Recombinant Coxiella burnetii Probable tRNA sulfurtransferase (thiI)

What is the primary function of thiI in Coxiella burnetii?

ThiI in C. burnetii, similar to its homolog in E. coli, likely functions as a tRNA sulfurtransferase involved in the formation of 4-thiouridine (s⁴U) at position 8 of tRNA molecules. In bacterial systems, thiI typically catalyzes the transfer of sulfur from L-cysteine to specific tRNA molecules through a two-step mechanism involving an adenylated intermediate. The enzyme contains a PP-loop motif characteristic of the ATP-pyrophosphatase family and utilizes ATP to activate the target uridine for subsequent thiolation . This modification enhances tRNA stability and may play roles in translational fidelity and the cellular response to environmental stresses.

How does the structure of C. burnetii thiI compare to characterized homologs?

While the specific crystal structure of C. burnetii thiI has not been directly reported in the provided search results, structural predictions can be made based on characterized homologs. The enzyme likely consists of multiple domains including a PP-loop-containing catalytic domain that binds ATP and the target tRNA, and potentially a rhodanese-like domain involved in sulfur mobilization. Like other thiouridylases, C. burnetii thiI probably contains conserved cysteine residues essential for persulfide formation and sulfur transfer. These may be functionally analogous to the conserved Cys199 and Cys102 residues identified in E. coli MnmA .

What role might thiI play in C. burnetii pathogenesis?

C. burnetii is a highly infectious bacterium capable of causing Q fever in humans . As an intracellular pathogen that replicates within modified phagocytic vacuoles , C. burnetii must adapt to environmental stresses within host cells. ThiI-mediated tRNA modifications may contribute to pathogen survival by enhancing translational efficiency under stress conditions encountered during infection. The enzyme could be particularly important during the transition from the metabolically dormant small cell variant (SCV) to the metabolically active large cell variant (LCV) of C. burnetii, when rapid protein synthesis is required for replication within the Coxiella-containing vacuole.

What expression systems are most effective for producing recombinant C. burnetii thiI?

For recombinant expression of C. burnetii thiI, E. coli-based systems are typically suitable with important modifications to account for potential toxicity and proper folding. Expression constructs should include a 6xHis or similar affinity tag for purification, preferably with a cleavable linker to remove the tag if it interferes with activity assays. For optimal expression:

• Use E. coli BL21(DE3) or Rosetta strains to accommodate potential rare codons in C. burnetii sequences

• Express at lower temperatures (16-20°C) after induction to enhance proper folding

• Include 0.1-0.5 mM IPTG for induction, with culture optical density at 0.6-0.8

• Supplement growth media with iron and cysteine if the protein binds an Fe-S cluster, as observed in some tRNA sulfurtransferases

• Consider anaerobic conditions during purification if Fe-S cluster stability is a concern

How can researchers effectively assay the enzymatic activity of recombinant C. burnetii thiI?

A comprehensive thiI activity assay should include multiple validation approaches:

Gel-based mobility shift assay:

• Prepare in vitro transcribed tRNA substrates or isolate bulk tRNA from an E. coli ΔthiI strain

• Incubate purified recombinant thiI with tRNA substrates, ATP, Mg²⁺, and a sulfur source (Na₂S or L-cysteine with IscS)

• Analyze products using denaturing urea PAGE containing N-acryloylaminophenylmercuric chloride (APM), which specifically retards migration of sulfur-containing tRNAs

HPLC-MS/MS nucleoside analysis:

• Following the enzymatic reaction, digest tRNA products to individual nucleosides

• Analyze by HPLC-MS/MS using s⁴U as a standard

• Quantify the formation of modified nucleosides compared to appropriate controls (no enzyme, no ATP, no sulfur source)

Controls should include:

• Reaction without enzyme

• Reaction with heat-inactivated enzyme

• Reaction omitting individual components (ATP, Mg²⁺, sulfur source)

• Comparison of holo-enzyme (with Fe-S cluster if required) versus apo-enzyme

What strategies can be employed to investigate the requirement of [4Fe-4S] clusters in C. burnetii thiI?

