Recombinant Pseudomonas putida Queuine tRNA-ribosyltransferase (tgt)

What is the biochemical reaction catalyzed by Queuine tRNA-ribosyltransferase in P. putida?

Queuine tRNA-ribosyltransferase (EC 2.4.2.29) in P. putida catalyzes the exchange of guanine at the wobble position (position 34) of tRNAs with GUN anticodons (specifically tRNA-Asn, tRNA-Asp, tRNA-His, and tRNA-Tyr) with the modified nucleobase queuine. The chemical reaction can be represented as:

[tRNA]-guanine + queuine → [tRNA]-queuine + guanine

This base exchange reaction is irreversible in vivo and results in the formation of queuosine (Q), a hypermodified nucleoside that influences translation efficiency and accuracy .

How does TGT structure in P. putida compare to other bacterial species?

The TGT enzyme in P. putida (B479_04660) is located on the chromosome (position 1053410-1054525 on the + strand) and encodes a protein of approximately 41.2 kDa with a predicted isoelectric point of 6.99 . Comparative analysis reveals that P. putida TGT shares structural similarities with other bacterial TGTs but has specific characteristics:

Unlike eukaryotic TGTs that function as heterodimers, bacterial TGTs including P. putida's function as monomers, which affects their substrate recognition and catalytic mechanism .

What expression systems are most effective for producing recombinant P. putida TGT?

The optimal expression system for recombinant P. putida TGT depends on research objectives, but the T7-like expression system has shown particular effectiveness. Recent research demonstrates that:

• T7-like expression system in P. putida KT2440: This system, mimicking the pET expression system in E. coli, shows a 1.4-fold improvement in protein expression when optimized .

• Site-specific integration: Integration of the T7 RNA polymerase gene at the phaC1 locus provides superior expression compared to other genomic locations .

• Induction parameters: Optimal expression is achieved with 0.5 mM IPTG added 4 hours after inoculation .

• Enhanced chassis options: The genome-reduced strain EM42 with T7-like expression system shows even higher expression levels (2.1-fold increase) compared to standard KT2440 strains .

When comparing expression systems in P. putida KT2440, the T7 and MmP1 expression systems outperform other inducible systems by nearly 3-fold .

How can researchers effectively detect and measure queuosine modification in tRNA?

Detection and quantification of queuosine modifications require specialized analytical methods:

• Chromatography-coupled tandem quadrupole mass spectrometry: This technique provides the most sensitive detection of queuosine in tRNA samples. The method involves:

  • Enzymatic digestion of tRNA to nucleosides

  • Resolution of nucleosides on a high-performance column (e.g., Hypersil aQ GOLD)

  • Detection using a triple quadrupole LC/MS with specific mass transitions

• Radioactive displacement assay: This alternative approach measures the exchange of [14C]-labeled guanine with queuine:

  • Preparation of tRNA containing [14C]-guanine at position 34

  • Incubation with TGT enzyme and potential substrates

  • Separation of tRNA and released [14C]-guanine using DEAE-cellulose

  • Quantification by liquid scintillation counting

• Incorporation assay for testing reversibility:

  • Modification of tRNA with non-labeled nucleobases

  • Incubation with TGT and [14C]-guanine

  • Analysis of [14C]-guanine incorporation to assess reversibility

How does TGT activity affect bacterial stress responses in P. putida?

The TGT enzyme and queuosine modification play significant roles in bacterial stress responses, though the specific mechanisms vary by species. In P. putida:

• Metal stress response: The queF gene (encoding an enzyme in the queuosine pathway) is induced by copper exposure, suggesting a role in metal stress adaptation, although queF mutants don't show copper sensitivity phenotypes .

• Oxidative stress: While not specifically demonstrated in P. putida, TGT-dependent queuosine modification is linked to oxidative stress resistance in related bacteria. In E. coli, TGT mutants show slightly increased sensitivity to oxidative stress, while in Streptococcus thermophilus, the sensitivity is more pronounced .

• Competitive fitness: Overexpression of queF in P. putida increases its ability to inhibit E. coli growth, suggesting a role in competitive fitness within microbial communities .

The specific mechanisms linking queuosine modification to stress responses likely involve translational reprogramming and altered expression of stress-responsive genes.

What is the connection between tgt function and antibiotic tolerance in Pseudomonas species?

Recent research indicates a complex relationship between queuosine modification and antibiotic tolerance:

• Aminoglycoside tolerance: In Vibrio cholerae, a detailed study showed that TGT engages translational reprogramming in response to aminoglycosides like tobramycin. This mechanism appears to involve codon-specific effects, particularly impacting tyrosine TAT and TAC codons .

• Translational regulation: The absence of queuosine modification alters the translation of specific transcripts, including the anti-SoxR factor RsxA, which has a codon bias toward tyrosine TAT. This affects oxidative stress responses, which are linked to aminoglycoside tolerance .

