Recombinant Bordetella bronchiseptica tRNA (Ile)-lysidine synthase (tilS)

What is the function of tRNA (Ile)-lysidine synthase (tilS) in Bordetella bronchiseptica?

tRNA (Ile)-lysidine synthase (tilS) in Bordetella bronchiseptica is responsible for catalyzing the conversion of cytidine (C34) to lysidine at the wobble position of the anticodon in tRNAIle (CAU). This post-transcriptional modification is crucial as it changes the codon recognition specificity from AUG to AUA and alters the amino acid specificity for synthetase activation from methionine to isoleucine . In Bordetella species, as in other bacteria, this enzymatic activity enables proper decoding of the isoleucine codon AUA during protein synthesis, distinguishing it from the methionine codon AUG . The tilS enzyme effectively discriminates between tRNAIle and tRNAMet, ensuring that once lysidine modification occurs (converting CAU to LAU), the tRNA can be correctly charged with isoleucine by isoleucyl-tRNA synthetase (IleRS) . This modification is essential for accurate protein synthesis and bacterial viability.

Why is lysidine modification considered essential for bacterial survival?

Lysidine modification is considered essential for bacterial survival because it directly impacts the fidelity of protein translation, which is fundamental to all cellular processes. The tilS gene, which encodes tRNA (Ile)-lysidine synthase, has been identified as essential for viability in Bacillus subtilis and is nearly universally present in eubacteria, including human pathogens . Without proper lysidine modification, tRNAIle cannot be correctly charged with isoleucine, leading to mistranslation where methionine may be incorporated instead of isoleucine at AUA codons . Such mistranslation can result in defective proteins with compromised function or stability, disrupting critical cellular processes. In experimental studies, partial inactivation of the tilS gene in E. coli leads to impaired AUA codon translation, further demonstrating its crucial role in bacterial protein synthesis . The evolutionary conservation of this modification system across bacterial species underscores its fundamental importance to bacterial physiology and survival.

What are the recommended methods for expressing and purifying recombinant Bordetella bronchiseptica tilS?

The expression and purification of recombinant Bordetella bronchiseptica tilS requires careful optimization of several parameters to ensure high yield and functional integrity. Based on established protocols for similar bacterial proteins, the recommended approach begins with gene cloning into an appropriate expression vector containing a histidine or other affinity tag for purification purposes . The expression system of choice can be E. coli, yeast, baculovirus, or mammalian cells, with E. coli being most commonly used for bacterial proteins due to its simplicity and cost-effectiveness . For optimal expression in E. coli, BL21(DE3) or similar strains designed for recombinant protein expression are recommended, with induction using IPTG at concentrations of 0.1-1.0 mM when cultures reach mid-log phase (OD600 of 0.6-0.8).
Purification typically follows a multi-step process beginning with cell lysis (using sonication or pressure-based methods) in a buffer containing protease inhibitors. Affinity chromatography using Ni-NTA resin for His-tagged proteins provides the initial purification step, followed by ion exchange chromatography to remove contaminants . Size exclusion chromatography serves as a final polishing step to achieve high purity. Throughout the purification process, maintaining protein stability is crucial; buffers containing 20-50 mM Tris-HCl (pH 7.5-8.0), 100-300 mM NaCl, 5-10% glycerol, and 1-5 mM DTT or 2-mercaptoethanol are typically effective for tilS enzymes. Activity assays using in vitro transcribed tRNA substrates should be performed to confirm that the purified protein retains its enzymatic function .

How can researchers design experiments to assess the catalytic activity of tilS?

