Recombinant Legionella pneumophila subsp. pneumophila Tyrosine--tRNA ligase (tyrS)

What is the primary function of tyrS in Legionella pneumophila?

Tyrosine--tRNA ligase (tyrS) in Legionella pneumophila is an essential enzyme that catalyzes the attachment of tyrosine to tRNA(Tyr) in a two-step reaction: tyrosine is first activated by ATP to form Tyr-AMP and then transferred to the acceptor end of tRNA(Tyr) . This aminoacylation process is crucial for protein synthesis and bacterial survival. As part of the translation machinery, tyrS ensures the correct incorporation of tyrosine into nascent peptides during translation.

How is tyrS expression regulated during different growth phases of Legionella?

Studies have shown that tyrS expression in L. pneumophila undergoes significant changes during different growth phases. Specifically, the tyrosyl-tRNA synthetase gene (lpp0628/lpl0611) shows approximately 1.9-fold downregulation during the transition from replicative to transmissive phase . This regulation appears to be linked to the stringent response mediated by ppGpp, suggesting that tyrS expression is tightly controlled as part of the bacterium's adaptation to changing environmental conditions and stresses.

What expression systems are most effective for recombinant Legionella tyrS production?

Multiple expression systems have been successfully employed for producing recombinant Legionella proteins, including tyrS:

• E. coli expression system: Most commonly used due to its simplicity and high yield potential

• Yeast expression system: Offers eukaryotic post-translational modifications that may be beneficial for certain studies

• Baculovirus/insect cell system: Particularly useful when higher-order folding or specific modifications are required

The choice of expression system should be determined by the specific research requirements, with consideration given to downstream applications and the need for proper folding and/or post-translational modifications.

What purification strategy provides the highest yield and purity of functional tyrS?

A successful purification strategy for recombinant tyrS typically involves:

• Addition of an affinity tag (such as His-tag) to facilitate initial capture

• Gravity-flow nickel resin chromatography for primary purification

• Ion exchange chromatography for secondary purification and removal of contaminants

• Optional size exclusion chromatography for higher purity requirements

This approach has been shown to yield 4-6 mg of highly pure protein per liter of culture for similar proteins . For optimal results, consider:

• Including protease inhibitors throughout the purification process

• Maintaining appropriate buffer conditions (pH 7.5-8.0, 150-300 mM NaCl)

• Adding stabilizing agents such as glycerol (10%) in storage buffers

How can researchers verify the activity of purified recombinant tyrS?

The enzymatic activity of recombinant tyrS can be verified through:

• Aminoacylation assay: Measuring the attachment of [14C]-tyrosine to tRNA(Tyr) over time

• ATP-PPi exchange assay: Monitoring the first step of the aminoacylation reaction

• Thermal shift assays: Evaluating protein stability in the presence of substrates and cofactors

For kinetic characterization, researchers can determine the Michaelis-Menten constants (Km and Vmax) using varying concentrations of tyrosine and tRNA substrates, similar to approaches used for other enzymes like tyrosinase (Km = 0.16±0.04 mM for monophenolase activity) .

What are the key structural domains of Legionella tyrS and their functions?

While the specific structure of Legionella pneumophila tyrS has not been fully characterized in the provided search results, based on homologous tyrosyl-tRNA synthetases, it likely contains:

• N-terminal catalytic domain: Contains the HIGH and KMSKS motifs essential for ATP binding and tyrosine activation

• C-terminal anticodon-binding domain: Responsible for specific recognition of tRNA(Tyr)

• Dimerization interface: tyrS typically functions as a dimer in solution

Understanding these structural elements is crucial for studies investigating enzyme mechanisms, inhibitor design, or protein engineering applications.

How does Legionella tyrS differ structurally from human tyrosyl-tRNA synthetase?

Bacterial tyrosyl-tRNA synthetases, including Legionella tyrS, differ from their human counterparts in several key aspects:

• Bacterial tyrS molecules typically lack the C-terminal EMAP II-like domain present in human YARS1

• The anticodon recognition mechanism involves different amino acid residues

• Bacterial enzymes generally show lower sensitivity to inhibitors that target human YARS1

These structural differences can be exploited for the development of selective inhibitors targeting bacterial tyrS without affecting the human enzyme, potentially serving as a basis for antibiotic development.

What cofactors are required for optimal tyrS activity?

