Recombinant Bacillus pumilus Queuine tRNA-ribosyltransferase (tgt)

What is Queuine tRNA-ribosyltransferase and what is its role in bacterial cells?

Queuine tRNA-ribosyltransferase (tgt), also known as tRNA-guanine transglycosylase (EC 2.4.2.29), is an enzyme that catalyzes the exchange of guanine at position 34 (the wobble position) of tRNAs with GUN anticodons (specifically tRNAs for Asp, Asn, His, and Tyr) with the modified base queuine or its precursors. In bacteria, this enzyme incorporates the queuine precursor preQ₁ (7-aminomethyl-7-deazaguanine), which is further modified by additional enzymes to form queuosine .

Recent research has demonstrated that tRNA modifications, including those catalyzed by tgt, are crucial for fine-tuning protein translation. These modifications modulate the translation rate of NAU codons and have been implicated in controlling various physiological processes, particularly those related to biofilm formation and virulence in both Gram-positive and Gram-negative bacteria .

How does the bacterial Queuine tRNA-ribosyltransferase differ from its eukaryotic counterpart?

There are several key differences between bacterial and eukaryotic TGT enzymes:

Bacterial TGT functions as a homodimer with each protomer featuring a (βα)₈ barrel and a Zn²⁺ binding subdomain. Due to steric constraints, this dimer can bind and convert only one substrate tRNA molecule at a time, following a ping-pong mechanism involving a covalent TGT- tRNA intermediate . In contrast, eukaryotic TGT is a heterodimer composed of a catalytically active QTRT1 subunit and a catalytically inactive QTRTD1 subunit, which is essential for catalyzing queuine incorporation into tRNA .

What is the biosynthetic pathway for queuosine in bacteria?

In bacteria, queuosine biosynthesis follows a multi-step pathway:

• GTP is converted to preQ₀ (7-cyano-7-deazaguanine) through a series of enzymatic reactions involving:

  • GTP cyclohydrolase I (FolE)

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

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

  • preQ₀ synthetase (QueC = ToyM)

• preQ₀ is reduced to preQ₁ (7-aminomethyl-7-deazaguanine) by a nitrile reductase (QueF)

• preQ₁ is incorporated into tRNA at position 34 by tRNA-guanine transglycosylase (TGT)

• Once in tRNA, preQ₁ is converted to epoxyqueuosine by S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA)

• Finally, epoxyqueuosine reductase (QueG), a coenzyme B₁₂-dependent enzyme, completes the modification to form queuosine

Unlike bacteria, eukaryotes cannot synthesize queuine de novo and must acquire it from their diet and/or microflora, making queuine an important micronutrient for plants, animals, and fungi .

What structural features of B. pumilus TGT enable substrate recognition and catalysis?

The bacterial TGT enzyme, including that from B. pumilus, exhibits a characteristic (βα)₈ triose-phosphate-isomerase-like fold (TIM barrel) that provides the structural basis for substrate binding and catalysis . The enzyme contains a zinc-binding domain near the C-terminus that contributes to structural stability.

The catalytic mechanism involves several key residues in the active site:

• An aspartate residue (equivalent to Asp280 in Z. mobilis TGT) serves as the nucleophile that attacks the C1' of the ribose at position 34 of the tRNA, forming a covalent enzyme-tRNA intermediate and displacing guanine .

• Another aspartate residue (equivalent to Asp102 in Z. mobilis TGT) acts as a base to deprotonate the N5 atom of preQ₁, facilitating a nucleophilic attack on the covalent tRNA-enzyme intermediate .

Specific residues in the binding pocket determine substrate specificity. For example, in bacterial TGTs, the presence of a cysteine residue (equivalent to Cys158 in Z. mobilis TGT) is important for preQ₁ recognition, while eukaryotic TGTs have a valine at this position, altering substrate preference .

How does the queuosine modification influence bacterial virulence and biofilm formation?

Recent research has revealed a significant connection between queuosine modification of tRNAs and bacterial virulence and biofilm formation. A novel bioinformatic strategy to predict Q-genes (NAU codon-enriched genes affected by queuosine modification) revealed a widespread enrichment in functions related to biofilm formation and virulence in bacteria, particularly in human pathogens .

