Recombinant Bacillus cereus Queuine tRNA-ribosyltransferase (tgt)

What is Queuine tRNA-ribosyltransferase (tgt) in Bacillus cereus and what is its primary function?

Queuine tRNA-ribosyltransferase (tgt) in Bacillus cereus is an enzyme that catalyzes the base-exchange of a guanine (G) residue with the queuine precursor 7-aminomethyl-7-deazaguanine (preQ1) at position 34 (the anticodon wobble position) of specific tRNAs, namely tRNAHis, tRNATyr, tRNAAsp, and tRNAAsn, all of which share a common uracil 33 – guanine 34 – uracil 35 sequence .

The preQ1 base is subsequently converted to the functional queuosine (Q) through additional enzymatic modifications. This process requires:

• PreQ1 incorporation by tgt

• Conversion to epoxyqueuosine by QueA (S-adenosylmethionine:tRNA ribosyltransferase-isomerase)

• Final reduction to queuosine by QueG (coenzyme B12-dependent epoxyqueuosine reductase)

While the precise function of queuosine has not been definitively determined, its strategic location in the tRNA anticodon strongly suggests involvement in regulating translational fidelity and speed, particularly in the reading of NAU codons .

How is the preQ1 precursor synthesized in Bacillus species?

In Bacillus species, like many bacteria capable of de novo Q synthesis, preQ1 is synthesized through a multi-step pathway starting from GTP. The complete biosynthetic pathway involves:

• GTP cyclohydrolase I (FolE) - Initial conversion of GTP

• 6-carboxy-5,6,7,8-tetrahydropterin synthase (QueD) - Further modification

• S-adenosyl-L-methionine-dependent organic radical-generating enzyme (QueE)

• PreQ0 synthetase (QueC/ToyM) - Formation of preQ0 intermediate

• Nitrile reductase (QueF) - Final conversion of preQ0 to preQ1

Researchers studying this pathway typically use genetic approaches such as gene knockouts (e.g., ΔqueD, ΔqueF) and complementation experiments to determine the role of each enzyme in the pathway . For example, to investigate the function of QueF, researchers have constructed E. coli DH10B ΔqueF strains using specific exchange of queF with an antibiotic resistance cassette through homologous recombination .

What are the recommended methods for cloning and expressing recombinant B. cereus tgt?

To successfully clone and express recombinant B. cereus tgt, researchers should follow these methodological steps:

• Gene amplification: Amplify the tgt gene from B. cereus genomic DNA using PCR with primers containing appropriate restriction sites for downstream cloning .

• Cloning vector selection: Based on the search results, vectors such as pBlueScript II SK(+) or pDR111 have been successfully used for similar proteins. For B. cereus tgt specifically, plasmid systems compatible with Gram-positive expression should be considered .

• Expression system: While E. coli is commonly used as a heterologous host, yeast expression systems have demonstrated success for expressing recombinant tgt proteins from various bacterial sources .

• Purification strategy: Incorporate a purification tag such as His-tag to facilitate protein isolation. Typical purification yields of >90% purity can be achieved with proper optimization .

• Activity validation: Confirm enzymatic activity using tRNA substrate assays that monitor the incorporation of preQ1 or radiolabeled queuine into appropriate tRNA substrates .

When optimizing expression, researchers should be aware that tgt activity can be affected by growth conditions, and expression in the native organism versus heterologous hosts may yield differences in post-translational modifications that could affect enzyme activity.

What experimental approaches can be used to measure tgt enzymatic activity?

Several experimental approaches can be used to measure B. cereus tgt enzymatic activity:

• Base incorporation assay: This method measures the incorporation of radiolabeled substrate (typically [3H]-queuine or radiolabeled preQ1) into tRNA substrates. The progress of the reaction can be monitored by taking aliquots at different time points, followed by precipitation of tRNA and scintillation counting .

• Covalent intermediate trapping: This technique involves incubating tgt with substrate tRNA in the presence of an uncompetitive inhibitor, which stabilizes the covalent tgt-tRNA intermediate. This complex can be visualized as retarded bands after SDS-PAGE. This method is particularly useful for investigating substrate specificity .

• Kinetic parameter determination: To determine kinetic constants such as Km and kcat, researchers can use varying concentrations of substrate (tRNA or base) and measure initial velocities. For B. cereus tgt, typical experiments would include:

  • Varying preQ1 concentration while keeping tRNA concentration fixed

  • Varying tRNA concentration while keeping preQ1 concentration fixed

• Competitive inhibition studies: These experiments can reveal insights about substrate binding mechanisms and can be used to investigate the affinity of tgt for different substrate analogues .

For accurate activity measurements, it's essential to use highly pure substrate preparations, as demonstrated in studies where trace contamination of preQ1 in queuine preparations led to misleading results .

How does tgt activity relate to biofilm formation and virulence in Bacillus cereus?