Researchers can design robust experiments to assess the catalytic activity of tilS by focusing on its ability to convert cytidine to lysidine in the tRNA anticodon. A comprehensive experimental approach should include both in vitro and potentially in vivo assays to fully characterize the enzyme's activity. For in vitro assessment, the primary assay involves incubating purified recombinant tilS with ATP, lysine, and in vitro transcribed tRNAIle2 substrate . The reaction mixture typically contains 50 mM HEPES-KOH (pH 7.5-8.0), 10 mM MgCl2, 1-5 mM ATP, 1-10 mM lysine, and 1-5 μM tRNA substrate. Formation of lysidine-modified tRNA can be detected through several complementary methods.
Gel electrophoresis under acidic conditions can separate modified from unmodified tRNAs based on charge differences . Mass spectrometry provides a more precise identification of the lysidine modification. Functional confirmation of successful modification can be assessed by testing the modified tRNA's ability to be aminoacylated by isoleucyl-tRNA synthetase (IleRS), as lysidine modification is both necessary and sufficient to convert tRNAIle2 into a substrate for IleRS . This functional assay involves incubating the modified tRNA with purified IleRS and radiolabeled isoleucine, followed by acid precipitation and scintillation counting to measure aminoacylation levels. Control experiments should include reactions without ATP, without lysine, or with catalytically inactive tilS mutants to confirm specificity. Kinetic parameters (Km and kcat) can be determined by varying substrate concentrations and measuring initial reaction rates. Additionally, testing lysine analogs as alternative substrates or inhibitors can provide insights into the enzyme's catalytic flexibility and substrate recognition mechanisms .

What approaches can be used to investigate the substrate specificity of Bordetella bronchiseptica tilS?

Investigating the substrate specificity of Bordetella bronchiseptica tilS requires multifaceted approaches focusing on both tRNA recognition and amino acid substrate utilization. To examine tRNA substrate specificity, researchers should prepare a series of in vitro transcribed tRNA variants with modifications to key recognition elements, particularly in the anticodon loop, acceptor stem, and D-arm regions . Each variant should be tested as a substrate in the standard tilS reaction assay, measuring the efficiency of lysidine formation. Comparative analysis with tRNAMet can identify the specific structural features that enable tilS to discriminate between tRNAIle and tRNAMet. Chimeric tRNAs containing elements from both tRNAIle and tRNAMet can pinpoint critical recognition domains.
For investigating amino acid substrate specificity, researchers should test a series of lysine analogs and related compounds as potential substrates or inhibitors in the tilS reaction . The panel should include compounds with modified side chains, altered amino or carboxyl groups, and structurally similar amino acids. For each analog, measure both its ability to serve as a substrate (resulting in modified tRNA) and its potential inhibitory effect on the canonical lysine-dependent reaction. Kinetic analysis comparing Km and kcat values for different substrates can quantify relative substrate preferences. Product characterization using mass spectrometry should confirm the identity of the modified nucleoside produced with each alternative substrate .
Complementary approaches include structural studies using X-ray crystallography or cryo-EM to visualize the enzyme-substrate interactions, computational docking and molecular dynamics simulations to predict binding interactions, and site-directed mutagenesis of key residues in the enzyme's active site to assess their contribution to substrate recognition and catalysis. This comprehensive approach can reveal the molecular basis for the unique substrate specificity of Bordetella bronchiseptica tilS compared to tilS enzymes from other bacterial species .

How does tilS contribute to antibiotic resistance mechanisms in Bordetella bronchiseptica?

The connection between tilS and antibiotic resistance in Bordetella bronchiseptica represents a complex and understudied area of research. As an essential enzyme for bacterial viability, tilS impacts fundamental cellular processes that can indirectly influence antibiotic susceptibility patterns . The primary mechanism through which tilS may contribute to antibiotic resistance involves its central role in protein translation fidelity. Since tilS ensures proper decoding of the isoleucine codon AUA, any modulation in its activity could alter the translation of proteins involved in antibiotic resistance, including efflux pumps, antibiotic-modifying enzymes, and cell wall components . When bacteria face antibiotic stress, precise protein synthesis becomes even more critical for survival and adaptation.
Experimental approaches to investigate this relationship should include comparative proteomics analysis of wildtype and tilS-deficient (or partially deficient) Bordetella strains under antibiotic pressure. Researchers should examine whether specific resistance-related proteins show altered expression or sequence accuracy in tilS-compromised strains. Additionally, studies should assess whether tilS upregulation occurs in response to specific antibiotics as part of the bacterial stress response. Another important consideration is whether the essential nature of tilS makes it a potential antibiotic target itself. Compounds that inhibit tilS activity could potentially sensitize Bordetella bronchiseptica to existing antibiotics by compromising its ability to synthesize resistance-related proteins accurately . This approach would represent a novel strategy in combating antibiotic resistance, particularly relevant for respiratory infections caused by Bordetella species.

