Recombinant Chlamydophila caviae Valine--tRNA ligase (valS), partial

What is the genomic context of the valS gene in Chlamydophila caviae?

Unlike some genes in C. caviae that show evidence of horizontal gene transfer, the valS gene is generally well-conserved due to its essential function in protein synthesis. Comparative genomic analyses using BLAST score ratio (BSR) techniques can identify the relative conservation of valS compared to orthologous genes in related species such as C. pneumoniae and C. muridarum .

How does the structure and function of C. caviae valS compare to other bacterial tRNA synthetases?

The Valine--tRNA ligase (valS) in C. caviae functions similarly to other bacterial aminoacyl-tRNA synthetases by catalyzing the attachment of valine to its cognate tRNA. While specific structural information for C. caviae valS is limited in the available literature, insights can be drawn from studies of related proteins. For instance, the valS gene from Lactobacillus casei encodes a protein of 901 amino acids that functionally complements temperature-sensitive growth in E. coli valS mutants .

Structurally, bacterial valS proteins typically contain several conserved domains: an N-terminal catalytic domain responsible for amino acid activation, a tRNA-binding domain that recognizes the appropriate tRNA^Val, and sometimes additional domains involved in editing functions to ensure translational fidelity. These structural features enable the enzyme to perform its critical function in protein biosynthesis by ensuring that valine is correctly attached to its cognate tRNA, thereby maintaining the accuracy of the genetic code translation.

What expression systems are most effective for producing recombinant C. caviae valS protein?

For recombinant expression of C. caviae valS, E. coli-based expression systems typically provide good yields and straightforward purification options. BL21(DE3) or similar strains carrying the pET vector system with a T7 promoter often produce satisfactory results for bacterial proteins. The effectiveness of expression can be enhanced by optimizing codon usage for E. coli, particularly given the differences in GC content between C. caviae (39%) and E. coli .

For functional studies, it may be beneficial to include affinity tags such as His6, which facilitates purification through nickel affinity chromatography. Temperature optimization is crucial, with lower temperatures (16-25°C) often favoring proper folding of complex proteins like tRNA synthetases. Expression screening should evaluate multiple conditions, including IPTG concentration (0.1-1.0 mM), induction time (3-18 hours), and growth media formulations to identify optimal parameters for both yield and activity.

When higher eukaryotic-like post-translational modifications are needed, systems such as insect cells (using baculovirus vectors) or mammalian cell lines may provide advantages, though with increased complexity and potential yield reductions compared to bacterial systems.

How can I design experiments to assess the enzymatic activity of recombinant C. caviae valS?

Designing robust enzymatic assays for recombinant C. caviae valS requires approaches that accurately measure its aminoacylation activity. The most direct method involves a two-step aminoacylation reaction that measures the attachment of radiolabeled valine to its cognate tRNA. In this assay, you would incubate purified recombinant valS with ATP, [14C]- or [3H]-labeled valine, and purified tRNA^Val (either isolated from C. caviae or produced through in vitro transcription). After the reaction, trichloroacetic acid precipitation captures the charged tRNAs, and scintillation counting quantifies the amount of labeled valine incorporated.

Alternative non-radioactive methods include the pyrophosphate release assay, where the pyrophosphate released during aminoacylation is coupled to enzymatic reactions that produce a colorimetric or fluorescent readout. For kinetic studies, stopped-flow techniques paired with fluorescently labeled tRNAs can monitor real-time changes in fluorescence as aminoacylation occurs. Additionally, thermal shift assays can provide valuable information about the stability of valS under various conditions, which is particularly relevant when assessing the impact of mutations or inhibitors.

When designing these experiments, controls should include reactions without enzyme, without substrate, and with known inhibitors of tRNA synthetases (such as non-hydrolyzable ATP analogs). Comparative analysis with valS enzymes from related species provides context for interpreting the kinetic parameters of C. caviae valS.

What approaches can be used to study the role of C. caviae valS in bacterial pathogenesis?