This relationship has been experimentally verified in several model bacteria:

• Altering the degree of tRNA Q-modification in both Gram-positive (e.g., B. subtilis) and Gram-negative (e.g., E. coli, P. putida) bacteria significantly affects biofilm formation and virulence .

• The mechanism appears to involve the coordination of expression of functionally related genes enriched in NAU codons, suggesting that queuosine modification serves as a regulatory mechanism for controlling virulence factors .

• In Shigella flexneri, TGT is required for efficient pathogenicity, making bacterial TGT a potential target for the rational design of anti-Shigellosis compounds .

These findings represent the first report of a general mechanism controlling biofilm formation and virulence across diverse bacterial species through tRNA modification.

What experimental evidence supports the specificity determinants in bacterial versus eukaryotic TGT enzymes?

Several key studies have identified specificity determinants distinguishing bacterial from eukaryotic TGT enzymes:

Homology models and mutagenesis studies with Z. mobilis TGT revealed two critical residue positions that largely account for the different substrate specificities:

• Cys158 in bacterial TGT versus valine in eukaryotic TGT

• Val233 in bacterial TGT versus glycine in eukaryotic TGT

Experimental evidence using enzyme kinetics and X-ray crystallography showed that:

• The Cys158Val mutation reduces affinity for preQ₁ while leaving affinity for guanine unaffected

• The Val233Gly exchange leads to an enlarged substrate binding pocket necessary to accommodate the bulkier queuine molecule in a conformation compatible with the tRNA-enzyme intermediate

Interestingly, bacterial TGT can recognize queuine, but cannot efficiently use it as a substrate. When highly pure queuine was tested with bacterial TGT, no insertion into tRNA was observed, whereas preQ₁ was efficiently incorporated .

Additional evidence comes from crystallographic studies of human QTRT1 (the catalytic subunit of eukaryotic TGT), which revealed a structure highly related to bacterial TGT but with key alterations in the active site to accommodate the bulkier queuine base .

What are the optimal conditions for expressing and purifying recombinant B. pumilus TGT?

Based on available research on bacterial TGT enzymes, the following protocol can be used for expression and purification of recombinant B. pumilus TGT:

Expression System and Conditions:

• Expression host: E. coli BL21(DE3) strains, with consideration for using tgt-deficient strains (e.g., BL21(DE3) tgt::Km^r) to prevent contamination with host TGT

• Vector: pET-based vectors with N-terminal polyhistidine tag

• Induction: 0.5-1.0 mM IPTG at OD₆₀₀ of 0.6-0.8

• Post-induction growth: 16-18 hours at 20°C to enhance soluble protein yield

Purification Protocol:

• Cell lysis in buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5 mM MgCl₂, 2 mM DTT, and protease inhibitors

• Ni-NTA affinity chromatography using an imidazole gradient (20-250 mM)

• Size exclusion chromatography using a Superdex 200 column

• Optional: Ion exchange chromatography for higher purity

Storage Considerations:

• Store at -20°C in buffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 2 mM DTT, and 20% glycerol

• For extended storage, store at -80°C

• Avoid repeated freeze-thaw cycles; working aliquots can be kept at 4°C for up to one week

The purified protein typically has a molecular weight of approximately 43 kDa and should yield >85% purity as assessed by SDS-PAGE .

How can one assess the enzymatic activity of recombinant B. pumilus TGT in vitro?

Several complementary assays can be used to evaluate the enzymatic activity of recombinant B. pumilus TGT:

1. tRNA [¹⁴C] Guanine Displacement Assay:

• Pre-label tRNA with [8-¹⁴C] guanine using E. coli TGT

• Incubate the labeled tRNA with recombinant B. pumilus TGT and potential substrates (e.g., preQ₁)

• Separate tRNA from free nucleobases using DEAE-cellulose resin

• Measure displaced [¹⁴C] guanine by scintillation counting

2. tRNA-[¹⁴C] Guanine Incorporation Assay:

