Recombinant Gloeobacter violaceus Queuine tRNA-ribosyltransferase (tgt)

What is Queuine tRNA-ribosyltransferase (tgt) and what function does it serve?

Queuine tRNA-ribosyltransferase (tgt, EC 2.4.2.29) is an enzyme that catalyzes the exchange of guanine (G) with queuine (q) at the wobble position (position 34) of tRNAs containing G34U35N36 anticodons (Asp, Asn, Tyr, and His) . This exchange results in queuosine (Q) modification of these tRNAs. The enzyme is crucial for translational efficiency and accuracy, as Q-modified tRNAs have been shown to control translational speed of Q-decoded codons as well as near-cognate codons . In bacteria like Gloeobacter violaceus, tgt is part of the machinery that maintains proper protein synthesis and may play roles in various physiological processes including biofilm formation and virulence in pathogenic species .

What expression systems are most effective for producing recombinant Gloeobacter violaceus tgt?

For recombinant expression of Gloeobacter violaceus tgt, Escherichia coli expression systems have proven effective, similar to the expression of other bacterial tgt enzymes. Typically, the gene coding for G. violaceus tgt can be amplified by PCR using primers containing appropriate restriction sites, then cloned into expression vectors such as pDR111 or similar plasmids designed for bacterial expression . For optimal expression, BL21(DE3) or similar E. coli strains grown in LB medium supplemented with appropriate antibiotics at temperatures ranging from 18-37°C after IPTG induction (0.1-1 mM) are commonly used. Purification can be facilitated by incorporating affinity tags (such as His6) and using metal affinity chromatography followed by size exclusion chromatography to obtain pure, active enzyme. Expression yields can be optimized by adjusting parameters such as temperature, IPTG concentration, and induction time based on empirical testing.

How do mutations in the active site of Gloeobacter violaceus tgt affect its substrate specificity and catalytic efficiency?

Mutations in the active site of G. violaceus tgt can significantly impact both substrate specificity and catalytic efficiency. Structure-function studies of bacterial tgt enzymes have revealed several critical residues involved in substrate binding and catalysis. For G. violaceus tgt, mutations targeting the binding pocket residues that interact with the preQ1 substrate might alter the enzyme's ability to recognize and bind this substrate versus queuine. To investigate this experimentally, researchers should employ site-directed mutagenesis to create specific amino acid substitutions in conserved regions of the active site, followed by enzymatic assays to measure:

Such studies would provide insights into whether G. violaceus tgt could be engineered to accept queuine directly (like eukaryotic enzymes) rather than requiring the preQ1 intermediate. Additionally, crystallographic analysis of mutant enzymes in complex with their substrates would elucidate the structural basis for any observed changes in specificity .

What are the implications of tgt-mediated tRNA modifications for stress response in Gloeobacter violaceus?

The implications of tgt-mediated tRNA modifications for stress response in G. violaceus likely parallel findings in other cyanobacteria and bacteria, where Q-modification of tRNAs has been linked to stress adaptation. In cyanobacteria, RNA binding proteins (RBPs) and RNA modifications play critical roles in adapting to environmental stressors such as light intensity changes, nutrient limitation, and temperature fluctuations .

To investigate this question experimentally, researchers should:

• Generate a tgt knockout strain of G. violaceus and compare its growth and survival under various stress conditions (high light, UV exposure, oxidative stress, nutrient limitation) to wild-type.

• Perform comparative transcriptomics and proteomics analyses between wild-type and Δtgt strains under normal and stress conditions, focusing on:

• Assess the Q-modification status of specific tRNAs under different stress conditions using APB-Northern blotting methodology, which can detect the presence of Q-modified tRNAs based on their slower migration pattern .

Understanding these relationships would provide insights into how ancient cyanobacteria like G. violaceus utilize tRNA modifications to respond to environmental challenges, potentially revealing evolutionary conserved mechanisms of stress adaptation.

How does Gloeobacter violaceus tgt activity influence the cyanobacterial RNA binding proteome?