For optimal enzymatic activity, Legionella tyrS requires:

• Magnesium ions (Mg²⁺): Essential for ATP binding and catalysis

• ATP: Required for amino acid activation

• Potassium ions (K⁺): Enhances catalytic efficiency

• Reducing environment: Typically maintained with DTT or β-mercaptoethanol to preserve cysteine residues

These requirements should be considered when designing assay conditions for enzymatic studies or when formulating storage buffers to maintain long-term stability.

How can tyrS be used as a tool to study Legionella pathogenesis?

Recombinant tyrS offers several applications for studying Legionella pathogenesis:

• Target for antibiotic development: As an essential enzyme for bacterial survival, tyrS inhibition could prevent Legionella growth

• Analysis of stress responses: Monitoring changes in tyrS expression during infection can provide insights into bacterial adaptation mechanisms

• Host-pathogen interaction studies: Investigating whether tyrS interacts with host components beyond its canonical role in translation

• Vaccine development: As a conserved bacterial protein, tyrS could potentially serve as an antigen in vaccine formulations

These applications can contribute to our understanding of Legionella infections and potentially lead to new therapeutic strategies.

What is known about tyrS expression during different stages of Legionella infection?

During Legionella infection cycles, tyrS expression appears to be regulated in conjunction with the bacterium's transition between replicative and transmissive phases:

• Replicative phase: Higher expression of translation-related proteins including tyrS

• Transmissive phase: Reduced expression of tyrS as the bacteria prepare for transmission to new hosts

• Stress conditions: Further downregulation in response to amino acid starvation, possibly mediated by the stringent response regulator RelA

This dynamic regulation suggests that tyrS may play a role in the bacterium's adaptation to different environments encountered during infection.

Could tyrS serve as a specific target for anti-Legionella therapeutics?

Legionella tyrS has potential as a therapeutic target for several reasons:

• It is essential for bacterial protein synthesis and survival

• Structural differences between bacterial and human tyrosyl-tRNA synthetases allow for selective targeting

• Successful precedents exist for aminoacyl-tRNA synthetase inhibitors as antibiotics (e.g., mupirocin)

Research approaches for developing tyrS inhibitors could include:

• Structure-based drug design using crystallographic data

• High-throughput screening of compound libraries

• Fragment-based drug discovery approaches

• Repurposing of known aminoacyl-tRNA synthetase inhibitors

What potential interactions might exist between tyrS and other Legionella virulence factors?

While direct interactions between tyrS and Legionella virulence factors have not been specifically documented in the provided search results, several intriguing possibilities exist:

• Dot/Icm Type IV secretion system (T4SS): Although tyrS is not known to be a T4SS substrate, it may indirectly support the production of effector proteins by ensuring efficient translation

• Effector protein synthesis: tyrS activity may be particularly important for efficient production of tyrosine-rich effector proteins

• Stress response coordination: tyrS regulation may be coordinated with virulence factor expression during host cell infection

Advanced proteomic approaches, such as proximity labeling or co-immunoprecipitation coupled with mass spectrometry, could help identify potential interaction partners of tyrS during infection.

How might host cell tyrosine phosphorylation events impact tyrS function during infection?

Legionella infection involves complex manipulation of host tyrosine phosphorylation pathways, which could potentially intersect with tyrS function:

• Legionella effectors like Lem4 exhibit phosphotyrosine phosphatase activity that modifies host signaling pathways

• SdeC-mediated phosphoribosyl-linked ubiquitination can modify tyrosine residues on host proteins

• These modifications could potentially affect host tRNA availability or other factors influencing tyrS function

Research in this area could employ phosphoproteomic approaches to simultaneously monitor bacterial tyrS activity and host tyrosine phosphorylation states during infection.

What methodological approaches can address the potential dual role of tyrS in both translation and pathogenesis?

To investigate potential non-canonical roles of tyrS beyond translation:

• Catalytically inactive mutants: Compare phenotypes of tyrS knockout complemented with wild-type versus catalytically inactive tyrS to distinguish translation-dependent and independent functions

• Protein-protein interaction studies: Employ BioID, APEX proximity labeling, or crosslinking mass spectrometry to identify non-canonical interaction partners

• Conditional expression systems: Develop systems for rapid tyrS depletion to distinguish primary from secondary effects

• Domain deletion/swapping: Create chimeric proteins to identify domains responsible for potential non-canonical functions

What are the critical parameters for designing a tyrS activity assay?