To determine whether C. burnetii thiI requires a [4Fe-4S] cluster for activity, researchers should:

• Reconstitution experiments:

  • Express recombinant protein under anaerobic conditions

  • Reconstitute with iron and sulfide in the presence of reducing agents

  • Measure iron and sulfide content using colorimetric assays

  • Perform UV-visible spectroscopy to detect the characteristic [4Fe-4S] absorbance around 390-420 nm

• Site-directed mutagenesis:

  • Identify conserved cysteine residues potentially involved in cluster coordination

  • Generate alanine substitution variants

  • Evaluate cluster binding and enzymatic activity of mutants

• EPR spectroscopy:

  • Perform electron paramagnetic resonance to characterize the nature of any Fe-S clusters

  • Compare spectra of oxidized and reduced forms of the enzyme

Evidence from related tRNA sulfurtransferases suggests that certain archaeal U8-tRNA sulfurases require [4Fe-4S] clusters for activity, while E. coli MnmA operates through a persulfide mechanism without Fe-S clusters . Characterization of C. burnetii thiI could reveal whether it follows the Fe-S cluster-dependent or independent pathway for tRNA thiolation.

What is the proposed catalytic mechanism of C. burnetii thiI?

Based on mechanistic studies of related tRNA sulfurtransferases, C. burnetii thiI likely follows a two-step reaction mechanism:

• Step 1: Activation - ThiI utilizes ATP to activate the target uridine at position 8 of tRNA, forming an adenylated tRNA intermediate. This reaction involves the PP-loop motif, which is characteristic of the ATP-pyrophosphatase family .

• Step 2: Sulfur transfer - The activated uridine undergoes nucleophilic attack by a sulfur species, which could be:

  • A persulfide formed on a conserved cysteine residue of thiI (similar to E. coli)

  • A [4Fe-4S] cluster-bound sulfide (as observed in some archaeal sulfurtransferases)

The source of sulfur is likely mobilized from L-cysteine through the action of a cysteine desulfurase such as IscS. The specific sulfur transfer pathway may involve direct interaction between thiI and IscS or may require intermediate sulfur carrier proteins, depending on whether C. burnetii utilizes a mechanism more similar to E. coli or alternative bacterial systems .

How does the sulfur mobilization pathway in C. burnetii compare to model systems like E. coli?

While specific details of the C. burnetii sulfur mobilization pathway are not explicitly described in the search results, comparative analysis with E. coli provides a framework for investigation:

E. coli pathway components:

• Initial sulfur mobilization from L-cysteine by IscS cysteine desulfurase

• Transfer via sulfur carrier proteins (TusA, TusBCD, TusE) in some thiolation pathways

• Final transfer to the tRNA via the catalytic enzyme (MnmA for 2-thiouridine or ThiI for 4-thiouridine)

The C. burnetii genome likely encodes homologs of these proteins, but the specific requirement for intermediate sulfur carriers may differ. For instance, some bacterial species like Bacillus subtilis can transfer sulfur directly from the cysteine desulfurase to the thiouridylase without intermediate carriers . Genomic analysis of C. burnetii should reveal which components of the sulfur relay system are present, informing experimental designs to characterize the pathway.

What regulatory mechanisms might control thiI activity in C. burnetii during infection?

Regulation of thiI activity in C. burnetii during infection likely responds to environmental stresses encountered within the host cell. Potential regulatory mechanisms include:

• Transcriptional regulation:

  • Expression levels may change in response to oxidative stress, pH changes, or nutrient limitation

  • Analysis of transcriptome data from C. burnetii isolated from infected cells (like those from Galleria mellonella hemocytes ) could reveal expression patterns

• Post-translational modifications:

  • Activity may be modulated by oxidation/reduction of critical cysteine residues

  • Phosphorylation or other modifications might regulate enzyme activity or stability

• Substrate availability:

  • Availability of ATP, which may be limited in the intracellular environment

  • Accessibility of target tRNAs, which may be sequestered during stress responses

  • Supply of sulfur, which might be restricted during host-imposed nutritional immunity

• Fe-S cluster status:

  • If C. burnetii thiI requires an Fe-S cluster, its assembly/disassembly could serve as a regulatory mechanism responsive to iron availability and oxidative stress

Understanding these regulatory mechanisms would provide insights into how C. burnetii adapts its translational machinery during different phases of infection and could reveal potential targets for therapeutic intervention.

How does C. burnetii thiI differ from E. coli thiI in terms of substrate specificity?

