectroscopy: Evaluate secondary structure content and thermal stability.

• DnaK binding assays: Verify the ability to form complexes with DnaK homologs using:

  • Co-immunoprecipitation

  • Surface plasmon resonance (SPR)

  • Microscale thermophoresis (MST)

• Nucleotide exchange activity assays: Measure the ability to catalyze nucleotide exchange on DnaK using fluorescently labeled nucleotides.

• ATP sensitivity tests: Confirm that the DnaK-grpE interaction is disrupted by ATP, as observed with E. coli grpE .

How can researchers investigate the role of B. thetaiotaomicron grpE in stress response and adaptation mechanisms?

B. thetaiotaomicron exhibits remarkable adaptability to environmental changes, including responses to oxidative stress during infection . To investigate grpE's role in these processes:

What experimental designs are most appropriate for studying the interaction network of B. thetaiotaomicron grpE?

To map the interaction network of B. thetaiotaomicron grpE:

• Affinity purification-mass spectrometry (AP-MS):

  • Express tagged grpE in B. thetaiotaomicron

  • Perform pull-downs under various conditions (normal growth, heat shock, other stresses)

  • Identify interacting proteins by mass spectrometry

  • Compare interactomes under different conditions

• Yeast two-hybrid screening:

  • Use grpE as bait against a B. thetaiotaomicron genomic library

  • Validate positive interactions with complementary methods

• Bacterial two-hybrid system:

  • Potentially more suitable for anaerobic bacterial proteins

  • Adapt existing systems for B. thetaiotaomicron proteins

• MS2 affinity purification and RNA-seq:

  • Based on methodologies used for identifying sRNA targets in B. thetaiotaomicron

  • Apply to investigate whether grpE interacts with RNA or RNA-binding proteins

• Cross-linking mass spectrometry:

  • Capture transient interactions within the native cellular environment

  • Provide structural insights into interaction interfaces

How does B. thetaiotaomicron grpE potentially contribute to carbohydrate metabolism and adaptation to dietary changes?

B. thetaiotaomicron is renowned for its sophisticated carbohydrate metabolism systems . To investigate potential roles of grpE in these processes:

• Expression correlation analysis:

  • Analyze grpE expression patterns across growth on different carbon sources

  • Compare with expression patterns of known carbohydrate metabolism genes

• Proteomic stability studies:

  • Determine if grpE contributes to the stability and proper folding of key carbohydrate-active enzymes

  • Test whether grpE depletion affects the functional capacity of polysaccharide utilization loci (PULs)

• Diet shift adaptation experiments:

  • Monitor grpE expression during adaptation to different dietary regimens

  • Assess whether grpE is involved in the adaptive response to changes in available carbohydrates

• Interaction with membrane proteins:

  • Investigate potential interaction with the multidomain carbohydrate-binding proteins involved in starch metabolism

  • Determine if grpE plays a role in the proper folding or localization of these membrane proteins

What methodologies are recommended for investigating the role of B. thetaiotaomicron grpE in microbiome resilience and stability?

Recent research shows that B. thetaiotaomicron populations undergo genetic adaptations during infection that increase fitness . To study grpE's potential role in this process:

• Longitudinal population studies:

  • Track genetic changes in grpE sequences in B. thetaiotaomicron populations over time

  • Compare populations in healthy vs. perturbed gut environments

• Competition assays:

  • Create isogenic strains with different grpE variants

  • Measure relative fitness in various conditions using barcoded strain tracking

• Host-microbe interaction models:

  • Use gnotobiotic mouse models colonized with defined B. thetaiotaomicron strains

  • Assess the impact of grpE variants on colonization success and resilience

• Community context experiments:

  • Investigate whether the presence of other bacterial species affects selection pressure on grpE variants

  • Examine potential metabolic interactions, such as those involving vitamin B6

• Stress resistance profiling:

  • Compare survival rates of different grpE variants under various stressors

  • Correlate with adaptability in complex gut environments

How can structural biology approaches advance our understanding of B. thetaiotaomicron grpE function?

Structural characterization would significantly enhance our understanding of B. thetaiotaomicron grpE:

• X-ray crystallography:

  • Determine the three-dimensional structure of B. thetaiotaomicron grpE

  • Compare with known bacterial grpE structures to identify unique features

  • Co-crystallize with DnaK to visualize interaction interfaces

• Cryo-electron microscopy:

  • Visualize larger complexes involving grpE and its partners

  • Capture different functional states of these complexes

• NMR spectroscopy:

  • Characterize dynamic regions and conformational changes

  • Study the interaction with ATP and how it disrupts the DnaK-grpE complex

• Molecular dynamics simulations:

  • Model structural dynamics under different conditions

  • Predict effects of mutations on protein function

• Structure-guided mutagenesis:

  • Design targeted mutations based on structural insights

  • Test functional consequences in vitro and in vivo

What are the best approaches for investigating potential horizontal gene transfer of grpE among gut microbiota?