• Pseudomonas-specific mechanisms: While direct evidence in Pseudomonas is limited, comparative genomics suggests similar mechanisms may operate. The manipulation of the mismatch repair system in P. putida affects mutation frequencies, which could potentially influence antibiotic resistance development .

These findings suggest that targeting TGT or queuosine modification pathways could potentially alter antibiotic susceptibility profiles in bacteria.

How does P. putida TGT compare functionally to eukaryotic TGT systems?

The TGT systems in P. putida and eukaryotes show important functional and structural differences:

Eukaryotes, including humans, cannot synthesize queuine de novo and must salvage it from dietary sources or gut microbiota, establishing a microbiome-to-translatome interaction that affects human health . This fundamental difference has implications for understanding host-bacterial interactions and potential competition for queuine as a micronutrient.

What evolutionary patterns exist in bacterial TGT enzymes across different species?

Evolutionary analysis of TGT reveals interesting patterns:

• High conservation: TGT is widely distributed across bacterial phyla. P. putida TGT belongs to a conserved ortholog group (POG001843) with 536 members across 614 genera, indicating strong evolutionary preservation .

• Specialized adaptations: Despite high conservation, three distinct pathways for queuosine acquisition have evolved:

  • Direct de novo synthesis (most free-living bacteria including P. putida)

  • Direct queuine salvage with altered TGT specificity (e.g., Chlamydia trachomatis)

  • Indirect queuine salvage through queuine lyase (QueL) that regenerates preQ₁ from queuine

• Gene clustering patterns: Comparative genomics reveals distinct clustering patterns of tgt with other genes:

  • In organisms with complete de novo synthesis, tgt clusters with queC/D/F genes

  • In organisms with indirect salvage, tgt clusters with queL and queuine transporters

  • In direct salvage organisms, tgt clusters with specialized transporters

These evolutionary patterns reflect adaptations to different ecological niches and nutrient availability.

How can researchers leverage P. putida TGT for studying translational regulation?

P. putida TGT offers several advantages for studying translational regulation:

• Codon-specific effects: The queuosine modification introduced by TGT affects translation in a codon-specific manner. Researchers can use this to study how tRNA modifications regulate translation efficiency and accuracy at specific codons .

• Stress response models: P. putida's robust stress response mechanisms make it an excellent model for studying how tRNA modifications remodel the translatome under various stresses. The connection between queuosine modification and oxidative/metal stress responses provides a framework for such studies .

• Reporter systems: Combining TGT manipulation with fluorescent reporters containing specific codon biases can reveal how queuosine modification affects the translation of different mRNA populations. For example:

  • Construct reporters enriched in NAU vs. NAC codons

  • Express in wild-type vs. tgt-deficient backgrounds

  • Measure translation efficiency under different conditions

• Ribosome profiling approaches: Applying ribosome profiling (RiboSeq) to P. putida wild-type and tgt mutants can provide genome-wide insights into how queuosine modification affects translation speed at every codon .

What are the current challenges in studying queuosine-dependent translation in model organisms?

Several significant challenges exist in studying queuosine-dependent translation:

How does bacterial queuosine modification research inform our understanding of human disease?

Research on bacterial TGT and queuosine modification has significant implications for human health:

• Microbiome-host interactions: Humans depend on bacterial sources for queuine, establishing a direct link between gut microbiome health and human tRNA modification status. This "gut-tRNA axis" connects bacterial queuosine research to human health .

• Neurodegenerative disease connections: Queuosine is essential for neurotransmitter synthesis and neuronal function, with links to neurodegenerative diseases. Understanding bacterial queuosine synthesis and salvage informs how these modifications affect brain health .

• Cancer implications: Enhanced expression of QTRT1 (the human TGT catalytic subunit) has been detected in lung adenocarcinoma (LUAD). Studies of QTRT1 in cancer contexts benefit from understanding the fundamental mechanisms elucidated in bacterial systems .

• Cardiovascular disease: Recent research shows that inhibition of QTRT1 ameliorates hyperlipidemia and atherosclerosis in mice by downregulating de novo lipogenesis. This suggests that tRNA modification has previously unrecognized roles in lipid metabolism .

• Competition for micronutrients: Bacteria, including many pathogens, compete with the host for queuine. Understanding bacterial queuine salvage pathways informs host-microbiome competition for this important micronutrient .

What methodological approaches bridge bacterial TGT research to translational medicine applications?

