Recombinant Bacteroides thetaiotaomicron Adenylate kinase (adk)

What is the role of adenylate kinase in Bacteroides thetaiotaomicron metabolism?

Adenylate kinase in B. thetaiotaomicron catalyzes the reversible phosphoryl transfer reaction to interconvert adenine nucleotides (ATP + AMP ⇌ 2ADP), playing a critical role in maintaining cellular energy homeostasis . This function is particularly important for B. thetaiotaomicron as a gut symbiont that must efficiently manage energy resources in the competitive gut environment. The enzyme helps maintain high levels of ATP through a combination of adenylate kinase activity and mitochondrial oxidative phosphorylation .

How does B. thetaiotaomicron adenylate kinase compare structurally to well-characterized homologs?

While the specific structure of B. thetaiotaomicron adenylate kinase has not been fully resolved, structural insights can be drawn from well-characterized homologs like E. coli adenylate kinase. These enzymes typically contain:

• A nucleoside monophosphate binding domain (NMPbd)

• A LID domain (LIDbd) that covers the ATP binding site

• A glycine-rich region for nucleoside triphosphate binding

• A distinct site for binding nucleoside monophosphates

Upon substrate binding, adenylate kinases undergo conformational changes where both the NMPbd and LIDbd transition from open to closed configurations .

What expression patterns does adenylate kinase show in B. thetaiotaomicron under different conditions?

The expression of adenylate kinase in B. thetaiotaomicron appears to be influenced by nutritional availability and environmental conditions. While specific expression data for B. thetaiotaomicron adenylate kinase is limited in the provided search results, research on microbial adenylate kinases suggests that expression may be upregulated during energy stress or carbon limitation . This would be consistent with the enzyme's role in energy homeostasis, helping the bacterium adapt to changing nutrient conditions in the gut environment.

What are the optimal conditions for recombinant expression of B. thetaiotaomicron adenylate kinase?

For recombinant expression of B. thetaiotaomicron adenylate kinase, researchers should consider:

Expression System Selection:

• E. coli BL21(DE3) is commonly used for initial attempts

• B. thetaiotaomicron's own expression system may be preferable for proper folding and post-translational modifications

Expression Parameters:

Genetic Design Considerations:
Specific integration sites have been validated for B. thetaiotaomicron genome integration, including attBT1-1 and attBT2-1/2 sites . Using a constitutive promoter such as P BT1311 has proven effective for stable protein expression in B. thetaiotaomicron .

What purification strategy yields the highest activity for recombinant B. thetaiotaomicron adenylate kinase?

A multi-step purification approach is recommended to obtain high-activity recombinant adenylate kinase:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using a His6-tag

• Intermediate Purification: Ion exchange chromatography

• Polishing Step: Size exclusion chromatography

Critical Buffer Considerations:

Activity Preservation:
Addition of 5-10% glycerol to the final storage buffer and flash-freezing in liquid nitrogen with storage at -80°C helps maintain enzymatic activity for extended periods.

What are the most sensitive assays for measuring B. thetaiotaomicron adenylate kinase activity?

Several methodologies can be employed to assess adenylate kinase activity with varying degrees of sensitivity:

Coupled Enzyme Assays:
The most widely used approach couples ADP production to NADH oxidation through pyruvate kinase and lactate dehydrogenase. The decrease in NADH absorbance at 340 nm directly correlates with adenylate kinase activity.

Direct ADP Measurement:

• HPLC-based quantification of reaction products

• Mass spectrometry for precise nucleotide quantification

• Luminescence-based assays that detect ADP formation

Real-time Conformational Monitoring:
Time-resolved X-ray solution scattering (TR-XSS) can be used to visualize structural changes in adenylate kinase upon ATP binding in real-time, providing insights into enzyme function beyond simple activity measurements .

How can researchers investigate the role of adenylate kinase in B. thetaiotaomicron gut colonization?