What role does tilS play in the pathogenesis and host adaptation of Bordetella bronchiseptica?

The role of tilS in Bordetella bronchiseptica pathogenesis and host adaptation remains largely unexplored but potentially significant due to its fundamental impact on protein synthesis. Bordetella bronchiseptica, unlike its more host-restricted relatives B. pertussis and B. parapertussis, exhibits a broader host range and environmental adaptability, which may be partly enabled by precise regulation of protein synthesis during different infection phases . As tilS ensures accurate translation of the isoleucine codon AUA, it potentially influences the expression of virulence factors and host adaptation proteins where isoleucine content at AUA codons is critical to function.
Several virulence mechanisms in Bordetella species involve proteins for adhesion to ciliated epithelial cells, including filamentous hemagglutinin, pertactin, fimbriae, and various toxins . The accurate translation of these virulence factors depends on proper tRNA modification by tilS. Researchers should investigate whether the expression or activity of tilS changes during different stages of infection or in response to different host environments, possibly reflecting a regulatory mechanism for virulence. This could be studied using transcriptomic and proteomic analysis of bacteria isolated from different infection stages or growth conditions.
Additionally, comparison of tilS sequence conservation and activity across different Bordetella species with varying host ranges could reveal correlations between tilS properties and host adaptation capabilities. Experimental infection models using tilS-modulated strains (either through controlled expression systems or point mutations affecting activity but not viability) could help determine whether altered tilS function impacts colonization efficiency, persistence, or immune evasion. Such studies would provide valuable insights into the fundamental connection between basic cellular processes like tRNA modification and the complex phenomenon of bacterial pathogenesis and host adaptation.

How can computational approaches aid in predicting the impact of tilS mutations on enzyme function?

Computational approaches offer powerful tools for predicting the impact of tilS mutations on enzyme function, providing guidance for experimental studies and insights into evolutionary patterns. A comprehensive computational analysis should begin with homology modeling of Bordetella bronchiseptica tilS if a crystal structure is unavailable, using structures from related organisms like E. coli or Aquifex aeolicus as templates . Once a reliable structural model is established, several computational methods can be applied to predict mutation effects.
Molecular dynamics (MD) simulations can assess how specific mutations affect protein stability, flexibility, and conformational changes during catalysis. By comparing the dynamics of wild-type and mutant tilS over nano- to microsecond timescales, researchers can identify altered movement patterns that might impact function. Binding site analysis using docking simulations with substrates (tRNA, ATP, and lysine) can predict how mutations might alter substrate recognition and binding affinity . This is particularly valuable for mutations near the catalytic site or at substrate interaction interfaces.
Free energy calculations can quantify the energetic consequences of mutations on protein stability and substrate binding. Methods such as free energy perturbation (FEP) or thermodynamic integration (TI) provide estimates of ΔΔG values associated with mutations. Machine learning approaches trained on existing mutation-function data from related enzymes can provide rapid predictions for novel mutations. Sequence conservation analysis across bacterial species can identify highly conserved residues likely essential for function, with mutations at these positions predicted to be more detrimental .
To implement these approaches effectively, researchers should establish a mutation classification system predicting functional impacts (e.g., catalytic efficiency, substrate specificity, structural stability) and validate computational predictions with experimental measurements for a subset of mutations. This integrated computational-experimental approach can accelerate understanding of structure-function relationships in Bordetella bronchiseptica tilS and guide protein engineering efforts for research applications.

How has tilS evolved across bacterial species, and what does this reveal about its functional importance?