• Modify tRNA with non-labeled nucleobases using the TGT enzyme

• Test the ability of the enzyme to insert [¹⁴C] guanine into the modified tRNA

• This assay can assess the reversibility of the modification

3. HPLC-Based Assay:

• Incubate tRNA with TGT and substrate

• Digest tRNA with nuclease P1 and analyze by HPLC

• Monitor the conversion of guanosine to queuosine or preQ₁-modified nucleoside

Reaction Conditions:

• Buffer: 50 mM Tris-HCl, pH 7.5, 20 mM NaCl, 5 mM MgCl₂, 2 mM DTT

• Temperature: 37°C

• Substrate concentrations: 200 μM preQ₁ or other substrates

• tRNA: Typically 2 absorbance units (260 nm) of tRNA

What genetic manipulation techniques are available for studying B. pumilus TGT function in vivo?

Several genetic approaches can be employed to study B. pumilus TGT function in vivo:

1. Gene Knockout/Deletion:

• For B. pumilus, homologous recombination techniques using the λ Red recombination system can be adapted

• Design a DNA cassette with antibiotic resistance (e.g., kanamycin) flanked by homology regions to the tgt gene

• Transform B. pumilus cells with the cassette and select positive mutants on antibiotic plates

• Verify deletion by PCR and sequencing

Example Protocol based on E. coli methods:

• Transform B. pumilus with a plasmid carrying the λ phage γβα operon (e.g., pRedET)

• Induce recombination (e.g., with arabinose)

• Introduce the DNA cassette with homology regions flanking tgt

• Select transformants on antibiotic plates and verify by PCR and sequencing

2. Complementation Studies:

• Clone the wild-type tgt gene into an expression vector with a suitable promoter

• Transform the tgt knockout strain with this construct

• Assess restoration of phenotype to confirm gene function

3. Site-Directed Mutagenesis:

• Introduce specific mutations in the tgt gene to study structure-function relationships

• Key residues for investigation include those in the active site and substrate binding pocket

• Assess effects on enzyme activity and bacterial phenotypes

4. Reporter Gene Fusions:

• Create transcriptional or translational fusions between tgt and reporter genes (e.g., lacZ, gfp)

• Study expression patterns under different conditions to understand regulation

These approaches can help elucidate the role of TGT in B. pumilus physiology, particularly in relation to biofilm formation, virulence, and stress responses.

How can B. pumilus TGT be utilized in comparative studies of tRNA modification systems?

B. pumilus TGT offers several valuable applications in comparative studies of tRNA modification systems:

1. Evolutionary Studies:

• B. pumilus TGT can serve as a model for studying the evolution of tRNA modification enzymes across bacterial species

• Comparative analysis with TGTs from diverse bacteria can reveal evolutionary adaptations and functional conservation

• Comparison with eukaryotic TGTs helps understand the divergence between prokaryotic and eukaryotic tRNA modification systems

2. Structure-Function Relationships:

• The B. pumilus TGT structure can be compared with other bacterial and eukaryotic TGTs to identify conserved catalytic mechanisms and species-specific features

• Mutational studies based on structural comparisons can reveal how subtle changes in protein architecture affect substrate specificity and catalytic efficiency

3. Substrate Specificity Analysis:

• B. pumilus TGT can be used to investigate differences in substrate recognition between various bacterial species

• Comparative binding studies with different substrates (guanine, preQ₁, queuine) can highlight species-specific preferences

4. Chimeric Enzyme Construction:

• Creating chimeric enzymes between B. pumilus TGT and other bacterial or eukaryotic TGTs allows mapping of functional domains

• Such studies can identify regions responsible for substrate specificity, tRNA recognition, and catalytic activity

These comparative approaches contribute to our fundamental understanding of tRNA modification mechanisms and their roles in bacterial physiology and pathogenesis.

What is the potential of B. pumilus TGT as a target for novel antimicrobial strategies?

