Recombinant Campylobacter concisus Queuine tRNA-ribosyltransferase (tgt)

What is Queuine tRNA-ribosyltransferase and what is its function in C. concisus?

Queuine tRNA-ribosyltransferase (TGT) is a critical enzyme in C. concisus responsible for catalyzing the incorporation of queuine into the wobble anticodon position of specific tRNAs - particularly those for amino acids tyrosine, histidine, asparagine, and aspartic acid. This enzyme plays a fundamental role in ensuring translation fidelity and efficiency from RNA to protein. In bacterial systems like C. concisus, TGT functions are often expanded beyond simple tRNA modification to include roles in cellular adaptation and pathogenicity. The enzyme is part of a system that maintains proper protein synthesis and cellular function under various environmental conditions encountered in the human oral-gastrointestinal tract .

How does C. concisus TGT relate to BisA, another important enzyme in this pathogen?

BisA in C. concisus represents an interesting parallel to TGT as both are multifunctional enzymes that optimize bacterial adaptation with limited genomic resources. While TGT primarily functions in tRNA modification, BisA demonstrates a dual role in both N-/S-oxide-supported respiration and protein repair, specifically of methionine sulfoxide residues. C. concisus has evolved to rely on versatile enzymes like BisA and TGT as part of its adaptation to diverse environments in the human oral-gastrointestinal tract. This adaptation strategy is particularly important given that C. concisus has a significantly smaller genome (1.8-2.1 Mb) compared to other gut bacteria like E. coli (~5 Mb) .

What distinguishes bacterial TGT from eukaryotic QTRT1?

The bacterial TGT system differs fundamentally from the eukaryotic system in several ways:

The eukaryotic enzyme, often referred to as QTRT1, forms a heterodimeric complex with QTRT2, whereas bacterial TGT typically functions as a single protein with the complete catalytic machinery .

What methods are recommended for cloning and expressing recombinant C. concisus TGT?

When cloning and expressing recombinant C. concisus TGT, researchers should consider the following methodology:

• Gene identification and primer design: Identify the TGT gene sequence from C. concisus genomic databases and design primers with appropriate restriction sites.

• PCR amplification: Extract genomic DNA from C. concisus using standard protocols and amplify the TGT gene using high-fidelity polymerase.

• Cloning strategy: Insert the amplified gene into an expression vector (such as pET systems for E. coli expression) with an appropriate tag (His-tag recommended for easy purification).

• Transformation: Transform the construct into an expression host, typically E. coli BL21(DE3) or similar strains optimized for protein expression.

• Expression optimization: Test multiple conditions including temperature (16-37°C), IPTG concentration (0.1-1 mM), and incubation times to maximize soluble protein yield.

• Purification: Employ affinity chromatography (Ni-NTA for His-tagged proteins) followed by size exclusion chromatography to obtain pure protein.

For initial validation, Western blotting with anti-His antibodies or activity assays using defined tRNA substrates should be performed .

How can researchers generate C. concisus TGT knockout strains?

Generation of C. concisus TGT knockout strains requires a three-step strategy similar to that used for other Campylobacter genes:

• Construct a knockout cassette by amplifying 400-850 bp DNA sequences flanking the TGT gene and joining them to an antibiotic resistance marker (such as a chloramphenicol resistance cassette with its own promoter).

• Methylate the knockout cassette DNA by incubating with C. concisus cell-free extract in the presence of S-adenosylmethionine (SAM) to protect it from restriction enzymes.

• Introduce the methylated construct into C. concisus using either natural transformation or electroporation (recommended parameters: 2,500 V/pulse).

• Plate transformed cells first on non-selective media for 8-12 hours, then transfer to media containing the appropriate antibiotic (8-10 μg/mL chloramphenicol is typically used).

• Confirm successful knockout by PCR amplification of genomic DNA from transformants using primers that flank the targeted region.

This approach has been successfully employed for generating various mutants in Campylobacter species, including disruption of genes like bisA and associated enzymes .

What assays are available to measure C. concisus TGT enzymatic activity?

Several assays can be employed to measure TGT enzymatic activity:

For the most direct assessment, researchers typically use purified recombinant TGT and synthetic or purified tRNA substrates in vitro, then analyze the incorporation of queuine or analogs using one of the methods above .