The evolutionary trajectory of tilS across bacterial species offers profound insights into its fundamental importance in bacterial physiology. Phylogenetic analysis reveals that tilS is nearly universally present in eubacteria, indicating its ancient origin and essential function in bacterial translation . This high conservation stands in contrast to the evolutionary plasticity observed in many other tRNA modification enzymes, underscoring the uniquely crucial role of lysidine modification in bacterial survival. Comparative genomic studies show that tilS homologs maintain significant sequence conservation in catalytic domains while exhibiting more variation in substrate recognition regions, reflecting adaptation to species-specific tRNA structures .
Interestingly, tilS appears to have undergone different selective pressures across bacterial lineages. In obligate intracellular bacteria with reduced genomes, tilS remains conserved despite extensive gene loss, highlighting its indispensable nature . Conversely, certain free-living bacteria show greater sequence divergence in tilS, potentially reflecting adaptation to different ecological niches or translation requirements. The Bordetella genus demonstrates interesting evolutionary patterns, with some species showing distinctive features in their tRNA recognition profiles compared to other bacteria . This suggests that even within a conserved essential function, tilS has undergone fine-tuning during bacterial evolution.
Coevolution analysis between tilS and its tRNA substrates reveals coordinated changes that maintain the critical enzyme-substrate interaction despite evolutionary drift. These complementary mutations in enzyme and substrate represent a fascinating example of molecular coevolution. Furthermore, the absence of eukaryotic homologs of tilS indicates that this modification system represents a bacterial-specific solution to the challenge of AUA codon recognition, making it both an interesting evolutionary case study and a potential antibiotic target . These evolutionary insights not only illuminate the history of bacterial translation systems but also provide context for understanding tilS function in contemporary bacterial species including Bordetella bronchiseptica.

How do differences in tRNA recognition by tilS impact bacterial adaptation to diverse environments?

The specialized tRNA recognition properties of tilS have profound implications for bacterial adaptation to diverse environments, representing a fascinating intersection of molecular mechanism and ecological strategy. The essential function of tilS in discriminating between tRNAIle and tRNAMet ensures accurate translation of AUA codons as isoleucine rather than methionine . This precision becomes particularly important under stressful environmental conditions where protein synthesis fidelity is critical for survival and adaptation. Differences in tRNA recognition by tilS across bacterial species, including the unique patterns observed in some Bordetella species, may reflect adaptations to specific environmental challenges .
In bacteria that must navigate diverse host environments, like Bordetella bronchiseptica, subtle variations in tilS-tRNA recognition could influence codon usage preferences during adaptation to different hosts or niches. Analysis of codon usage patterns in environmentally responsive genes could reveal correlations between AUA usage and habitat-specific protein requirements. The observed difficulty in classifying certain Bordetella bronchiseptica tRNAs due to their divergent features compared to related bacteria suggests unique evolutionary adaptations that may contribute to this pathogen's broad host range and environmental versatility .
Beyond codon usage, tilS-mediated translation fidelity likely impacts bacterial stress responses and environmental adaptation through several mechanisms. Accurate translation of stress-response proteins containing AUA-encoded isoleucines would be essential during temperature fluctuations, pH changes, nutrient limitation, or host immune pressures. Experimental approaches to investigate this connection could include measuring tilS expression and activity under various environmental stresses, assessing the environmental fitness of strains with modified tilS activity, and comparative genomic analysis of tilS and tRNA sequences from Bordetella isolates from different hosts or environments. Such studies would illuminate how a fundamental aspect of translation machinery contributes to the remarkable adaptability of bacterial pathogens across diverse ecological contexts.

What potential exists for developing tilS-targeted antimicrobial strategies against Bordetella bronchiseptica?

The essential nature of tilS for bacterial viability positions it as a promising target for novel antimicrobial strategies against Bordetella bronchiseptica and potentially other bacterial pathogens. Targeting tilS would represent an approach distinct from conventional antibiotics, potentially helping to address the growing challenge of antimicrobial resistance . Several lines of evidence support the feasibility of this approach. First, the essentiality of tilS has been demonstrated in multiple bacterial species, including the related organism B. subtilis, suggesting that inhibition would have lethal consequences for the pathogen . Second, the absence of tilS homologs in eukaryotes presents an opportunity for selectivity, potentially reducing off-target effects on host cells.
Development of tilS inhibitors could take multiple strategic approaches. Structure-based drug design utilizing crystallographic data or homology models of B. bronchiseptica tilS could identify compounds that bind to and inhibit the enzyme's active site. High-throughput screening of chemical libraries against purified recombinant tilS could identify lead compounds with inhibitory activity . The known catalytic flexibility of tilS with respect to lysine analogs suggests that substrate-competitive inhibitors might be a productive avenue for exploration . Mechanistic inhibitors could target the ATP-binding site, lysine-binding pocket, or tRNA interaction surface.
Several experimental approaches would be essential for evaluating potential inhibitors. In vitro enzyme assays measuring lysidine formation in the presence of candidate compounds would provide initial screening data . Cellular assays examining growth inhibition of B. bronchiseptica and cytotoxicity to mammalian cells would assess both efficacy and selectivity. Animal models of Bordetella infection would ultimately be needed to evaluate in vivo efficacy, pharmocokinetics, and toxicity profiles. While significant challenges exist in developing clinically viable tilS inhibitors, including optimization of cell permeability and metabolic stability, this approach represents an innovative direction for addressing respiratory infections caused by Bordetella species and potentially other bacterial pathogens.

