Recombinant Bacteroides thetaiotaomicron tRNA dimethylallyltransferase 1 (miaA1)

What is the function of tRNA dimethylallyltransferase 1 (miaA1) in Bacteroides thetaiotaomicron?

tRNA dimethylallyltransferase 1 (miaA1) catalyzes the transfer of a dimethylallyl group from dimethylallyl pyrophosphate (DMAPP) to the N6 position of adenosine at position 37 adjacent to the anticodon in specific tRNAs. This modification produces N6-(dimethylallyl)adenosine (i6A), which enhances codon-anticodon interactions and improves translational fidelity.

In B. thetaiotaomicron specifically, this enzyme contributes to proper protein synthesis, which is essential for metabolic versatility and adaptation to the changing nutritional landscape of the intestinal environment. This is particularly important given B. thetaiotaomicron's role as a prominent member of the human gut microbiota and its capacity to utilize diverse dietary polysaccharides .

Methodological approaches to study this function include:

• Gene knockout studies comparing wildtype and miaA1-deficient strains

• Ribosome profiling to assess translation efficiency changes

• tRNA modification profiling using liquid chromatography-mass spectrometry

• Comparative proteomics to identify proteins most affected by miaA1 deficiency

How should Recombinant Bacteroides thetaiotaomicron tRNA dimethylallyltransferase 1 (miaA1) be stored and handled for optimal activity?

Proper storage and handling of Recombinant Bacteroides thetaiotaomicron tRNA dimethylallyltransferase 1 (miaA1) is critical for maintaining enzymatic activity. Based on standard protocols for similar enzymes :

• Store long-term at -20°C or -80°C in appropriate storage buffer containing glycerol (20-50%)

• For working aliquots, store at 4°C for up to one week

• Avoid repeated freezing and thawing cycles as they significantly reduce enzyme activity

• Keep the enzyme on ice during experiments and return to appropriate storage promptly

• Use enzyme activity buffer typically containing:

  • 50 mM Tris-HCl (pH 7.5-8.0)

  • 10 mM MgCl₂

  • 5 mM DTT

  • 0.1 mM EDTA

For activity assessment, regular testing using suitable tRNA substrates and dimethylallyl pyrophosphate is recommended, with activity quantified by measuring modified tRNA products through radiometric assays or mass spectrometry-based approaches.

What experimental methods can be used to assess the role of miaA1 in B. thetaiotaomicron gut colonization?

The role of miaA1 in B. thetaiotaomicron gut colonization can be investigated through multiple complementary approaches:

Genetic manipulation strategies:

• Generate miaA1 knockout strains using CRISPR-Cas9 or homologous recombination

• Create point mutants with reduced enzyme activity but maintained protein structure

• Develop complemented strains to confirm phenotype specificity

• Engineer strains with inducible/repressible miaA1 expression for temporal control

In vivo colonization experiments:

• Single-strain colonization in germ-free mice comparing wildtype and miaA1-deficient strains

• Competitive colonization assays with barcoded strains to assess relative fitness

• Temporal sampling to monitor population dynamics during adaptation phases

• Analysis of strain persistence following perturbations (diet changes, inflammation)

Mechanistic investigations:

• Transcriptomics to identify genes differentially expressed in miaA1-deficient strains during colonization

• Metabolomics to assess changes in metabolic capabilities

• tRNA modification profiling during different colonization phases

• Ribosome profiling to evaluate translation efficiency of colonization-related genes

B. thetaiotaomicron undergoes significant metabolic adaptations during colonization, shifting from amino acid biosynthesis to polysaccharide utilization . The miaA1 enzyme likely plays a crucial role in ensuring translational fidelity during these transitions, particularly in the competitive environment of the gut where capsular polysaccharides and metabolic versatility contribute to fitness .

How does the activity of Bacteroides thetaiotaomicron tRNA dimethylallyltransferase 1 (miaA1) compare to miaA2 and orthologs in other Bacteroides species?

A comparative analysis of B. thetaiotaomicron miaA1 with miaA2 and orthologs in related species provides insights into functional specialization within the Bacteroides genus. Research approaches include:

Biochemical characterization:

• Recombinant expression of multiple enzyme variants

• Determination of substrate specificity profiles

• Measurement of kinetic parameters under standardized conditions

• Analysis of temperature and pH optima

Structural comparisons:

• X-ray crystallography or cryo-EM structural determination

• Homology modeling if experimental structures are unavailable

• Identification of conserved and variable regions

• Correlation of structural differences with functional parameters

The following table summarizes predicted differences based on comparative studies:

This comparative approach reveals evolutionary adaptations in tRNA modification systems across Bacteroides species, potentially correlating with their distinct ecological niches within the gut microbiome .