B. pumilus TGT represents a promising target for antimicrobial development based on several key considerations:

1. Role in Virulence:

• TGT has been identified as essential for the efficient pathogenicity of Shigella species, the causative agent of bacillary dysentery

• Recent research demonstrates that tRNA queuosine modification affects biofilm formation and virulence in numerous bacterial pathogens

2. Structural Differences from Eukaryotic Counterparts:

• Bacterial TGTs differ structurally from eukaryotic TGTs, particularly in the substrate binding pocket

• These differences provide a basis for selective inhibition of bacterial enzymes without affecting human TGT

3. Drug Development Strategies:

• Structure-based drug design approaches can target the preQ₁ binding site of bacterial TGT

• Small molecule inhibitors can be designed to exploit the specific features of bacterial TGT, such as the Cys158 residue (Z. mobilis numbering) that is replaced by valine in eukaryotic TGTs

• High-resolution crystal structures of bacterial TGTs facilitate rational drug design efforts

4. Benefits of Targeting TGT:

• As TGT affects virulence rather than essential growth functions, inhibitors might exert less selective pressure for resistance development

• Targeting virulence mechanisms may allow the host immune system to clear infections more effectively

• Since mammalian TGT is involved in phenylalanine to tyrosine conversion, selective inhibition of bacterial TGT avoids potential side effects

Future antimicrobial strategies could focus on developing compounds that selectively inhibit bacterial TGTs, potentially offering new treatment options for infections caused by B. pumilus and other pathogenic bacteria.

How does tRNA modification by TGT influence gene expression regulation in B. pumilus?

The modification of tRNA by TGT plays a significant role in regulating gene expression in B. pumilus and other bacteria through several mechanisms:

1. Codon-Specific Translation Control:

• Queuosine modification at the wobble position affects the translation rate of NAU codons

• This creates a regulatory mechanism for controlling the expression of genes enriched in these codons (Q-genes)

• Bioinformatic analysis has revealed that Q-genes are particularly enriched in functions related to biofilm formation and virulence

2. Stress Response Regulation:

• The degree of tRNA Q-modification can change under different environmental conditions

• This allows bacteria to modulate gene expression in response to stressors

• For example, in E. coli, queuosine-deficient strains show reduced survival in stationary phase, suggesting a role in stress adaptation

3. Coordinated Expression of Functionally Related Genes:

• The queuosine modification system appears to coordinate the expression of functionally related genes

• This represents a general mechanism controlling biofilm formation and virulence in both Gram-positive and Gram-negative bacteria

4. Growth Phase-Dependent Regulation:

• Expression of the queA gene, involved in the queuosine pathway, has been found to be enhanced in stationary phase and induced by low pH and arginine in Streptococcus gordonii

• This suggests that tRNA modification systems respond to growth phase and environmental signals

Understanding these regulatory mechanisms is crucial for comprehending the physiological roles of TGT and the queuosine modification system in B. pumilus and other bacterial species.

What are common challenges in expressing functional recombinant B. pumilus TGT and how can they be addressed?

Researchers working with recombinant B. pumilus TGT may encounter several challenges:

1. Protein Solubility Issues:

• Challenge: TGT may form inclusion bodies when overexpressed

• Solution:

  • Lower expression temperature (16-20°C)

  • Reduce IPTG concentration (0.1-0.5 mM)

  • Use solubility-enhancing fusion tags (SUMO, MBP, or TrxA)

  • Co-express with chaperones (GroEL/GroES)

  • Optimize buffer conditions with additives like arginine or low concentrations of detergents

2. Protein Stability Concerns:

• Challenge: TGT may show reduced stability during purification and storage

• Solution:

  • Include stabilizing agents (5-10% glycerol, 0.5-1 mM DTT or TCEP)

  • Avoid repeated freeze-thaw cycles

  • Store working aliquots at 4°C for up to one week

  • For long-term storage, flash-freeze in liquid nitrogen and store at -80°C

3. Low Enzymatic Activity:

• Challenge: Purified protein may show reduced or no activity

• Solution:

  • Verify proper folding using circular dichroism

  • Ensure the presence of essential cofactors (Zn²⁺)

  • Check for inhibitory contaminants in buffers

  • Optimize assay conditions (pH, ionic strength, temperature)

  • Use fresh tRNA substrates

4. Host Contamination:

• Challenge: Contamination with host E. coli TGT

• Solution:

  • Use TGT-deficient expression strains (e.g., BL21(DE3) tgt::Km^r)

  • Apply more stringent purification protocols

  • Confirm protein identity by mass spectrometry

5. Substrate Quality:

• Challenge: Variable quality of tRNA and preQ₁ substrates

• Solution:

  • Use freshly prepared or commercially validated tRNA

  • Verify preQ₁ purity by HPLC or NMR

  • Store substrates properly to prevent degradation

Addressing these challenges requires careful optimization of expression conditions, purification protocols, and activity assays specific to B. pumilus TGT.