How does TGT activity in C. concisus contribute to its pathogenesis in inflammatory bowel disease?

The relationship between C. concisus TGT activity and inflammatory bowel disease (IBD) pathogenesis involves several interconnected mechanisms:

• Translation efficiency and stress response: TGT-mediated tRNA modification ensures proper translation of proteins necessary for C. concisus survival in the inflammatory gut environment.

• Epithelial barrier effects: Research shows that altered expression of QTRT1 (the eukaryotic equivalent) affects intestinal epithelial cell junctions and proliferation. In a similar manner, C. concisus TGT activity may influence bacterial proteins that interact with host epithelial barriers.

• Inflammatory signaling: Properly modified tRNAs ensure accurate translation of bacterial proteins involved in host-microbe interactions, potentially influencing inflammatory cascades.

Expression studies on QTRT1 have shown significant downregulation in ulcerative colitis and Crohn's disease patients, with associated changes in epithelial junction proteins including β-catenin, claudin-5, and claudin-2. This suggests that tRNA modification processes are disrupted during intestinal inflammation .

What structural features of C. concisus TGT are essential for its catalytic activity?

While specific structural data for C. concisus TGT is not provided in the search results, we can infer essential catalytic features based on related enzymes:

• Active site architecture: TGT enzymes generally contain a catalytic pocket that accommodates the tRNA anticodon loop and the queuine substrate.

• RNA recognition motifs: Specific protein domains recognize and bind to the tRNA substrate with high specificity.

• Zn2+ binding domain: Many TGT enzymes require zinc coordination for structural integrity and function.

• Catalytic residues: Key amino acids positioned to facilitate the ribosyltransferase reaction, typically including aspartate residues that enable nucleophilic attack.

These structural features work together to catalyze the precise exchange of guanine with queuine at the wobble position of tRNA anticodons. Further structural studies through X-ray crystallography or cryo-EM would be valuable to confirm these features specifically in C. concisus TGT .

How does queuine availability affect C. concisus TGT function in the human gut environment?

In the complex ecosystem of the human gut, C. concisus TGT function is likely influenced by queuine availability through several mechanisms:

• Competitive dynamics: Eukaryotic cells and other bacteria compete for available queuine, potentially creating microenvironments where queuine is limited.

• Adaptive responses: C. concisus may modulate TGT expression or activity based on queuine availability to optimize resource utilization.

• Cross-feeding relationships: Some bacteria produce queuine that may be utilized by C. concisus, creating interdependencies within the gut microbiome.

• Host inflammation effects: Inflammatory conditions alter bacterial populations and potentially queuine availability, creating feedback loops that affect C. concisus TGT function.

Research has established that queuine starvation can cause changes in protein synthesis patterns and may interfere with other nutrient factors including vitamin B12, tetrahydrobiopterin, and epidermal growth factor, which could all impact C. concisus survival and virulence .

What RNA isolation methods are optimal for studying TGT-modified tRNAs from C. concisus?

For optimal isolation of TGT-modified tRNAs from C. concisus, a two-step RNA isolation protocol using TRIzol followed by column purification is recommended:

• Harvest C. concisus cells from culture at mid-logarithmic phase.

• Resuspend cell pellet in TRIzol reagent (1 mL per 10^7 cells), lyse cells by repetitive pipetting or homogenization.

• Add chloroform (0.2 mL per 1 mL TRIzol), shake vigorously, and centrifuge to separate phases.

• Transfer the aqueous phase to a new tube and precipitate RNA with isopropanol.

• Wash RNA pellet with 75% ethanol, air-dry briefly, and resuspend in RNase-free water.

• Further purify the RNA using Qiagen RNeasy kit following manufacturer's instructions.

• For tRNA-specific isolation, consider size fractionation or enrichment using custom oligonucleotide probes.

This method effectively preserves RNA modifications while removing protein and DNA contaminants that could interfere with downstream analysis .

How can researchers differentiate between TGT activity and other RNA-modifying enzymes in C. concisus?

Distinguishing TGT activity from other RNA-modifying enzymes requires a multi-faceted approach:

• Substrate specificity analysis: Use defined tRNA substrates with specific anticodon sequences (targeting tRNA^Tyr, tRNA^His, tRNA^Asn, and tRNA^Asp) as TGT specifically modifies these tRNAs.