G. violaceus tgt activity likely influences the cyanobacterial RNA binding proteome through modulation of translation efficiency and accuracy. In cyanobacteria, RNA binding proteins (RBPs) play crucial roles in various cellular processes including RNA processing, stability, localization, and translation . The Q-modification of tRNAs catalyzed by tgt affects the translation of specific codons, potentially creating regulatory effects on the expression of RBPs themselves.

To investigate this relationship experimentally:

• Compare the RNA binding proteome between wild-type and Δtgt G. violaceus strains using techniques such as RNA interactome capture or GradSeq (gradient profiling by sequencing) .

• Analyze the codon usage patterns in genes encoding RBPs to identify those enriched in NAU codons (potentially regulated by Q-modified tRNAs).

• Perform ribosome profiling to measure translation efficiency of RBP-coding mRNAs in the presence and absence of functional tgt.

• Examine whether Q-modification influences the assembly or function of ribonucleoprotein complexes involved in RNA metabolism.

Expected findings might include altered expression of specific RBPs in the Δtgt strain, particularly those with NAU codon enrichment, potentially affecting downstream RNA-dependent processes such as photosystem assembly, tRNA maturation, or sRNA-mediated regulation .

What are the optimal conditions for assaying Gloeobacter violaceus tgt enzymatic activity in vitro?

For optimal assaying of G. violaceus tgt enzymatic activity in vitro, researchers should consider the following conditions and protocols:

Reaction Buffer Components:

• 100 mM HEPES or Tris-HCl (pH 7.5-8.0)

• 5-10 mM MgCl₂ (essential cofactor)

• 1-5 mM DTT (reducing agent to maintain enzyme stability)

• 0.1-0.5 mg/ml BSA (stabilizing agent)

• 20-100 mM KCl or NaCl (ionic strength)

Substrate Preparation:

• Synthetic tRNA substrates containing G34U35N36 anticodons (Asp, Asn, Tyr, or His tRNAs)

• Purified preQ₁ or queuine at concentrations ranging from 1-100 μM

• Radiolabeled substrates (³H-guanine or ³H-preQ₁) for sensitive detection

Assay Conditions:

• Temperature: 30-37°C (depending on G. violaceus optimal growth temperature)

• Incubation time: 15-60 minutes

• Enzyme concentration: 10-100 nM purified recombinant tgt

Detection Methods:

• Filter-binding assay: Measure incorporation of radiolabeled substrate into tRNA

• HPLC analysis: Detect guanine release or preQ₁/queuine incorporation

• Mass spectrometry: Identify modified tRNAs with high precision

The assay should be validated using known inhibitors of tgt (such as specific tgt inhibitors or competitive substrate analogs) as negative controls. Additionally, the enzymatic parameters (Km and kcat) should be determined under these conditions to establish a baseline for comparing mutant enzymes or different reaction conditions .

What strategies can be employed to improve the solubility and stability of recombinant Gloeobacter violaceus tgt?

Improving the solubility and stability of recombinant G. violaceus tgt requires multiple approaches targeting expression, purification, and storage conditions:

Expression Optimization:

• Lower induction temperature (16-20°C) to slow protein synthesis and improve folding

• Use specialized E. coli strains (e.g., Arctic Express, Rosetta, or SHuffle) designed for challenging proteins

• Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

• Test different fusion tags beyond conventional His6-tag:

  • Solubility-enhancing tags: MBP, SUMO, Thioredoxin, or GST

  • Cleavable tags with precise proteases (TEV, PreScission)

Buffer Optimization Table:

Purification Strategies:

• Multi-step purification including affinity chromatography followed by ion exchange and size exclusion

• Addition of mild detergents (0.05-0.1% Triton X-100 or 0.01-0.05% Tween-20) during lysis

• Inclusion of protease inhibitors during all purification steps

• Maintaining constant cold temperature (4°C) throughout purification

Storage Conditions:

• Flash-freeze purified enzyme in liquid nitrogen in small aliquots

• Store at -80°C in buffer containing 25-50% glycerol

• Avoid repeated freeze-thaw cycles

• For short-term storage, keep at 4°C with protease inhibitors

Empirical testing of these conditions with activity assays after each optimization step will help determine the optimal combination for maintaining functional G. violaceus tgt .