Investigating the role of valS in C. caviae pathogenesis requires integrating molecular genetics with infection models. One approach involves creating conditional knockdowns or temperature-sensitive mutants of valS, as complete deletion may be lethal given its essential function. This can be challenging in Chlamydia species due to their obligate intracellular lifestyle, but recent advances in genetic manipulation of Chlamydia make targeted genetic approaches increasingly feasible.

The guinea pig model provides an excellent system for studying C. caviae pathogenesis, as it closely resembles chlamydial infection in humans, particularly regarding transmission mechanisms and disease progression . In this model, intranasal or intravaginal challenge with C. caviae strains carrying valS mutations (if viable) or strains with altered valS expression could reveal the impact on infection establishment, bacterial burden, and upper reproductive tract pathology.

Additionally, in vitro infection systems using guinea pig or human cell lines can investigate the effects of valS inhibitors on chlamydial growth and development. These studies should monitor elementary body formation, inclusion size, and bacterial replication rates. Proteomic approaches comparing wild-type and valS-manipulated strains could identify downstream effects on the bacterial proteome, potentially revealing connections between translation fidelity and virulence factor expression.

How does the amino acid sequence of C. caviae valS compare with orthologs from other Chlamydiaceae species?

Comparative sequence analysis of valS across Chlamydiaceae species reveals both conserved domains crucial for aminoacylation function and species-specific variations that may reflect adaptation to different hosts or niches. While detailed sequence comparisons specific to valS are not provided in the search results, insights can be drawn from genome-wide comparative studies.

When analyzing the valS sequence across species, attention should be paid to: (1) the ATP-binding motifs essential for amino acid activation, (2) the amino acid binding pocket that determines specificity for valine, (3) the tRNA recognition elements that ensure correct tRNA selection, and (4) editing domains that prevent mischarging. Using tools like BLAST combined with structural modeling can highlight functionally significant differences between C. caviae valS and orthologs from human pathogens like C. trachomatis.

What methodologies can resolve contradictory data about C. caviae valS enzymatic properties?

When confronting contradictory data regarding C. caviae valS enzymatic properties, a multi-faceted approach combining biochemical, structural, and computational methods is necessary. Begin by rigorously standardizing experimental conditions across laboratories, as variations in buffer composition, pH, temperature, and metal ion concentrations significantly impact aminoacyl-tRNA synthetase activity. Implement enzyme preparations of defined purity using multiple orthogonal purification techniques followed by analytical size exclusion chromatography to ensure conformational homogeneity.

Structural studies provide critical context for interpreting contradictory kinetic data. X-ray crystallography of C. caviae valS in various states (apo, bound to ATP, bound to valine, and bound to tRNA) can reveal conformational changes during catalysis. Where crystal structures prove challenging, hydrogen-deuterium exchange mass spectrometry (HDX-MS) offers insights into protein dynamics and ligand-induced conformational changes.

For complex kinetic discrepancies, pre-steady-state kinetics using rapid quench-flow or stopped-flow techniques can deconvolute multi-step enzymatic mechanisms, resolving contradictions in steady-state measurements. Additionally, single-molecule studies using fluorescence resonance energy transfer (FRET) between labeled valS and tRNA can directly visualize the conformational dynamics during aminoacylation at unprecedented resolution.

Computational approaches including molecular dynamics simulations can bridge experimental techniques by modeling how sequence variations affect enzyme behavior under different conditions. This integrated approach ensures that contradictions are systematically addressed rather than simply averaged or dismissed.

How might directed evolution be employed to engineer C. caviae valS with modified substrate specificity?

Directed evolution of C. caviae valS to alter its substrate specificity requires a sophisticated experimental pipeline combining random mutagenesis with powerful selection strategies. The process begins with creating a diverse valS mutant library using error-prone PCR, DNA shuffling, or site-saturation mutagenesis targeting the amino acid binding pocket. For optimal library diversity, multiple rounds of mutagenesis with controlled mutation rates (2-5 mutations per gene) provide a balance between innovation and maintenance of basic enzyme function.