E. coli thiI is known to specifically catalyze the formation of 4-thiouridine (s⁴U) at position 8 of tRNA molecules. While specific substrate characterization data for C. burnetii thiI is not directly provided in the search results, several comparative aspects warrant investigation:

• tRNA recognition elements:

  • C. burnetii thiI may recognize different structural features in tRNA compared to E. coli thiI

  • The enzyme may have evolved specificity for tRNAs with sequences optimized for C. burnetii's codon usage patterns

• Position specificity:

  • While E. coli thiI targets U8, C. burnetii thiI should be tested for activity at this and potentially other positions

  • Some tRNA sulfurtransferases have evolved to modify different positions within tRNA molecules

• Nucleoside preference:

  • C. burnetii thiI should be assayed for its ability to form different thiolated nucleosides beyond s⁴U

  • Testing with various tRNA substrates would reveal whether the enzyme has broader or narrower specificity than its E. coli counterpart

Experimental approaches to determine substrate specificity should include in vitro modification assays using different tRNA substrates, followed by nucleoside analysis to identify the specific modifications introduced.

How does the evolutionary conservation of thiI across bacterial pathogens inform functional studies?

Phylogenetic analysis of thiI across bacterial pathogens provides context for functional studies of the C. burnetii enzyme:

This evolutionary perspective can guide the design of functional studies by highlighting specific features unique to C. burnetii thiI that may be particularly relevant to its pathogenic lifestyle.

How might genetic manipulation of thiI inform C. burnetii pathogenesis studies?

Genetic manipulation of thiI in C. burnetii could provide valuable insights into pathogenesis through several approaches:

• Gene knockout/knockdown studies:

  • Construction of a thiI deletion mutant or conditional expression strain

  • Evaluation of growth kinetics in axenic media and cell culture systems

  • Assessment of virulence in infection models like Galleria mellonella or mammalian cells

  • Analysis of transcriptome and proteome changes in thiI-deficient strains

• Site-directed mutagenesis:

  • Generation of point mutations in catalytic residues to create enzymatically inactive variants

  • Creation of chimeric proteins with domains from other bacterial thiI proteins to assess domain functions

  • Introduction of tagged versions for localization studies within bacterial cells

• Overexpression studies:

  • Analysis of the effects of thiI overexpression on tRNA modification levels

  • Assessment of whether increased thiI activity enhances stress resistance or virulence

• Application to infection models:

  • Utilization of established C. burnetii infection models, including Galleria mellonella , to assess the role of thiI in pathogenesis

  • Comparative analysis of wild-type and thiI-mutant strains in terms of replication efficiency and host responses

These approaches would help define the contribution of tRNA sulfurtransferase activity to C. burnetii's ability to establish and maintain infection within host cells.

What potential does C. burnetii thiI hold as a therapeutic target for Q fever?

The evaluation of C. burnetii thiI as a potential therapeutic target requires consideration of several factors:

• Essentiality assessment:

  • Determination of whether thiI is essential for C. burnetii growth or virulence

  • Validation through genetic approaches (see 5.1) or chemical inhibition studies

• Structural uniqueness:

  • Identification of structural features unique to C. burnetii thiI compared to host enzymes

  • Analysis of active site architecture to identify "druggable" pockets

• Inhibitor development strategy:

  • Design of high-throughput screening assays to identify small molecule inhibitors

  • Structure-based drug design approaches if crystal structures become available

  • Repurposing of known inhibitors of related enzymes from other bacterial systems

• Therapeutic considerations:

  • Evaluation of whether thiI inhibition would be bactericidal or bacteriostatic

  • Assessment of potential for resistance development

  • Consideration of pharmacokinetic requirements for compounds to reach intracellular bacteria

The highly infectious nature of C. burnetii and its intracellular lifestyle make effective treatments challenging. If thiI proves to be essential for bacterial survival or virulence, targeting this enzyme could offer a new approach to treating Q fever infections, particularly chronic cases that respond poorly to current therapies.

How might tRNA modification patterns change during different phases of C. burnetii infection?