What role does miaA1 play in the dynamic genetic adaptation of B. thetaiotaomicron during colonization?

B. thetaiotaomicron undergoes remarkable genetic adaptation during gut colonization, with significant temporal changes in gene expression patterns . The miaA1 enzyme likely plays a pivotal role in this process through several mechanisms:

Translational regulation during metabolic shifts:
During initial colonization, B. thetaiotaomicron transitions from amino acid biosynthesis to polysaccharide utilization . This shift requires rapid adaptation of the translational machinery to efficiently produce new metabolic enzymes. The i6A modification catalyzed by miaA1 enhances translation of specific codons, potentially facilitating this adaptive response.

Methodological approach to investigate this role:

• Create conditional miaA1 mutants that can be regulated during colonization

• Perform temporal transcriptomics and ribosome profiling during colonization phases

• Analyze codon usage bias in genes upregulated during different adaptation stages

• Quantify tRNA modification levels at each stage using LC-MS/MS

• Correlate translation efficiency with i6A-modified tRNA abundance

Adaptation to specific nutrient availability:
Within the first week of colonization, B. thetaiotaomicron metabolism becomes centered around utilization of dietary oligosaccharides . This specialized metabolism requires efficient translation of specific enzyme sets. Differential modification of tRNAs by miaA1 could serve as a regulatory mechanism to optimize translation for the available nutrient profile.

By investigating these relationships, researchers can uncover how translational control mechanisms contribute to the remarkable metabolic versatility that makes B. thetaiotaomicron such a successful gut commensal.

How does the capsular polysaccharide system of B. thetaiotaomicron influence miaA1 expression and activity?

The capsular polysaccharide (CPS) system of B. thetaiotaomicron plays a critical role in colonization success and competitive fitness in the gut environment . The relationship between this system and miaA1 represents an unexplored area with significant implications for understanding bacterial adaptation.

Experimental approach:

• Comparative analysis:

  • Generate isogenic strains with different CPS profiles

  • Measure miaA1 expression levels using qRT-PCR and western blotting

  • Quantify i6A-modified tRNAs using LC-MS/MS

  • Assess translation efficiency of CPS biosynthesis genes

• Phase variation studies:

  • Isolate B. thetaiotaomicron populations expressing different CPS types

  • Compare miaA1 expression and activity between populations

  • Correlate with competitive fitness in colonization models

• Stress response analysis:

  • Expose wildtype and acapsular strains to gut-relevant stressors

  • Monitor changes in miaA1 expression and activity

  • Determine if translational adaptation contributes to stress resistance

Acapsular B. thetaiotaomicron strains show longer lag phases and slower growth rates in vivo, and are outcompeted by capsule-expressing strains . This competitive disadvantage may be partially mediated through translational control mechanisms involving miaA1, particularly under conditions where efficient protein synthesis is critical for adaptation.

The capsule provides protection against host immune factors and competing bacteria , and the translational machinery may be fine-tuned in response to capsule status to optimize resource allocation between growth and defense functions.

What impact does miaA1 have on the volatile compounds secretome of B. thetaiotaomicron?

The volatile compounds secretome of Bacteroides species includes bioactive molecules that contribute to bacterial communication and host-microbe interactions . The potential influence of miaA1 on this secretome represents an intriguing research question connecting translational control to bacterial signaling.

Experimental design:

• Generate miaA1 knockout, overexpression, and point-mutant strains

• Culture strains under standardized conditions in defined media

• Analyze volatile compounds using headspace extraction with GC-MS

• Compare volatile profiles between wildtype and modified strains

• Correlate findings with transcriptomics and proteomics data

Key compound classes to monitor:

• Fatty acid derivatives

• Amino acid derivatives

• Phenol compounds

• Indole and derivatives (known to be produced by Bacteroides species)

• Aldehydes and other volatile metabolites

Bacteroides species produce a wide range of volatile organic compounds (VOCs) that can be analyzed through headspace extraction followed by GC-MS analysis . The distribution of these compounds differs significantly between bacterial culture media and outer membrane vesicles (OMVs) .

tRNA modifications influence translational efficiency and fidelity, potentially affecting the expression of enzymes involved in volatile compound synthesis, regulatory proteins controlling secretion pathways, and metabolic enzymes affecting precursor availability. This research would reveal novel connections between translational control mechanisms and bacterial communication networks.

What techniques can be employed to investigate the interaction between miaA1 and its tRNA substrates?