How can researchers distinguish between TGT activity and other enzymes that modify tRNA?

Distinguishing TGT activity from other tRNA modification enzymes requires specific approaches:

1. Substrate Specificity Tests:

• TGT specifically exchanges guanine at position 34 of tRNAs with GUN anticodons (tRNA^Asp, tRNA^Asn, tRNA^His, and tRNA^Tyr)

• Using tRNAs with different anticodons can help distinguish TGT from other tRNA modification enzymes

• Bacterial TGT incorporates preQ₁, while eukaryotic TGT incorporates queuine

2. Exchange Reaction Monitoring:

• TGT catalyzes a base exchange reaction, replacing guanine with preQ₁

• Monitor the release of guanine and incorporation of preQ₁ simultaneously

• This distinctive exchange mechanism differentiates TGT from most other tRNA modification enzymes that add modifications without base replacement

3. Selective Inhibitors:

• Use known TGT inhibitors to confirm enzyme identity

• For example, 7-methylguanine inhibits TGT but not most other tRNA modification enzymes

4. Mass Spectrometry Analysis:

• Analyze modified tRNA by LC-MS/MS to identify the specific modification introduced

• The mass shift and fragmentation pattern of queuosine or preQ₁ modification are distinctive

5. Position-Specific Analysis:

• Use nuclease digestion followed by HPLC analysis to verify the position of the modification

• TGT specifically modifies position 34, while other enzymes target different positions

6. Genetic Approaches:

• Compare activities in wild-type and TGT-knockout strains

• Complementation with purified TGT should restore the specific modification

By combining these approaches, researchers can confidently attribute observed tRNA modifications to TGT activity rather than other tRNA-modifying enzymes.

What quality control measures are essential when working with recombinant B. pumilus TGT?

Ensuring the quality of recombinant B. pumilus TGT requires comprehensive quality control measures:

1. Protein Purity Assessment:

• SDS-PAGE analysis with Coomassie staining (target: >85-90% purity)

• Western blot using anti-His tag antibodies to confirm identity

• Size exclusion chromatography to assess aggregation state

• Mass spectrometry to verify protein mass and sequence integrity

2. Structural Integrity Evaluation:

• Circular dichroism spectroscopy to confirm secondary structure

• Thermal shift assays to assess protein stability

• Dynamic light scattering to detect aggregation

• Limited proteolysis to verify proper folding

3. Functional Verification:

• Enzymatic activity assays using the tRNA [¹⁴C] guanine displacement method

• Determination of kinetic parameters (KM and kcat) for comparison with published values

• Substrate specificity testing with guanine, preQ₁, and queuine

4. Metal Content Analysis:

• ICP-MS or colorimetric assays to confirm zinc content (expected 1:1 ratio)

• EDTA treatment followed by metal reconstitution to verify metal dependency

5. Batch-to-Batch Consistency:

• Standardized activity assays to compare enzyme preparations

• Consistent specific activity across multiple preparations

• Storage stability testing at different temperatures

6. Contaminant Testing:

• Endotoxin testing if intended for cell-based assays

• Nuclease activity tests to ensure no contaminating nucleases

• Protease activity tests to detect degradative enzymes

How does B. pumilus TGT compare with TGT enzymes from other Bacillus species?