Recent research has established a significant connection between tRNA Q-modification (mediated by tgt) and biofilm formation and virulence in bacteria, including Bacillus species. The relationship works through the following mechanisms:

To investigate this relationship in B. cereus specifically, researchers can:

• Create tgt knockout mutants and analyze their biofilm-forming capacity

• Perform comparative transcriptomic/proteomic analyses between wild-type and tgt-deficient strains

• Identify and characterize the NAU codon-enriched genes in B. cereus that are affected by Q-modification

• Use infection models to assess virulence differences between strains with varying levels of tgt activity

What structural features determine substrate specificity in B. cereus tgt?

Substrate specificity in tgt enzymes is determined by key structural features within the active site. Based on studies of related bacterial tgt enzymes, the following factors influence substrate specificity in B. cereus tgt:

To investigate these structural features in B. cereus tgt specifically, researchers should consider:

• Site-directed mutagenesis of conserved residues predicted to be involved in substrate binding

• X-ray crystallography studies of B. cereus tgt with various substrates

• Molecular docking and simulation studies to predict substrate interactions

How can researchers investigate the relationship between tgt function and the SOS response in Bacillus species?

Investigating the relationship between tgt function and the SOS response in Bacillus species requires a multifaceted approach:

• Gene expression analysis: The SOS response in B. subtilis (a model for B. cereus) involves RecA and LexA (DinR) proteins that regulate the expression of DNA damage-inducible genes. Researchers can use promoterless reporter gene fusions (such as lacZ) to monitor the expression of SOS genes under various conditions, including when tgt function is altered .

• Regulatory network characterization: In B. subtilis, LexA represses the expression of 63 genes in 26 operons. Researchers can use chromatin immunoprecipitation (ChIP) techniques to identify if tgt or Q-modification-related genes are directly regulated by LexA .

• DNA damage response experiments: Techniques to investigate the relationship include:

  • Exposing wild-type and tgt-deficient strains to DNA-damaging agents and monitoring survival rates

  • Measuring mutation frequencies in the presence and absence of functional tgt

  • Assessing RecA-ssDNA filament formation and LexA cleavage in various genetic backgrounds

• Translational efficiency analysis: Since tgt affects tRNA modification and potentially translational efficiency, researchers can use ribosome profiling to determine if SOS genes are differentially translated in tgt mutants compared to wild-type strains.

• Prophage induction: In B. subtilis, the SOS response can lead to prophage induction. Researchers can monitor prophage activity in tgt mutants to determine if tRNA modification status affects this aspect of the SOS response .

It's important to note that while the regulatory mechanism of the SOS pathway is conserved between B. subtilis and E. coli, approximately 85% of the genes comprising the SOS regulon in B. subtilis appear distinct from those in E. coli .

How does B. cereus tgt differ from tgt in other bacterial species?

Comparative genomic analysis reveals several important differences between B. cereus tgt and tgt in other bacterial species:

• Sequence conservation: While the core catalytic domain of tgt is generally conserved across bacterial species, B. cereus tgt (typically around 379 amino acids in length) shows distinct sequence features compared to tgt from Gram-negative bacteria like E. coli .

• Substrate specificity determinants: Based on studies of other bacterial tgt enzymes, key residues involved in substrate recognition may differ between B. cereus and other bacteria. For example, the Cys158 and Val233 residues (in Z. mobilis numbering) that affect preQ1 binding may have different counterparts or conformations in B. cereus tgt .

• Regulatory context: The genomic neighborhood and regulatory elements controlling tgt expression vary across bacterial species. In B. cereus, as a Gram-positive bacterium, the regulation may more closely resemble that of B. subtilis than E. coli .

• Q-modification pathway integration: The queuosine modification pathway shows variations across bacterial species. Some bacteria, including certain Bacillus species, can synthesize Q de novo, while others rely on salvage pathways. The specific integration of tgt within these pathways can differ between species .

To systematically investigate these differences, researchers should:

• Perform multiple sequence alignments of tgt proteins from diverse bacterial species

• Analyze crystal structures (when available) to identify structural differences

• Conduct heterologous expression experiments to test functional conservation

• Use genetic complementation assays to determine if tgt from one species can substitute for another

What evidence exists for horizontal gene transfer of tgt genes among Bacillus species and other bacteria?

While the search results don't directly address horizontal gene transfer (HGT) of tgt genes, several lines of evidence could be used to investigate this possibility:

How can recombinant B. cereus tgt be utilized to study queuosine modification in complex microbial communities?