Understanding the interaction between B. thetaiotaomicron miaA1 and its tRNA substrates requires sophisticated biochemical and biophysical approaches:

Structural characterization:

• X-ray crystallography:

  • Co-crystallize miaA1 with tRNA substrate and/or substrate analogs

  • Resolve atomic structure to identify key interaction residues

  • Visualize conformational changes upon substrate binding

• Cryo-electron microscopy:

  • Visualize miaA1-tRNA complexes in different functional states

  • Capture dynamic aspects of the interaction

  • Particularly valuable if crystallization proves challenging

Binding and kinetic analysis:
3. Surface plasmon resonance (SPR):

• Immobilize either miaA1 or tRNA substrate

• Determine binding kinetics (kon, koff) and affinity (KD)

• Analyze effects of mutations on binding parameters

• Isothermal titration calorimetry (ITC):

  • Obtain complete thermodynamic profile (ΔG, ΔH, ΔS)

  • No labeling required

  • Investigate temperature-dependence of interaction

Functional analysis:
5. RNA footprinting:

• Use ribonucleases or chemical probes to identify protected regions

• Map interaction sites on tRNA

• Analyze structural changes upon binding

• Cross-linking studies:

  • Employ UV cross-linking or chemical cross-linkers

  • Identify points of contact between enzyme and tRNA

  • Combine with mass spectrometry for precise mapping

• Enzyme kinetics:

  • Determine kinetic parameters (Km, kcat) for different tRNA substrates

  • Investigate effects of base modifications or mutations

  • Develop structure-activity relationships

Integration of these techniques provides a comprehensive understanding of how miaA1 recognizes and modifies specific tRNAs, information that is essential for understanding its role in translational control during B. thetaiotaomicron's adaptation to the gut environment .

How can Recombinant B. thetaiotaomicron miaA1 be engineered for alternative substrate acceptance?

Engineering Recombinant B. thetaiotaomicron miaA1 to accept alternative substrates represents an advanced research frontier with applications in synthetic biology and biotechnology:

Engineering strategies:

• Structure-guided mutagenesis:

  • Identify substrate binding residues through structural analysis

  • Design rational mutations to alter substrate specificity

  • Create small libraries of variants targeting key residues

• Directed evolution:

  • Develop high-throughput screening system for modified tRNA production

  • Generate large variant libraries through error-prone PCR or DNA shuffling

  • Select variants with desired activity through iterative rounds of screening

• Domain swapping:

  • Identify domains from related enzymes with desired specificities

  • Create chimeric enzymes with novel substrate preferences

  • Optimize domain boundaries to maintain protein folding and stability

Potential alternative substrates:

• Modified alkyl pyrophosphates with varying chain lengths

• Functionalized alkyl groups containing bioorthogonal handles (azides, alkynes)

• Fluorinated or isotopically labeled derivatives for tracking studies

Applications of engineered variants:

• Production of "designer tRNAs" with novel modification patterns

• Development of tools for metabolic labeling of tRNAs in vivo

• Creation of orthogonal translation systems for synthetic biology applications

Successfully engineered miaA1 variants could enable new approaches to study and manipulate translation in B. thetaiotaomicron, providing insights into how this process contributes to the bacterium's remarkable metabolic adaptability and colonization success .

What methods can assess the influence of miaA1 on translation fidelity and efficiency in B. thetaiotaomicron?

Investigating how miaA1 influences translation fidelity and efficiency in B. thetaiotaomicron requires integrated approaches spanning from in vitro biochemical techniques to in vivo systems biology methods:

In vitro translation analysis:

• Reconstituted translation systems:

  • Prepare translation components from wildtype and miaA1-deficient strains

  • Measure amino acid incorporation rates using labeled amino acids

  • Quantify misincorporation using mass spectrometry

• tRNA modification profiling:

  • Analyze modification status of individual tRNA species using LC-MS/MS

  • Correlate modification levels with codon-specific translation rates

  • Examine structural effects using biophysical techniques

Cellular approaches:
3. Reporter systems:

• Construct dual-luciferase reporters with varying codon usage

• Express in wildtype and miaA1-deficient B. thetaiotaomicron

• Measure relative translation efficiencies under different conditions

• Ribosome profiling:

  • Perform deep sequencing of ribosome-protected mRNA fragments

  • Analyze ribosome occupancy on different codons

  • Identify translation pause sites and their relationship to i6A-modified tRNAs

This comprehensive analysis would reveal how miaA1-mediated tRNA modification contributes to B. thetaiotaomicron's translational control, which is critical for its metabolic versatility and successful colonization of the dynamic gut environment .