• Competitive inhibition studies: Employ known TGT inhibitors versus inhibitors of other RNA-modifying enzymes to differentiate activities.

• Mass spectrometry profiling: Analyze the specific mass shifts associated with queuosine versus other modifications.

• Genetic approaches: Create knockout strains for TGT and other RNA-modifying enzymes, then analyze the resulting tRNA modification profiles to identify specific enzyme contributions.

• Biochemical separation: Use chromatographic techniques to separate different enzymatic activities from cell extracts before assaying.

This comprehensive strategy helps establish which modifications are specifically attributed to TGT activity versus other enzymes that may modify similar or adjacent positions in tRNA molecules .

What are the implications of C. concisus TGT research for developing new antimicrobial strategies?

Research on C. concisus TGT offers several promising avenues for antimicrobial development:

• Targeted inhibition: TGT could serve as a selective target for antimicrobials that disrupt tRNA modification without affecting human enzymes, potentially reducing bacterial fitness or virulence.

• Biofilm disruption: If TGT influences biofilm formation (as other tRNA modifications do in some bacteria), targeting this enzyme could help disrupt C. concisus persistence.

• Host-microbe interaction modulation: Understanding how TGT-modified tRNAs affect bacterial protein expression might reveal ways to disrupt pathogenic interactions with host cells.

• Combination therapy approaches: TGT inhibitors could potentially sensitize C. concisus to existing antibiotics by altering stress responses or membrane permeability.

The proven dual functionality of the similar enzyme BisA in C. concisus suggests that targeting tRNA-modifying enzymes could simultaneously disrupt multiple bacterial processes, potentially creating more effective antimicrobial approaches with reduced resistance development .

How might C. concisus TGT activity influence microbiome-host interactions in intestinal disease?

C. concisus TGT activity likely influences microbiome-host interactions through multiple mechanisms:

Research has demonstrated that QTRT1-related processes significantly impact intestinal epithelial cell junctions and proliferation. Knockdown of QTRT1 in intestinal epithelial cells results in downregulation of proliferating cell nuclear antigen (PCNA), β-catenin, and claudin-5, while upregulating claudin-2. These changes mirror some of the epithelial disruptions seen in inflammatory bowel disease, suggesting that tRNA modification processes—including those potentially influenced by bacterial TGT—play important roles in maintaining intestinal homeostasis .

What emerging technologies will advance our understanding of C. concisus TGT function?

Several cutting-edge technologies are poised to revolutionize our understanding of C. concisus TGT:

• CRISPR-Cas9 genome editing: Enables more precise and efficient genetic manipulation of C. concisus to study TGT in its native context.

• Nanopore direct RNA sequencing: Allows detection of tRNA modifications without prior conversion or amplification, providing a more accurate picture of the tRNA modificationome.

• Single-cell transcriptomics: Facilitates studies of TGT expression and activity at the individual bacterial cell level, revealing population heterogeneity.

• Cryo-electron microscopy: Enables high-resolution structural analysis of TGT-tRNA complexes in near-native states.

• Microfluidic co-culture systems: Creates controlled environments to study how TGT activity influences interactions between C. concisus and host epithelial cells in real-time.

These technologies will help researchers address current knowledge gaps regarding TGT's specific role in C. concisus pathogenicity and its potential as a therapeutic target .

How can computational approaches enhance experimental studies of C. concisus TGT?

Computational approaches offer powerful complementary methods to experimental studies:

• Homology modeling and molecular dynamics: Predict structural features and substrate interactions of C. concisus TGT based on related enzymes with known structures.

• Codon usage analysis: Identify genes particularly dependent on queuosine-modified tRNAs based on codon bias.

• Systems biology approaches: Model how TGT activity influences broader metabolic and protein interaction networks in C. concisus.

• Machine learning algorithms: Predict potential inhibitors by analyzing chemical features of compounds that interact with related enzymes.

• Comparative genomics: Identify conservation patterns and evolutionary relationships among TGT enzymes across Campylobacter species and strains.

These computational methods can guide experimental design, help interpret results, and generate new hypotheses about TGT function that might not be immediately apparent from experimental data alone .