Recombinant Pseudomonas syringae pv. tomato tRNA 2-thiocytidine biosynthesis protein TtcA (ttcA)

Basic Research Questions

• What is the function of TtcA in Pseudomonas syringae pv. tomato and how does it relate to bacterial physiology?

  TtcA (tRNA 2-thiocytidine biosynthesis protein) catalyzes the post-transcriptional thiolation of cytosine 32 (C32) to 2-thiocytidine (s2C32) in specific tRNA molecules. This modification is crucial for proper tRNA function and translation efficiency. In P. syringae, like its relative P. aeruginosa, TtcA plays a role in the response to oxidative stress and potentially impacts virulence mechanisms .

  Unlike typical modification enzymes, TtcA employs a unique mechanism requiring an iron-sulfur ([Fe-S]) cluster to catalyze a non-redox reaction . This represents a specialized case in tRNA modification biochemistry where an iron-sulfur protein participates in thiolation via a non-radical mechanism.

• What are the structural characteristics of TtcA and how do they relate to its function?

  TtcA belongs to group I of the TtcA family, characterized by:

  • Two CXXC motifs that are essential for iron-sulfur cluster binding

  • A PP-loop motif (commonly 39SGGKDS45 in bacterial species) that binds ATP

  • A [4Fe-4S] cluster chelated by only three cysteine residues (typically C115, C118, and C206 in Pseudomonas species)

  The [4Fe-4S] cluster is oxygen-sensitive and redox-active, which may explain TtcA's involvement in oxidative stress responses. Site-directed mutagenesis studies have confirmed that the conserved cysteine motifs involved in [Fe-S] cluster ligation are absolutely required for TtcA function .

  Table 1: Key Structural Elements of TtcA Protein in Pseudomonas Species

  Structural Element	Position	Function
  PP-loop motif	39SGGKDS45	ATP binding for adenylate formation
  First CXXC motif	C115, C118	[Fe-S] cluster ligation
  Conserved cysteine	C184	Structural integrity
  Conserved cysteines	C203, C206	[Fe-S] cluster ligation
  Adjacent cysteine	C38	Near PP-loop, may have auxiliary role

• How is ttcA expression regulated in Pseudomonas syringae pv. tomato?

  Expression of ttcA is upregulated in response to oxidative stress conditions. Similar to findings in P. aeruginosa, ttcA expression in P. syringae likely increases upon exposure to reactive oxygen species such as hydrogen peroxide (H2O2) . This regulation suggests that TtcA's function becomes particularly important during bacterial stress responses, potentially through its impact on translation efficiency of specific proteins involved in stress tolerance.

  The regulation may occur at both transcriptional and post-transcriptional levels, as observed in other bacterial species where tRNA modification enzymes are part of coordinated stress response networks . While the specific transcription factors regulating ttcA in P. syringae have not been fully characterized, comparison with related species suggests potential involvement of oxidative stress-responsive regulators.

• What tRNA molecules are targeted by TtcA in Pseudomonas syringae pv. tomato?

  TtcA specifically modifies position C32 of certain tRNAs. While the exact complement of target tRNAs in P. syringae has not been fully characterized, research in related bacteria suggests TtcA typically modifies tRNAs with a C32 position in the anticodon loop .

  Characterization of TtcA target tRNAs typically involves techniques such as:

  • High-performance liquid chromatography (HPLC) of tRNA hydrolysates

  • Mass spectrometry (MS) analysis to confirm the s2C32 modification by its characteristic mass signature (MH+ = 260.03)

  • Comparison of tRNA modification profiles between wild-type and ttcA deletion mutants

  The modification status can be identified through cDNA misincorporation and termination sequencing analysis, as these modified nucleosides can cause specific signature patterns during reverse transcription .

• How is TtcA related to other tRNA-thiolating enzymes in bacteria?

  TtcA belongs to a broader family of tRNA-thiolating enzymes but has distinctive characteristics:

  • Unlike ThiI (responsible for s4U8 synthesis) and MnmA (responsible for s2U34 synthesis), TtcA requires an [Fe-S] cluster

  • Similar to ThiI and MnmA, TtcA contains a PP-motif used for ATP binding and likely forms an adenylate intermediate during the thiolation process

  • Unlike MiaB (which also contains an [Fe-S] cluster), TtcA catalyzes a non-redox reaction

  This positions TtcA as a unique hybrid enzyme that shares features of both ATP-dependent thiolation enzymes and [Fe-S] cluster-dependent tRNA modification enzymes .