How can biotechnological applications leverage the unique properties of tilS?

The unique catalytic properties and substrate specificity of tilS offer exciting opportunities for biotechnological applications beyond antimicrobial development. The enzyme's ability to perform site-specific modification of tRNA molecules with lysine or lysine analogs provides a powerful tool for synthetic biology and biotechnology . One of the most promising applications involves expanding the genetic code through the incorporation of non-canonical amino acids into proteins. By engineering the tilS-tRNA system, researchers could potentially create novel tRNAs capable of incorporating designer amino acids at specific codons, enabling the production of proteins with enhanced or entirely new functionalities .
The demonstrated catalytic flexibility of tilS with respect to lysine analogs is particularly valuable for this purpose. Research has shown that tilS can accept various lysine analogs and even simpler primary amines as alternative substrates for tRNA modification . This opens possibilities for creating tRNAs modified with a range of chemical groups, potentially expanding the chemical diversity accessible in protein engineering. Methodology for such applications would involve expressing recombinant tilS, generating in vitro transcribed tRNA substrates, performing modification reactions with selected lysine analogs, and verifying the modified tRNAs through analytical techniques such as mass spectrometry .
Another promising biotechnological application involves using tilS as a biocatalyst for the site-specific modification of RNA molecules beyond tRNAs. If the enzyme's substrate specificity could be engineered to accept designed RNA structures, it could potentially be used to introduce specific modifications at targeted positions in various RNA molecules, with applications in RNA therapeutics and synthetic biology. Additionally, the essential nature of tilS makes it a potential selection marker for bacterial expression systems, where functional tilS expression could complement tilS-deficient strains. These various biotechnological applications highlight how fundamental research on bacterial tRNA modification enzymes can lead to innovative tools for synthetic biology, protein engineering, and RNA therapeutics.

What are the most promising directions for structural studies of Bordetella bronchiseptica tilS?

Structural studies of Bordetella bronchiseptica tilS represent a critical frontier that could accelerate both fundamental understanding and applied research in this field. Despite its essential role in bacterial physiology, detailed structural information specifically for B. bronchiseptica tilS remains limited, presenting several high-priority research opportunities . The most promising approach would begin with obtaining a high-resolution crystal structure of B. bronchiseptica tilS, ideally in multiple states: the apo enzyme, enzyme bound to ATP and lysine, and the complete ternary complex with tRNA substrate. Such structures would reveal the molecular basis for catalysis and substrate recognition specific to this organism.
Given the challenges associated with crystallizing protein-RNA complexes, complementary structural biology techniques should be employed. Cryo-electron microscopy (cryo-EM) has emerged as a powerful method for visualizing macromolecular complexes and could be particularly valuable for capturing the tilS-tRNA interaction . Nuclear magnetic resonance (NMR) spectroscopy could provide insights into the dynamics of specific domains during catalysis, particularly for regions that may be flexible in crystal structures. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) could map conformational changes that occur upon substrate binding, providing insights into the enzyme's mechanism.
To understand the structural basis for the unique tRNA recognition properties observed in Bordetella species, comparative structural studies with tilS enzymes from other bacteria would be invaluable . This could include examining differences in the tRNA binding interface that might explain the distinctive tRNA discrimination patterns in Bordetella. Additionally, structural studies of tilS variants with mutations at key residues could connect sequence variations to functional differences. Time-resolved structural studies, if technically feasible, could capture the enzyme during the catalytic cycle, potentially revealing transient states critical for function. These structural insights would not only advance fundamental understanding of tilS biology but also provide crucial information for structure-based drug design efforts targeting this essential bacterial enzyme.