Investigating adenylate kinase's role in gut colonization requires a multi-faceted approach:

Genetic Manipulation Strategies:

• Gene deletion mutants (Δadk)

• Point mutations in catalytic residues

• Complementation studies with wild-type and mutant variants

In Vivo Colonization Models:

• Gnotobiotic mouse models with defined diets

• Competitive colonization assays (wild-type vs. mutant)

• Epithelial cell adherence models

Experimental Design Considerations:
When conducting colonization experiments, it's critical to evaluate performance across multiple diet conditions, as colonization fitness effects may be diet-specific . The ribose utilization phenotype observed in B. thetaiotaomicron, for example, shows diet-specific competitive advantage .

What approaches can be used to assess adenylate kinase's contribution to B. thetaiotaomicron energy metabolism?

Researchers can employ several complementary approaches:

Metabolic Flux Analysis:
Using isotope-labeled substrates (¹³C-glucose or ¹³C-ribose) to track carbon flow through central metabolism and quantify the impact of adenylate kinase activity on energy production pathways.

Nucleotide Pool Measurement:
Quantifying intracellular ATP:ADP:AMP ratios using:

• HPLC analysis

• Luciferase-based ATP detection

• LC-MS/MS for comprehensive nucleotide profiling

Growth Phenotyping:
Comparing growth kinetics of wild-type and adenylate kinase mutants under various nutrient limitations, particularly focusing on carbon-limited conditions where significant transcriptional changes occur in response to nutrient stress .

How can researchers determine if B. thetaiotaomicron adenylate kinase is regulated by the BT4338 master regulator?

BT4338 is a major transcriptional regulator in B. thetaiotaomicron that controls hundreds of genes involved in carbohydrate utilization and is essential for gut colonization . To determine if adenylate kinase is under BT4338 regulation:

Transcriptional Analysis:

• RNA-seq comparing wild-type and BT4338 mutant strains

• qRT-PCR validation of adenylate kinase expression levels

• Promoter-reporter fusion assays to measure transcriptional activity

DNA-Binding Studies:

• Chromatin immunoprecipitation (ChIP) to identify BT4338 binding to the adenylate kinase promoter

• Electrophoretic mobility shift assays (EMSA) with purified BT4338 protein

• DNase I footprinting to map precise binding sites

In Vivo Validation:
Constructing mutations in potential BT4338 binding sites within the adenylate kinase promoter to confirm functional relevance.

The experimental design should include carbon limitation conditions, as BT4338 has been shown to activate gene expression during carbon limitation .

What structural analysis techniques are most applicable to B. thetaiotaomicron adenylate kinase conformational studies?

Multiple techniques can provide insights into the conformational dynamics of adenylate kinase:

X-ray Crystallography:
Provides high-resolution static structures of different conformational states, particularly effective when trapping the enzyme with inhibitors like Ap5A that stabilize the closed conformation .

Time-Resolved X-ray Solution Scattering (TR-XSS):
Enables visualization of structural changes in real-time following substrate binding, allowing researchers to track the conformational changes with millisecond temporal resolution .

Nuclear Magnetic Resonance (NMR):
Allows for the study of protein dynamics in solution, providing insights into domain movements and conformational exchange rates.

Molecular Dynamics Simulations:
Computational approaches that can model the transition between open and closed states, particularly useful when paired with experimental structural data.

How can adenylate kinase be integrated into synthetic biology applications in B. thetaiotaomicron?

B. thetaiotaomicron has emerging potential as a chassis for gut microbiome therapeutic delivery . Adenylate kinase could be integrated into synthetic biology applications through:

Energy-Sensing Genetic Circuits:

• Using adenylate kinase-dependent promoters as sensors for cellular energy status

• Designing circuits that respond to ATP:ADP ratios as metabolic indicators

Genetic Circuit Design:
The Cello circuit design automation software has been implemented for B. thetaiotaomicron , allowing for the creation of complex genetic circuits. Adenylate kinase could be incorporated as:

• A reporter gene to monitor cellular energy states

• Part of a metabolic toggle switch responding to changing energy demands

• A component in circuits distinguishing between different host environments

Integration Considerations:
When designing synthetic circuits in B. thetaiotaomicron, genome integration at specific sites (attBT1-1 or attBT2-1/2) has shown stable expression for at least 12 days under laboratory conditions .

What are the main challenges in expressing active recombinant B. thetaiotaomicron adenylate kinase?