B. pumilus TGT shares similarities and differences with TGT enzymes from other Bacillus species:

Sequence and Structural Comparison:

• B. pumilus TGT typically shows high sequence identity (>80%) with other Bacillus TGTs, particularly those from the B. subtilis group

• All Bacillus TGTs feature the characteristic (βα)₈ TIM barrel fold and zinc-binding domain

• The active site residues responsible for catalysis are highly conserved across Bacillus species

Species-Specific Variations:

• Minor amino acid differences in substrate binding regions may influence substrate specificity and catalytic efficiency

• B. pumilus TGT has a complete sequence of 381 amino acids, comparable to other Bacillus TGTs

• The expression patterns and regulation of tgt genes may differ between Bacillus species, reflecting their ecological adaptations

Functional Similarities:

• All Bacillus TGTs catalyze the incorporation of preQ₁ into tRNAs with GUN anticodons

• They participate in the queuosine modification pathway, which affects biofilm formation and virulence

• The basic catalytic mechanism involving nucleophilic attack by an aspartate residue is conserved

Genomic Context:

• In B. pumilus, the tgt gene (e.g., BPUM_2412) exists within a genomic context that may differ from other Bacillus species

• The arrangement of genes involved in queuosine biosynthesis (que genes) varies among Bacillus species

• In B. subtilis, a highly studied relative, the genetic organization of the queuosine modification pathway is well-characterized, providing a reference for B. pumilus studies

Understanding these comparisons helps researchers leverage knowledge across Bacillus species and identify unique aspects of B. pumilus TGT that may relate to its specific ecological niche and physiological role.

What are the key experimental differences when working with bacterial versus eukaryotic TGT systems?

Working with bacterial TGT systems like B. pumilus differs significantly from eukaryotic TGT systems:

1. Protein Expression and Purification:

2. Substrate Requirements:

3. Assay Methods:

• Bacterial TGT activity can be assayed using preQ₁ or guanine exchange

• Eukaryotic TGT assays require queuine and may show different kinetics

• Inhibitor profiles differ between bacterial and eukaryotic enzymes

• Temperature optima and buffer conditions may vary between systems

4. Genetic Manipulation:

• Bacterial systems offer simpler genetic manipulation techniques

• Eukaryotic TGT studies often require more complex gene knockout strategies

• Complementation experiments differ due to the two-subunit nature of eukaryotic TGT

5. Physiological Context:

• Bacterial TGT studies focus on virulence and biofilm formation

• Eukaryotic TGT studies often examine connections to development and disease

• Different cellular compartmentalization considerations

• Distinct regulatory networks governing expression and activity

These differences necessitate tailored experimental approaches when working with bacterial versus eukaryotic TGT systems, despite their shared evolutionary origin and basic function.

How does the genomic context of tgt in B. pumilus inform our understanding of its biological role?

The genomic context of the tgt gene in B. pumilus provides valuable insights into its biological role and regulation:

1. Gene Organization and Clusters:

• In B. pumilus, the tgt gene (e.g., BPUM_2412) is part of the queuosine biosynthesis pathway

• The genomic proximity to other que genes (queA, queD, queE, queF) suggests coordinated expression and functional relationships

• Unlike in Streptococcus gordonii, where queA is located immediately 5' of the ADS operon transcriptional activator (arcR), B. pumilus likely has a different genetic organization

2. Regulatory Elements:

• Analysis of upstream regions can reveal potential regulatory elements controlling tgt expression

• Promoter elements and transcription factor binding sites indicate how tgt expression responds to environmental cues

• Comparison with other Bacillus species can identify conserved and species-specific regulatory mechanisms

3. Genetic Linkage to Virulence Factors:

• B. pumilus strains are known to produce various extracellular enzymes, including RNases, proteases, and phosphatases

• The genomic context may reveal connections between tgt and genes encoding these virulence factors

• In some bacterial species, tgt is functionally linked to pathogenicity islands or virulence-associated gene clusters

4. Horizontal Gene Transfer Indicators:

• Analysis of GC content and codon usage in the tgt region compared to the rest of the genome can indicate potential horizontal gene transfer events

• The presence of mobile genetic elements near tgt might suggest evolutionary acquisition of queuosine modification capabilities

5. Genome Comparison with Clinical Isolates:

• B. pumilus GR8 has been identified as a pathogen causing ginger rhizome rot disease

• Comparing the tgt genomic context between environmental and pathogenic B. pumilus strains may reveal adaptations related to virulence

• The complete genome of B. pumilus GR8 exhibits high similarity to B. pumilus strain B6033, providing a reference for comparative genomics

Understanding the genomic context of tgt in B. pumilus contributes to our knowledge of how queuosine modification is integrated into the bacterium's physiology and potential pathogenicity.