Recombinant B. cereus tgt offers several methodological approaches for studying queuosine modification in complex microbial communities:

• In vitro tRNA modification assays: Purified recombinant B. cereus tgt can be used to modify bulk tRNA extracted from microbial communities. By comparing the Q-modification status before and after treatment, researchers can:

  • Determine the baseline Q-modification in the community

  • Identify which tRNA species are undermodified in natural settings

  • Assess the impact of environmental factors on Q-modification potential

• Metabolic labeling experiments: Using recombinant tgt along with labeled preQ1 or queuine, researchers can trace the incorporation of these precursors into tRNAs within complex communities. This approach could reveal:

  • Differential uptake of Q-precursors among community members

  • Competition dynamics for limited Q-resources

  • Metabolic interdependencies related to Q-modification

• Functional complementation studies: By introducing recombinant B. cereus tgt into communities with varying Q-modification capabilities, researchers can:

  • Assess the impact of enhanced Q-modification on community structure and function

  • Identify species that benefit most from external Q-modification support

  • Evaluate potential probiotic applications of Q-modification enhancement

• Methodological framework:

  • Extract bulk tRNA from microbial communities (e.g., gut microbiome samples)

  • Perform in vitro modification with recombinant tgt and appropriate precursors

  • Analyze modification status using mass spectrometry or other sensitive detection methods

  • Correlate modification patterns with community composition and functionality

This approach is particularly valuable given the evidence that Q-modification influences biofilm formation and virulence, suggesting that tgt activity may be a significant factor in microbial community dynamics and host-microbe interactions .

What are the current methodological challenges in studying the impact of tgt-mediated tRNA modifications on bacterial translation efficiency?

Researchers face several methodological challenges when investigating how tgt-mediated tRNA modifications affect bacterial translation efficiency:

• Quantification of modified tRNAs:

  • Challenge: Accurate quantification of Q-modified versus unmodified tRNAs at single-nucleotide resolution

  • Current approaches: Mass spectrometry and high-performance liquid chromatography

  • Limitation: These methods typically require substantial amounts of purified tRNA and may not be sensitive enough for low-abundance tRNA species

  • Methodological solution: Development of more sensitive detection methods, such as nanopore sequencing technologies that can directly detect modified nucleotides

• Codon-specific translation effects:

  • Challenge: Determining how Q-modification affects translation of specific NAU codons in a context-dependent manner

  • Current approaches: Ribosome profiling and reporter systems with codon-optimized sequences

  • Limitation: Difficulty in isolating the effect of Q-modification from other factors affecting translation

  • Methodological solution: Engineered minimal translation systems with defined components to directly measure the impact of Q-modification on specific codons

• Temporal dynamics:

  • Challenge: Understanding how Q-modification status changes in response to environmental conditions or stress

  • Current approaches: Time-course experiments with bulk measurements

  • Limitation: Poor temporal resolution and inability to track modifications in single cells

  • Methodological solution: Development of real-time sensors for tRNA modification status

• Integration with other tRNA modifications:

  • Challenge: Q-modification coexists with numerous other tRNA modifications that may have synergistic or antagonistic effects

  • Current approaches: Genetic manipulation of individual modification pathways

  • Limitation: Difficulty in dissecting complex interactions between multiple modification types

  • Methodological solution: Systematic combinatorial studies using synthetic biology approaches to recreate defined modification patterns

• Physiological relevance:

  • Challenge: Connecting observed in vitro effects to physiological outcomes in bacterial populations

  • Current approaches: Phenotypic characterization of modification-deficient strains

  • Limitation: Pleiotropic effects of genetic manipulations

  • Methodological solution: Development of specific inhibitors of tgt that allow acute manipulation of modification status without genetic perturbation

How might structural studies of B. cereus tgt inform the development of new antimicrobial strategies?

Structural studies of B. cereus tgt could significantly inform the development of novel antimicrobial strategies through several avenues:

• Structure-based inhibitor design:

  • Detailed knowledge of the active site architecture of B. cereus tgt could enable the rational design of specific inhibitors

  • X-ray crystallography and cryo-EM studies of tgt in complex with substrates and substrate analogs would reveal key binding interactions

  • Comparative analysis with human tgt homologs would allow the design of selective inhibitors targeting bacterial tgt while sparing human enzymes

• Targeting biofilm formation pathways:

  • Since tgt-mediated Q-modification affects biofilm formation, structural insights could reveal how this modification alters the translation of biofilm-related proteins

  • Understanding the structural basis of this connection could lead to new approaches for disrupting biofilm formation in B. cereus and related pathogens

  • This would be particularly valuable for addressing biofilm-associated infections, which are notoriously resistant to conventional antibiotics

• Exploiting species-specific features:

  • Structural comparisons between tgt enzymes from different bacterial species could reveal B. cereus-specific features that could be targeted

  • Identification of allosteric sites unique to B. cereus tgt might provide opportunities for highly selective inhibition

  • Such approaches could lead to narrow-spectrum antimicrobials with reduced impact on beneficial microbiota

• Methodological framework for structure-guided antimicrobial development:

  • Obtain high-resolution structures of B. cereus tgt using X-ray crystallography or cryo-EM

  • Perform molecular dynamics simulations to identify conformational changes during catalysis

  • Use virtual screening and fragment-based approaches to identify initial hit compounds

  • Optimize hits through structure-guided medicinal chemistry

  • Validate candidates using in vitro enzyme assays and bacterial growth/biofilm inhibition studies

  • Assess effects on complex bacterial communities to evaluate ecological impact of tgt inhibition