Given the genomic plasticity of Bacteroides species , investigating horizontal gene transfer (HGT) involving grpE is relevant:

• Comparative genomic analysis:

  • Compare grpE sequences across Bacteroides and other gut microbiota

  • Identify signatures of horizontal gene transfer through:

    • Phylogenetic incongruence

    • Unusual codon usage patterns

    • GC content analysis

• Mobile genetic element association:

  • Examine genomic context of grpE in different strains

  • Look for proximity to transposable elements, prophages, or other mobile genetic elements

• Experimental HGT models:

  • Design laboratory systems to track potential transfer events

  • Use fluorescently tagged grpE constructs to visualize transfer

• Metagenomic analysis:

  • Analyze grpE diversity in human microbiome datasets

  • Look for chimeric variants suggesting recombination events

How can researchers leverage the Theta-Base web browser and other resources to facilitate B. thetaiotaomicron grpE research?

Recently developed resources provide valuable tools for B. thetaiotaomicron research:

• Theta-Base utilization strategies:

  • Access transcriptome data across 15 in vivo-relevant growth conditions

  • Analyze expression patterns of putative grpE genes and potential interaction partners

  • Identify co-regulated genes that may function in the same pathways

• Integration with fitness data:

  • Combine expression data with published transposon mutant fitness data

  • Predict condition-specific importance of grpE

  • Compare with patterns observed for other heat shock proteins

• Regulon analysis:

  • Identify stress- and carbon source-specific transcriptional regulons that include grpE

  • Map potential regulatory elements controlling grpE expression

• Small RNA connections:

  • Explore potential regulation by sRNAs using expanded sRNA annotations

  • Investigate whether grpE is subject to post-transcriptional regulation

• Data visualization approaches:

  • Create integrated visualizations combining expression, fitness, and interaction data

  • Develop custom tracks for genome browsers focusing on chaperone systems

What strategies can resolve issues with low solubility of recombinant B. thetaiotaomicron grpE?

Solubility challenges are common when working with recombinant proteins from anaerobic bacteria:

• Expression condition optimization:

  • Reduce induction temperature to 16-20°C

  • Lower inducer concentration for slower, more controlled expression

  • Test different media formulations, particularly those mimicking anaerobic conditions

• Fusion tag screening:

  Tag	Size (kDa)	Potential Benefit for grpE
  MBP	42	High solubility enhancement
  SUMO	12	Promotes correct folding
  Thioredoxin	12	Assists disulfide bond formation
  GST	26	Solubility and affinity purification

• Chaperone co-expression:

  • Co-express with E. coli DnaK-DnaJ-GrpE system

  • Test B. thetaiotaomicron native chaperones if available

• Solubilization agents:

  • Screen mild detergents (0.05-0.1% Triton X-100, NP-40)

  • Test arginine (50-100 mM) as a stabilizing additive

  • Consider osmolytes like glycerol (5-10%) or sucrose (5%)

How can researchers address difficulties in detecting protein-protein interactions involving B. thetaiotaomicron grpE?

Detecting interactions with anaerobic bacterial proteins can be challenging:

• Buffer optimization for complex stability:

  • Based on E. coli grpE studies, ensure buffers maintain the integrity of complexes

  • Test different salt concentrations (the DnaK-grpE complex is stable even in 2M KCl)

• Crosslinking approaches:

  • Use formaldehyde or DSS for in vivo crosslinking

  • Apply SPINE (Strep-protein interaction experiment) for selective capture

• ATP-dependent dissociation considerations:

  • Control ATP levels carefully when studying DnaK-grpE interactions

  • Use non-hydrolyzable ATP analogs to trap specific interaction states

• Detection method selection:

  • For transient interactions, consider FRET-based assays

  • For stable complexes, co-immunoprecipitation or pull-down assays

  • For structural characterization, apply crosslinking mass spectrometry

• In vivo proximity labeling:

  • Adapt BioID or APEX2 systems for B. thetaiotaomicron

  • Use spatially restricted enzymatic tagging to identify neighboring proteins

What considerations are important when analyzing contradictory data regarding B. thetaiotaomicron grpE function?

When faced with contradictory results:

• Strain and genetic background considerations:

  • Verify the exact strains used in different studies

  • Determine if genetic differences beyond the targeted protein exist

  • Check for potential suppressor mutations

• Environmental condition variations:

  • Precisely document and control growth conditions

  • Consider that B. thetaiotaomicron adapts rapidly to environment changes

  • Test whether observed differences are condition-specific

• Technical approach differences:

  • Compare methodological details between contradictory studies

  • Evaluate sensitivity and specificity of different detection methods

  • Consider whether in vitro versus in vivo approaches explain differences

• Experimental design validation:

  • Revisit experimental research design fundamentals

  • Ensure proper controls and variables are in place

  • Verify that time factors in establishing cause-effect relationships are considered

• Reconciliation strategies:

  • Design experiments that directly address contradictions

  • Test whether differences reflect distinct functional states of grpE

  • Consider whether contradictory results reveal novel regulatory mechanisms