A robust tyrS activity assay should include these key parameters:

• Substrate preparation:

  • Purified tRNA(Tyr) (either extracted from Legionella or produced by in vitro transcription)

  • L-tyrosine (typically 0.1-1.0 mM)

  • ATP (2-5 mM)

• Reaction conditions:

  • Buffer: Typically HEPES or Tris at pH 7.5-8.0

  • Magnesium concentration: 5-10 mM MgCl₂

  • Temperature: 30-37°C to reflect physiological conditions

  • Time course: Multiple time points to ensure linearity

• Detection methods:

  • Radioactive assay: Using [¹⁴C] or [³H]-labeled tyrosine

  • Colorimetric pyrophosphate detection

  • HPLC-based detection of aminoacylated tRNA

Michaelis-Menten kinetic analysis should be conducted to determine Km and Vmax values for both tyrosine and tRNA substrates, allowing comparison with tyrS enzymes from other bacterial species.

How can researchers effectively study tyrS regulation during Legionella infection cycles?

To study tyrS regulation during different stages of infection:

• Transcriptional analysis:

  • qRT-PCR to measure tyrS mRNA levels at different infection timepoints

  • RNA-seq for genome-wide context of tyrS regulation

  • Reporter constructs (e.g., tyrS promoter driving fluorescent protein expression)

• Translational regulation:

  • Western blotting with anti-tyrS antibodies

  • Ribosome profiling to assess translation efficiency

  • Pulse-chase labeling to determine protein turnover rates

• Environmental triggers:

  • Controlled amino acid limitation to simulate host environments

  • Temperature shifts to mimic environmental-to-host transition

  • pH changes reflecting different intracellular compartments

These approaches can reveal how tyrS expression is coordinated with other virulence factors during infection progression.

What approaches can address potential cross-talk between tyrS and the stringent response in Legionella?

The stringent response, mediated by ppGpp, appears to influence tyrS expression during growth phase transitions . To investigate this relationship:

• Genetic approaches:

  • Compare tyrS expression and activity in wild-type versus RelA-deficient strains

  • Engineer strains with constitutive or regulated ppGpp production

  • Create tyrS promoter variants with modified stringent response elements

• Biochemical approaches:

  • Test direct effects of ppGpp on tyrS enzymatic activity

  • Analyze tyrS protein modifications under stringent response conditions

  • Measure aminoacylated vs. non-aminoacylated tRNA(Tyr) pools during stringent response

• Structural studies:

  • Investigate potential ppGpp binding sites on tyrS

  • Examine structural changes in tyrS under stringent response conditions

This research direction could reveal how Legionella coordinates protein synthesis with stress adaptation during host infection.

How might comparative studies of tyrS across Legionella strains provide insights into host adaptation?

Comparative analysis of tyrS sequences and activities across Legionella strains that differ in host range or virulence could:

• Identify amino acid variations that correlate with host preference

• Reveal adaptations related to differences in intracellular replication efficiency

• Uncover potential coevolution with strain-specific tRNA modifications

• Provide insights into selective pressures driving aminoacyl-tRNA synthetase evolution

Such studies could employ whole-genome sequencing data from clinical and environmental isolates , combined with recombinant expression and biochemical characterization of representative tyrS variants.

What potential non-canonical functions of tyrS might exist beyond aminoacylation?

Some aminoacyl-tRNA synthetases in other organisms exhibit functions beyond their canonical roles. For Legionella tyrS, potential non-canonical functions could include:

• Signaling roles: Similar to human YARS1, which can act as a positive regulator of poly-ADP-ribosylation independent of its aminoacylation activity

• Regulatory functions: Potential binding to mRNA or regulatory elements

• Protein-protein interactions: Possible interactions with components of stress response pathways

• Moonlighting activities: Alternative enzymatic functions or structural roles

Approaches to investigate these possibilities could include pull-down assays coupled with mass spectrometry, RNA immunoprecipitation, or genetic screens for synthetic interactions.

Could tyrS be part of a coordinated network of tRNA-related pathways manipulated during Legionella infection?

Recent research has revealed that Legionella uses small RNAs, including tRNAs, to manipulate host defense signaling pathways . This suggests a potential broader role for tRNA metabolism in pathogenesis:

• Legionella translocates bacterial small RNAs into host cells via extracellular vesicles

• tRNA-Phe can bind host mRNAs including ddx58 and irak1, reducing expression of RIG-I and IRAK1

• This miRNA-like regulation represents a general mechanism for bacterial host-pathogen communication

These findings raise the question of whether tyrS might participate in this process by:

• Influencing the pool of available tRNA(Tyr) for potential regulatory functions

• Potentially interacting with extracellular vesicle components

• Contributing to the regulation of tyrosine-rich effector protein synthesis