Researchers face several technical challenges:

Codon Usage Optimization:
B. thetaiotaomicron has distinct codon preferences compared to common expression hosts like E. coli, potentially leading to translation inefficiencies and reduced protein yields.

Protein Solubility:
Adenylate kinases often form inclusion bodies when overexpressed. Optimization strategies include:

• Lower induction temperatures (16-18°C)

• Co-expression with chaperone proteins

• Fusion tags that enhance solubility (MBP, SUMO)

Protein Stability:
Adenylate kinases may exhibit reduced stability in vitro. Stabilization approaches include:

• Buffer optimization with osmolytes (glycerol, trehalose)

• Structure-guided mutagenesis to enhance thermostability

• Ligand addition to stabilize specific conformations

How can researchers differentiate between the direct and indirect metabolic effects of adenylate kinase in B. thetaiotaomicron?

Distinguishing direct from indirect effects requires sophisticated experimental approaches:

Catalytically Inactive Mutants:
Engineering point mutations in catalytic residues creates proteins that maintain structural integrity but lack enzymatic activity, allowing separation of structural from catalytic effects.

Complementation Studies:

• Complementing adk knockout with heterologous adenylate kinases

• Using inducible expression systems for controlled titration of enzyme levels

• Creating chimeric enzymes to map functional domains

Multi-Omics Integration:
Combining transcriptomics, proteomics, and metabolomics data to build comprehensive metabolic models that can predict direct versus indirect effects of adenylate kinase perturbation.

What experimental controls are essential when studying the ribose scavenging connection to adenylate kinase function?

B. thetaiotaomicron possesses ribose utilization systems (RUSs) that confer a competitive advantage during gut colonization . When investigating potential connections to adenylate kinase function:

Essential Controls:

• Genetic Controls:

  • Clean deletion mutants with confirmed lack of polar effects

  • Complemented strains to verify phenotype restoration

  • Multiple independent mutant clones to ensure consistency

• Metabolic Precursors:

  • Testing defined nucleoside substrates versus complex ribose sources

  • Including intermediate metabolites to pinpoint pathway bottlenecks

  • Isotope-labeled substrates to track metabolic fates

• Competition Experiments:

  • In vitro growth curves in defined media with various carbon sources

  • In vivo competition with wild-type in gnotobiotic models

  • Diet variation to identify condition-specific phenotypes

The connection between ribose metabolism and nucleoside phosphorylases should be carefully considered, as these pathways interact during colonization on plant fiber-rich diets .

How might adenylate kinase from B. thetaiotaomicron be engineered for improved catalytic properties?

Adenylate kinase engineering could enhance various properties:

Catalytic Efficiency Enhancement:

• Structure-guided mutagenesis targeting active site residues

• Directed evolution using high-throughput activity screening

• Computational design to optimize substrate binding

Substrate Specificity Modification:
Altering the enzyme to accept alternative nucleotides or nucleotide analogs could enable:

• Novel biosynthetic pathways

• Metabolic engineering applications

• Diagnostic tools for gut microbiome research

Thermostability Improvement:
Enhanced thermostability would benefit purification processes and potential applications, achievable through:

• Consensus design approaches

• Disulfide bridge engineering

• Rigidifying flexible regions identified through molecular dynamics simulations

What is the potential role of adenylate kinase in B. thetaiotaomicron's adaptation to environmental stresses in the gut?

Adenylate kinase likely plays a crucial role in stress adaptation through:

Energy Homeostasis During Nutrient Fluctuation:
The gut environment undergoes constant changes in nutrient availability. Adenylate kinase may help B. thetaiotaomicron maintain energy charge during transitions between feast and famine conditions.

Oxidative Stress Response:
ATP levels influence cellular responses to oxidative stress. Adenylate kinase's role in maintaining ATP pools may indirectly affect oxidative stress tolerance.

Integration with Specialized Translation Factors:
B. thetaiotaomicron coordinates cellular metabolism with protein synthesis via specialized translation factors to promote survival in the mammalian gut . Adenylate kinase may interface with this system by regulating energy availability for translation.

Research Approaches:

• Stress exposure experiments (oxidative, pH, bile acid) with adk mutants

• Transcriptional profiling under various stresses

• Integration with BT4338 regulatory network analysis