C. burnetii undergoes significant physiological changes during its developmental cycle and in response to host environments. The dynamics of tRNA modifications catalyzed by thiI during infection may reveal important aspects of bacterial adaptation:

• Developmental regulation:

  • Comparison of tRNA modification patterns between small cell variants (SCVs) and large cell variants (LCVs)

  • Analysis of whether thiI expression and activity changes during the transition between these forms

• Response to host cell environment:

  • Examination of tRNA modifications under conditions mimicking the acidified phagolysosome

  • Assessment of changes in response to oxidative stress, nutrient limitation, or other host defense mechanisms

• Temporal dynamics during infection:

  • Time-course analysis of tRNA modification patterns throughout the intracellular replication cycle

  • Correlation with changes in bacterial gene expression and protein synthesis rates

• Methodological approaches:

  • RNA-seq techniques adapted for tRNA modification mapping

  • Mass spectrometry-based methods for comprehensive tRNA modification analysis

  • Reporter systems to monitor thiI activity in living bacteria during infection

Understanding these dynamics could reveal how C. burnetii fine-tunes its translational machinery during different stages of infection, potentially identifying critical periods when the bacterium might be most vulnerable to therapeutic intervention.

What are the major challenges in purifying active recombinant C. burnetii thiI?

Several technical challenges may arise when purifying active recombinant C. burnetii thiI:

• Protein solubility issues:

  • C. burnetii proteins may form inclusion bodies when overexpressed in E. coli

  • Solution: Optimize expression conditions (lower temperature, reduced inducer concentration), use solubility-enhancing fusion tags (SUMO, MBP), or explore alternative expression hosts

• Fe-S cluster instability:

  • If C. burnetii thiI contains an Fe-S cluster like some archaeal homologs , it may be oxygen-sensitive

  • Solution: Perform purification under anaerobic conditions, include reducing agents (DTT, β-mercaptoethanol), and consider reconstitution of the Fe-S cluster post-purification

• Contaminating activities:

  • E. coli expression hosts contain endogenous tRNA modification enzymes that may co-purify

  • Solution: Design purification schemes with multiple chromatography steps and validate enzyme purity by mass spectrometry

• Loss of cofactors:

  • Essential cofactors may dissociate during purification

  • Solution: Supplement purification buffers with stabilizing agents and analyze cofactor content of purified enzyme

• Post-translational modifications:

  • C. burnetii may utilize specific post-translational modifications not reproduced in E. coli

  • Solution: Consider expression in more closely related hosts or cell-free systems derived from C. burnetii extracts

Addressing these challenges requires systematic optimization of expression and purification protocols, with regular activity assays to confirm retention of enzymatic function.

What experimental controls are essential when studying the cellular impacts of thiI modification?

Rigorous experimental controls are critical when investigating the cellular impacts of thiI-mediated tRNA modifications:

For genetic studies:

• Complementation controls: Reintroduction of wild-type thiI into knockout strains to verify phenotype rescue

• Catalytically inactive mutants: Point mutations in catalytic residues to distinguish enzymatic from structural roles

• Conditional expression systems: To control the timing and level of thiI expression

For biochemical analyses:

• Substrate controls: Unmodified tRNA prepared under identical conditions

• Enzyme controls: Heat-inactivated enzyme and catalytically inactive mutants

• Reaction component controls: Omission of ATP, sulfur source, or other cofactors

For phenotypic studies:

• Growth condition controls: Standard vs. stress conditions to reveal condition-specific effects

• Comparative controls: Related but distinct tRNA modification enzymes to identify thiI-specific effects

• Host cell controls: Uninfected cells and cells infected with wild-type bacteria for comparison with thiI mutants

For infection models:

• Dose-matched controls: Ensuring equal initial bacterial loads

• Time-matched sampling: Collecting samples at consistent time points post-infection

• Host variation controls: Using sufficient biological replicates to account for host variability

How should researchers interpret heterogeneity in tRNA modification patterns?

When analyzing tRNA modification patterns in C. burnetii, researchers may encounter heterogeneity that requires careful interpretation:

• Sources of heterogeneity:

  • Partial modification: Not all tRNA molecules of a given type may be modified

  • Mixed modifications: Different modifications may occur at the same position in different tRNA molecules

  • Condition-dependent changes: Modification patterns may vary with growth conditions

• Analytical approaches:

  • Quantitative analysis: Determine the proportion of modified vs. unmodified tRNAs

  • Position-specific analysis: Map modifications to specific positions within tRNA sequences

  • Isoacceptor-specific analysis: Compare modification patterns across different tRNAs

• Interpretation framework:

  • Regulatory significance: Heterogeneity may reflect regulatory mechanisms rather than inefficient modification

  • Functional consequences: Partial modification may enable fine-tuning of translation

  • Technical artifacts: Distinguish genuine heterogeneity from method-induced variability

• Validation strategies:

  • Multiple detection methods: Combine approaches (e.g., APM gel shift , HPLC-MS/MS , RNA-seq)

  • Biological replicates: Establish reproducibility across independent samples

  • Controlled modification: In vitro modification of tRNA to generate standards

Understanding heterogeneity in tRNA modification patterns can provide insights into how C. burnetii adapts its translational machinery to different environments, potentially revealing mechanisms that contribute to pathogen survival during infection.