The critical challenge lies in selection system design. One effective approach involves complementation of an E. coli valS temperature-sensitive strain, similar to methods used with L. casei valS . By supplementing the growth medium with valine analogs while limiting natural valine, selection pressure favors valS variants that can utilize the non-canonical amino acid. Alternatively, a positive selection system could link cell survival to the incorporation of a valine analog at specific positions in essential proteins.

For high-throughput screening approaches, fluorescence-activated cell sorting (FACS) can be employed by linking valS activity to fluorescent reporter expression. This typically requires a secondary reporter system where successful charging of tRNA with the target amino acid allows read-through of a premature stop codon in the fluorescent protein gene.

After isolation of promising variants, detailed biochemical characterization must include:

• Kinetic parameters (kcat, KM) for both natural and target substrates

• Fidelity measurements to ensure accurate tRNA charging

• Structural analysis to understand the molecular basis for the altered specificity

This iterative process of mutagenesis, selection, and characterization typically requires 5-10 rounds of evolution to achieve significant changes in substrate specificity while maintaining catalytic efficiency.

What are the methodological considerations for studying the potential role of valS in modulating C. caviae stress responses?

Investigating valS involvement in stress responses requires sophisticated methodologies that link translation to broader bacterial physiology. Begin by establishing stress response models for C. caviae using defined stressors relevant to its lifecycle: oxidative stress (H₂O₂ or superoxide generators), nutrient limitation, temperature shifts, and host immune factors. For each condition, monitor valS expression using RT-qPCR with carefully validated reference genes stable under stress conditions.

To establish causality between valS modulation and stress response, develop inducible expression systems for C. caviae that allow controlled overexpression or depletion of valS. While genetic manipulation of Chlamydia species is challenging, recent advances permit conditional gene expression. Alternatively, chemical genetics approaches using small-molecule inhibitors with varying selectivity for valS can provide mechanistic insights when genetic approaches prove difficult.

Proteomic analyses using stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry can quantify global changes in protein synthesis during stress with and without valS modulation. This should be complemented by ribosome profiling to examine translation efficiency across the transcriptome, potentially revealing valS-dependent translational recoding during stress.

For in vivo relevance, the guinea pig infection model offers a system to examine how valS modulation affects C. caviae survival during host-induced stress . Samples collected at defined timepoints post-infection can be analyzed for bacterial load, valS expression, and stress response markers.

The relationship between valS and the stringent response deserves particular attention, focusing on whether altered valS activity influences (p)ppGpp synthesis and subsequent stress adaptation. This connection can be examined through quantification of charged and uncharged tRNA^Val pools during stress, combined with metabolomic analysis of alarmone levels.

What purification strategies yield the highest activity of recombinant C. caviae valS?

Obtaining high-activity recombinant C. caviae valS requires a purification strategy that preserves the native conformation and enzymatic function. A recommended multi-step approach begins with affinity chromatography, typically using an N-terminal His6-tag and nickel-NTA resin, which provides good initial purification. Critical buffer components during this stage include 20-50 mM HEPES or Tris-HCl (pH 7.5-8.0), 100-300 mM NaCl, 5-10% glycerol for stability, and 5-10 mM β-mercaptoethanol or DTT to maintain reduced cysteines.

Following initial capture, ion exchange chromatography (typically on a Q-Sepharose column) can separate differentially charged variants and remove contaminants with similar affinity for the initial resin. The final polishing step should employ size exclusion chromatography to isolate monomeric, properly folded valS and remove aggregates or degradation products. Throughout purification, samples should be maintained at 4°C and handled rapidly to minimize activity loss.

Activity preservation requires special attention to several factors:

• Metal ions: Include 1-5 mM MgCl₂ in all buffers as magnesium is essential for aminoacyl-tRNA synthetase activity

• ATP: Consider adding 0.1-0.5 mM ATP to stabilize the enzyme's active conformation

• Protease inhibitors: Use a complete protease inhibitor cocktail during initial lysis and early purification steps

• Storage: Flash-freeze purified enzyme in small aliquots with 20-25% glycerol and store at -80°C to prevent freeze-thaw cycles

Enzyme quality should be verified through multiple analytical methods including SDS-PAGE for purity (>95%), dynamic light scattering for monodispersity, and thermal shift assays to confirm proper folding. Activity assays comparing fresh and stored enzyme preparations help establish optimal storage conditions for maintaining catalytic function.