Several methodological approaches connect basic TGT research to translational applications:

• Comparative genomics and systems biology:

  • Sequence similarity networks identify novel enzyme families involved in queuosine metabolism

  • Comparative analysis of queuosine pathways across pathogens and commensals reveals potential intervention points

• Metabolic engineering approaches:

  • Engineered bacterial strains with enhanced or depleted queuosine modification capacity

  • Controlled expression systems for queuosine-related enzymes to manipulate tRNA modification levels

• Structural biology methods:

  • Over 36 structures have been solved for TGT enzymes, providing templates for structure-based drug design

  • Inhibitor development targeting pathogen-specific features of TGT

• Genetic and biochemical screening:

  • High-throughput screens for compounds that modulate TGT activity

  • Functional genomics approaches to identify genetic interactions with queuosine modification pathways

• Animal models and human studies:

  • Germ-free animal models to study the impact of queuine derivation

  • Clinical studies examining queuosine modification status in various disease states

These methodological bridges facilitate the translation of basic TGT research into potential therapeutic applications for human disease.

What emerging technologies will advance our understanding of P. putida TGT function?

Several cutting-edge technologies promise to deepen our understanding of P. putida TGT:

• Single-molecule approaches:

  • Single-molecule fluorescence to track TGT-tRNA interactions in real-time

  • Optical tweezers to measure the kinetics and thermodynamics of TGT catalysis

• CRISPR-based technologies:

  • CRISPR interference for precise temporal control of TGT expression

  • Base editing technologies for introducing specific mutations in TGT

  • CRISPRi screens to identify genetic interactions with queuosine pathways

• Advanced mass spectrometry:

  • Nanopore direct RNA sequencing for detection of tRNA modifications

  • Improvements in chromatography-coupled tandem quadrupole mass spectrometry for more sensitive detection of queuosine

• Systems biology integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Machine learning models predicting the impact of queuosine modification on translation

• Synthetic biology platforms:

  • Expanded genetic code systems incorporating queuosine derivatives

  • Engineered ribosomes with altered specificity for queuosine-modified tRNAs

What are the critical unanswered questions about P. putida TGT that require further investigation?

Despite decades of research, several critical questions about P. putida TGT remain unanswered:

• Regulatory mechanisms:

  • How is TGT expression regulated in P. putida under different environmental conditions?

  • What transcription factors control tgt gene expression?

  • How does queuosine modification status feedback to regulate its own synthesis?

• Physiological roles:

  • Why does P. putida maintain the complete queuosine synthesis pathway?

  • What specific advantages does queuosine modification confer to P. putida in its environmental niches?

  • How does queuosine modification interface with P. putida's robust stress response systems?

• Evolutionary questions:

  • Why have some bacteria evolved to use queuine directly while others maintain the full synthesis pathway?

  • What selective pressures have shaped the diversification of queuosine-related enzymes?

• Mechanistic details:

  • How does the P. putida TGT achieve specificity for certain tRNAs?

  • What is the precise catalytic mechanism of the base exchange reaction?

  • How does queuosine modification alter tRNA structure and function at the molecular level?

• Ecological significance:

  • Does P. putida compete with other microorganisms for queuine or its precursors?

  • How does queuosine modification affect P. putida's interactions with plants and other organisms in its environment?

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, genetics, and systems biology.

What are common challenges in expressing and purifying active recombinant P. putida TGT?

Researchers frequently encounter several challenges when working with recombinant P. putida TGT:

• Protein solubility issues:

  • TGT can form inclusion bodies in heterologous expression systems

  • Solution: Expression at lower temperatures (16-20°C), use of solubility tags (MBP, SUMO), or co-expression with chaperones

• Activity preservation:

  • TGT may lose activity during purification processes

  • Solution: Include reducing agents (DTT, β-mercaptoethanol) in buffers, optimize purification speed, and add stabilizing agents like glycerol

• Substrate availability:

  • Limited commercial availability of queuine and its precursors

  • Solution: Chemical synthesis of substrates or extraction from natural sources, collaboration with chemistry groups

• Expression optimization:

  • Finding the optimal expression system and conditions

  • Solution: Systematic testing of different promoters, host strains, and induction parameters as detailed in section 2.1

• Activity assays:

  • Developing reliable assays for TGT activity

  • Solution: Use radioisotope-based assays or mass spectrometry-based methods as described in section 2.2

How can researchers address contradictory findings in queuosine modification studies?

The literature on queuosine modification contains apparent contradictions that researchers must navigate:

• Species-specific differences:

  • The effects of queuosine modification vary between organisms

  • Solution: Clearly define the model system and avoid generalizing findings across species without validation

• Context-dependent effects:

  • The same modification can have different effects depending on experimental conditions

  • Solution: Standardize growth conditions, carefully control media composition, and report all relevant parameters

• Technical variations:

  • Different detection methods can yield varying results

  • Solution: Use multiple complementary techniques and include appropriate controls

• Strain background effects:

  • Genetic background can influence the phenotypic consequences of tgt mutation

  • Solution: Use isogenic strains, perform complementation studies, and validate key findings in multiple strain backgrounds

• Interpretation frameworks:

  • Theoretical models for how queuosine affects translation continue to evolve

  • Solution: Consider multiple mechanistic models when interpreting data, and design experiments that can distinguish between alternative hypotheses