What statistical approaches are appropriate for analyzing thiI-dependent phenotypes?

Proper statistical analysis is crucial for interpreting phenotypes associated with thiI function or mutation:

• For growth and survival studies:

  • Repeated measures ANOVA for growth curves

  • Log-rank test for survival analysis

  • Mixed effects models to account for experimental batch effects

• For gene expression studies:

  • Differential expression analysis with multiple testing correction

  • Enrichment analysis for pathway and functional impacts

  • Time-series analysis for dynamic expression changes

• For infection models:

  • Power analysis to determine appropriate sample sizes

  • Non-parametric tests if normality assumptions are violated

  • Statistical controls for host variability factors

• For biochemical activity assays:

  • Enzyme kinetics modeling (Michaelis-Menten, allosteric models)

  • Replicate averaging with standard error reporting

  • Comparison of initial rates rather than endpoint measurements

• For high-throughput data:

  • Dimensionality reduction techniques to identify patterns

  • Clustering approaches to group similar phenotypes

  • Machine learning models to predict thiI-dependent effects

The appropriate statistical approach depends on the specific experimental design and data type, but should generally include:

• Proper replication (minimum n=3, preferably more)

• Appropriate controls as described in section 6.2

• Clear reporting of statistical methods and significance thresholds

• Visualization that accurately represents the data and its variability

What emerging technologies might advance our understanding of C. burnetii thiI function?

Several emerging technologies hold promise for deepening our understanding of C. burnetii thiI:

• CRISPR-Cas9 genome editing in C. burnetii:

  • Precise genetic manipulation to create clean deletions or point mutations

  • CRISPRi approaches for conditional knockdown studies

  • CRISPR-based screens to identify genetic interactions with thiI

• Advanced structural biology techniques:

  • Cryo-electron microscopy to visualize thiI-tRNA complexes

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic protein regions

  • Single-molecule FRET to observe conformational changes during catalysis

• Transcriptome-wide tRNA modification mapping:

  • Nanopore direct RNA sequencing to detect modifications without chemical treatments

  • NAIL-MS (Nucleic Acid Isotope Labeling coupled with Mass Spectrometry) for dynamic modification analysis

  • RiboMeth-seq adaptations for mapping thiolation sites

• In situ techniques:

  • Proximity labeling approaches to identify thiI interaction partners in living bacteria

  • Live-cell imaging of fluorescently tagged thiI to track localization during infection

  • Bio-orthogonal labeling of modified tRNAs to track their fate in cells

• Host-pathogen interaction models:

  • Organoid systems to model tissue-specific aspects of C. burnetii infection

  • Humanized mouse models to better reflect human Q fever pathology

  • Multi-omics approaches to integrate transcriptomic, proteomic, and metabolomic data

These technologies could help resolve outstanding questions about thiI function, regulation, and contribution to C. burnetii pathogenesis, potentially opening new avenues for therapeutic intervention in Q fever.

How might comparative studies across bacterial species inform our understanding of thiI evolution?

Comparative studies of thiI across bacterial species can provide valuable evolutionary insights:

• Phylogenetic analysis:

  • Reconstruction of thiI evolutionary history across bacterial lineages

  • Identification of conserved vs. rapidly evolving regions

  • Detection of horizontal gene transfer events that may have shaped thiI function

• Structure-function relationships:

  • Mapping of sequence conservation onto structural models to identify functional hotspots

  • Correlation of sequence variations with differences in substrate specificity or catalytic mechanism

  • Identification of lineage-specific insertions or deletions that may confer specialized functions

• Coevolution with tRNA substrates:

  • Analysis of whether thiI evolution correlates with changes in tRNA sequence or structure

  • Investigation of whether thiI adaptations reflect changes in translation or codon usage

• Adaptation to ecological niches:

  • Comparison of thiI from intracellular pathogens vs. free-living bacteria

  • Correlation of thiI features with environmental factors (temperature, pH, oxygen availability)

  • Analysis of whether pathogenic bacteria show distinctive patterns of thiI evolution