How can researchers effectively analyze the interaction between C. caviae valS and its cognate tRNA?

For more precise thermodynamic parameters, isothermal titration calorimetry (ITC) directly measures heat changes during binding, providing enthalpy (ΔH), entropy (ΔS), and Gibbs free energy (ΔG) values alongside the Kd. Surface plasmon resonance (SPR) offers an alternative approach for real-time binding kinetics, where immobilized valS or tRNA on a sensor chip allows determination of association (kon) and dissociation (koff) rate constants.

Structural insights into the interaction can be gained through several complementary techniques:

• Chemical footprinting using reagents like dimethyl sulfate (DMS) can identify nucleotides in tRNA^Val protected upon valS binding

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) reveals regions of valS that undergo conformational changes upon tRNA binding

• Cryo-electron microscopy can visualize the complete valS-tRNA complex, particularly advantageous when crystallization proves challenging

• Small-angle X-ray scattering (SAXS) provides low-resolution structural information about the complex in solution

For functional analysis, aminoacylation assays measuring the rate of valine attachment to tRNA^Val should be conducted with wild-type and mutant forms of both valS and tRNA^Val. This structure-function correlation helps identify critical residues and nucleotides in the recognition interface.

What are the key technical challenges in studying valS function in the context of C. caviae infection models?

Studying valS function during active C. caviae infection poses significant technical challenges stemming from the bacterium's obligate intracellular lifestyle. Primary among these is the difficulty of genetic manipulation of Chlamydia species, though recent advances have improved transformation efficiencies. Targeted mutagenesis of valS is particularly challenging as it is likely essential for bacterial survival, necessitating conditional approaches such as inducible expression systems or temperature-sensitive mutants.

The guinea pig model, while physiologically relevant for studying C. caviae infections as it closely resembles human chlamydial infections, adds complexity to experimental design and analysis . When using this model, researchers must consider:

• Variability in infection progression between individual animals

• Ethical considerations limiting sample sizes and experimental interventions

• Technical challenges in isolating sufficient bacterial material from infected tissues for molecular analysis

For in vitro studies, maintaining the developmental cycle of C. caviae in cell culture requires careful optimization of infection protocols. The biphasic lifecycle involving infectious elementary bodies (EBs) and replicative reticulate bodies (RBs) complicates the timing of experimental interventions targeting valS. Synchronization of infection is critical for reproducible results, particularly when studying temporal aspects of valS expression and function.

Isolating and analyzing valS-specific effects presents another significant challenge as aminoacyl-tRNA synthetases are integrated into complex cellular networks. Approaches to address this include:

• Using specific inhibitors of valS with minimal off-target effects

• Employing ribosome profiling to identify translational changes specifically at valine codons

• Developing reporter systems that selectively respond to changes in tRNA^Val charging levels

Finally, the host-pathogen interface adds complexity, as host cells may influence bacterial translation processes through nutrient limitation, oxidative stress, or direct targeting of bacterial components. Disentangling these interactions requires sophisticated co-culture systems and careful experimental controls.

How does C. caviae valS compare functionally to the orthologous enzyme in C. trachomatis?

Functional comparison between C. caviae valS and its C. trachomatis ortholog reveals insights into both conserved aminoacylation mechanisms and species-specific adaptations. While the search results don't provide direct comparative data on these specific enzymes, extrapolation from genome-wide studies and related tRNA synthetases suggests several key points of comparison.

The core catalytic functions of valS are likely highly conserved between these species, reflecting the essential nature of accurate tRNA charging for protein synthesis. Both enzymes would share the fundamental two-step reaction mechanism: first activating valine with ATP to form valyl-adenylate, then transferring the activated amino acid to the 3' end of tRNA^Val. Kinetic parameters such as kcat and KM for shared substrates (ATP, valine, and tRNA^Val) would be expected to show similarity, though potentially with optimizations reflecting the different growth temperatures and host environments of each species.