How can researchers accurately assess the incorporation of queuosine in tRNAs modified by Gloeobacter violaceus tgt?

Researchers can accurately assess the incorporation of queuosine in tRNAs modified by G. violaceus tgt using several complementary methodologies:

1. APB-Northern Blot Analysis:
This technique exploits the fact that Q-modified tRNAs migrate more slowly on polyacrylamide gels containing 3-(acrylamido)phenylboronic acid (APB) compared to unmodified tRNAs. The specific steps include:

• Separation of total RNA on APB-containing gels

• Transfer to nylon membranes

• Hybridization with biotinylated probes specific for target tRNAs (e.g., tRNA^Asp^GUC)

• Detection of Q-modified tRNAs by their characteristic mobility shift

2. Mass Spectrometry Methods:

• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of digested tRNAs to directly detect and quantify queuosine-containing nucleosides

• Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry of intact tRNAs or RNase digestion products

3. Radiolabeling Assays:

• Measure incorporation of radiolabeled queuine or preQ₁ into purified tRNAs

• Quantify by scintillation counting or phosphorimaging

4. Functional Complementation:
Using genetic systems to test Q-modification status:

• Express G. violaceus tgt in Q-deficient bacterial strains (e.g., E. coli ΔqueD)

• Assess restoration of Q-modification using the above methods

• Compare with controls including empty vector and known functional tgt genes

5. Reverse Transcription-based Methods:
Q-modification can cause specific patterns of reverse transcriptase stops or misincorporations, which can be detected by:

• Primer extension assays

• High-throughput sequencing techniques that capture RT errors or stops at modified sites

A combination of these approaches provides robust assessment of Q-incorporation, with APB-Northern blotting serving as a readily accessible method for routine analysis and mass spectrometry providing the gold standard for definitive modification identification and quantification .

How does Gloeobacter violaceus tgt activity correlate with photosystem assembly and function?

The correlation between G. violaceus tgt activity and photosystem assembly/function represents an intriguing research area, particularly given the unique characteristics of this ancient cyanobacterium. G. violaceus lacks thylakoid membranes, with photosystems located directly in the cytoplasmic membrane, making it an evolutionary important model for studying photosynthetic machinery.

Based on research in other cyanobacteria, tgt-mediated Q-modification likely influences the translation of specific proteins involved in photosystem assembly and function. Evidence from studies in Synechocystis sp. PCC 6803 has shown that RNA binding proteins (RBPs) with RNA recognition motifs (RRMs) are involved in transporting photosystem-related mRNAs to thylakoid membranes, and that the Rbp3 protein is critical for maintaining photosystem I levels .

To investigate the specific relationship between G. violaceus tgt and photosystems:

• Generate tgt knockout strains and analyze:

  • Photosystem I and II protein composition and stoichiometry

  • Photosynthetic electron transport rates

  • Chlorophyll fluorescence parameters

• Perform translatome analysis to identify specific photosystem-related transcripts whose translation is affected by Q-modification status.

• Examine whether genes encoding key photosystem components are enriched in NAU codons (potentially regulated by Q-modified tRNAs).

Expected results might include altered assembly or reduced levels of specific photosystem components in tgt-deficient strains, particularly affecting those proteins encoded by mRNAs rich in NAU codons. This could manifest as growth defects under specific light conditions or altered photosynthetic efficiency .

What role might Gloeobacter violaceus tgt play in primitive RNA-based regulatory networks?

G. violaceus, as one of the most ancient lineages of cyanobacteria, offers unique insights into primitive RNA-based regulatory networks. The tgt enzyme likely plays a significant role in these networks through modulation of translation via Q-modification of tRNAs.

In primitive regulatory systems, tRNA modifications may have served as early forms of translational regulation before the evolution of more complex regulatory mechanisms. G. violaceus tgt might represent an evolutionary conserved component of these primitive systems.

The research approach to understand this role should include:

• Comparative genomics analysis of tgt and associated genes across cyanobacterial lineages, with special focus on ancient lineages versus more modern forms.

• Analysis of codon usage patterns in G. violaceus, particularly examining whether genes in specific functional categories show enrichment or depletion of NAU codons (potentially regulated by Q-modified tRNAs).

• Investigation of potential interactions between tgt, modified tRNAs, and other components of RNA-based regulatory networks, including:

  • Small regulatory RNAs

  • RNA binding proteins

  • RNA processing enzymes

• Examination of whether tgt-dependent translation regulation interfaces with nitrogen metabolism regulation, as research in other cyanobacteria has revealed connections between sRNAs and nitrogen metabolism .

This research could provide important insights into how primitive translation regulation systems evolved and how they interface with other cellular processes in one of Earth's most ancient photosynthetic organisms .

How can comparative studies between Gloeobacter violaceus tgt and tgt from pathogenic bacteria inform antibiotic development?

Comparative studies between G. violaceus tgt and tgt from pathogenic bacteria can significantly inform antibiotic development strategies, especially as tgt has been linked to biofilm formation and virulence in several human pathogens .

The value of G. violaceus tgt in this context stems from its representation of an ancient, non-pathogenic form of the enzyme that can serve as a reference point for understanding how tgt function has been adapted in pathogenic bacteria.

Key research approaches should include:

• Structural comparisons between G. violaceus tgt and pathogenic bacterial tgt enzymes (from organisms like E. coli, Bacillus subtilis, Pseudomonas putida, and Chlamydia trachomatis) to identify:

  • Conserved catalytic residues across all forms

  • Pathogen-specific structural features that could be targeted selectively

  • Differences in substrate binding pockets

• Functional analysis comparing:

  • Substrate preferences (preQ₁ vs. queuine)

  • Enzymatic kinetics and efficiency

  • Impact on translation of virulence factors

• Development of a screening platform using recombinant G. violaceus tgt alongside pathogenic tgt variants to identify compounds that:

  • Selectively inhibit pathogenic tgt forms

  • Spare the ancient, non-pathogenic form

  • Modulate enzyme activity rather than completely inhibiting it

This comparative approach would enable the development of narrow-spectrum antibiotics targeting pathogen-specific features of tgt, potentially disrupting virulence without affecting beneficial microbiota. Additionally, since Q-modification affects the expression of NAU codon-enriched genes related to biofilm formation and virulence in bacteria, tgt inhibitors could serve as anti-virulence compounds rather than traditional antibiotics, potentially addressing concerns about antimicrobial resistance .

What are the current limitations in our understanding of Gloeobacter violaceus tgt and future research directions?

Current limitations in our understanding of G. violaceus tgt include insufficient structural characterization, limited knowledge of its natural substrates and enzymatic properties, and unclear connections to the unique physiology of this ancient cyanobacterium. Future research should focus on solving the crystal structure of G. violaceus tgt, characterizing its kinetic parameters with various substrates, and investigating its role in the context of the organism's unique cellular architecture lacking thylakoid membranes. Additionally, exploring the evolutionary significance of tgt in this early-branching cyanobacterium could provide insights into the development of tRNA modification systems and their roles in primitive translation regulation .

How might synthetic biology approaches utilize Gloeobacter violaceus tgt for novel applications?

Synthetic biology approaches could utilize G. violaceus tgt for several novel applications. The enzyme could be engineered to accept non-natural substrates for incorporating novel modifications into tRNAs, potentially creating ribosomes with expanded capabilities. G. violaceus tgt might also serve as a component in synthetic circuits designed to regulate gene expression through controlled modification of specific tRNAs, enabling condition-specific protein synthesis. Additionally, the enzyme could be incorporated into minimal synthetic cells as part of primitive translation quality control mechanisms. The ancient origin of G. violaceus tgt makes it particularly valuable for understanding the minimal requirements for functional translation systems in synthetic biology applications .