More significant differences may appear in regulatory features and responses to environmental stressors. C. trachomatis, as a human pathogen, may have evolved specific adaptations in valS to respond to host-induced stresses such as iron limitation or interferon-gamma exposure. In contrast, C. caviae valS may be optimized for the guinea pig host environment, where different immune responses and nutrient availabilities prevail .

The genome sequence of C. caviae reveals both highly conserved regions and species-specific adaptations compared to other Chlamydiaceae . Detailed sequence analysis of the valS genes from both species, combined with structural modeling and biochemical characterization, would be necessary to fully elucidate the functional implications of any sequence divergence.

What experimental approaches best demonstrate the specificity of C. caviae valS compared to other aminoacyl-tRNA synthetases?

Demonstrating the specificity of C. caviae valS requires methodologies that examine both its amino acid and tRNA selection fidelity. A comprehensive approach begins with in vitro mischarging assays, where purified valS is incubated with various amino acids (particularly those structurally similar to valine, such as isoleucine, leucine, and threonine) and tRNA^Val. Liquid chromatography-mass spectrometry (LC-MS) analysis of the aminoacylation products can quantify the relative incorporation of different amino acids, establishing the amino acid discrimination capacity of valS.

tRNA specificity can be evaluated through competitive binding assays where valS is incubated with its cognate tRNA^Val and other tRNA species at varying ratios. Techniques such as filter binding assays or electrophoretic mobility shift assays (EMSAs) can determine relative binding affinities, while aminoacylation assays with mixed tRNA populations measure functional selectivity.

For more detailed structural insights, X-ray crystallography or cryo-electron microscopy of valS in complex with valine, non-cognate amino acids, and various tRNAs can reveal the molecular determinants of specificity. Complementary approaches include:

• Molecular dynamics simulations to examine the energetics of binding different substrates

• Site-directed mutagenesis of predicted specificity-determining residues followed by kinetic analysis

• In vivo complementation assays testing whether C. caviae valS can rescue growth of E. coli with temperature-sensitive mutations in valS or other aminoacyl-tRNA synthetase genes

How does research on C. caviae valS contribute to understanding broader tRNA synthetase evolution in bacterial pathogens?

Research on C. caviae valS offers a valuable window into the evolutionary trajectories of tRNA synthetases in bacterial pathogens, particularly those with specialized intracellular lifestyles. The Chlamydiaceae family, with its reduced genome size of approximately 1.17 Mb compared to free-living bacteria, provides insights into how essential translation machinery adapts during genome streamlining . Comparative genomic analyses revealed that of the 1,009 annotated genes in C. caviae, 798 are conserved across all sequenced Chlamydiaceae genomes, suggesting these represent the core functions necessary for the chlamydial lifestyle .

By examining the sequence, structure, and function of valS across multiple chlamydial species that infect different hosts (C. caviae in guinea pigs versus C. trachomatis in humans), researchers can identify signature adaptations that reflect host-specific selective pressures. These adaptations may manifest as changes in substrate affinity, temperature optima, or regulatory responses that optimize valS function in different intracellular environments.

The evolution of aminoacyl-tRNA synthetases is particularly intriguing due to their ancient origins and central role in the translation apparatus. Phylogenetic analysis of valS sequences across bacterial pathogens can reveal horizontal gene transfer events, gene duplication and specialization, and convergent evolution in response to similar selective pressures. Such analyses benefit from the complete genome sequence of C. caviae, which facilitates accurate placement of valS in its genomic context and identification of syntenic relationships with other species .

The replication termination region (RTR) or plasticity zone of the C. caviae genome has been identified as a hotspot for genome variation, containing several genes of particular interest including toxin genes and evidence of ancestral bacteriophage insertions . Whether valS shows any relationship to genes in this region could provide insights into the coevolution of translation machinery with virulence factors.