Advanced Research Questions

• What is the proposed mechanism of TtcA-catalyzed tRNA thiolation in Pseudomonas species?

  The mechanism of TtcA-catalyzed thiolation involves several steps that combine features of different tRNA modification pathways:

  1. ATP-dependent activation: TtcA uses its PP-loop motif to bind Mg-ATP and activate the oxygen at position 2 of cytidine, likely forming a tRNA-OAMP (adenylated) intermediate

  2. Sulfur mobilization: The cysteine/cysteine desulfurase (IscS) system provides sulfur atoms for the reaction

  3. [Fe-S] cluster involvement: The [4Fe-4S] cluster is essential for activity, though its exact role remains debated. Two potential mechanisms have been proposed:

     • The [Fe-S] cluster may serve as a sulfur donor

     • The cluster may play a structural role in positioning substrates or as an electron transfer mediator

  4. Thiolation: The activated cytidine undergoes nucleophilic attack by the mobilized sulfur atom, resulting in the formation of s2C32

  The reaction requires not only ATP but also reducing agents like DTT in vitro, suggesting that maintaining the [Fe-S] cluster in its proper redox state is critical for enzyme function .

• How does deletion of ttcA affect pathogenicity and stress response in Pseudomonas species?

  Deletion of ttcA has significant impacts on bacterial physiology and pathogenicity:

  In P. aeruginosa:

  • ttcA-deleted mutants show hypersensitivity to oxidative stress, particularly H2O2 treatment

  • Decreased catalase activity, suggesting impaired antioxidant defense mechanisms

  • Attenuated virulence in host models such as Drosophila melanogaster

  Expected in P. syringae pv. tomato (based on homology):

  • Potential impairment of plant infection processes

  • Reduced fitness during host-pathogen interactions

  • Altered expression of virulence factors

  The effects of ttcA deletion appear to manifest at the post-transcriptional level, affecting translation of specific proteins involved in stress response and virulence . Research suggests that TtcA-mediated tRNA modifications ensure proper translation of these proteins under stress conditions.

• What methods are most effective for expressing and purifying active recombinant TtcA from Pseudomonas syringae pv. tomato?

  Obtaining active recombinant TtcA requires special considerations due to its [Fe-S] cluster:

  Expression system:

  • Homologous expression in Pseudomonas or related bacteria may preserve native folding

  • E. coli-based systems with co-expression of iron-sulfur cluster assembly machinery can be effective

  • Expression vectors with N-terminal His-tags have been shown to produce functional protein

  Purification conditions:

  • Anaerobic or low-oxygen conditions are critical to preserve the oxygen-sensitive [4Fe-4S] cluster

  • Buffer systems typically include:

    • Reducing agents (DTT, β-mercaptoethanol)

    • Iron and sulfide sources during purification

    • Chelating agents to prevent non-specific metal binding

  Activity verification:

  • In vitro enzyme assays requiring ATP, DTT, and the cysteine/cysteine desulfurase system

  • Spectroscopic characterization (UV-visible absorption, EPR, Mössbauer) to confirm [Fe-S] cluster integrity

  • HPLC analysis of tRNA modifications to confirm functional activity

  The purified enzyme exists as a dimer containing the iron-sulfur cluster, with the [4Fe-4S] form being the catalytically active state .

• How can site-directed mutagenesis be used to study TtcA function in Pseudomonas syringae pv. tomato?

  Site-directed mutagenesis is a powerful approach to investigate TtcA structure-function relationships:

  Key residues for targeted mutagenesis:

  • Cysteine residues involved in [Fe-S] cluster coordination (typically C115, C118, C206)

  • PP-loop residues involved in ATP binding

  • Other conserved cysteines (C38, C184, C203)

  Mutation strategies:

  • Cysteine to serine substitutions preserve size but eliminate sulfur coordination

  • Conservative mutations in the PP-loop to assess ATP binding requirements

  Functional assays after mutagenesis:

  • Complementation of ttcA deletion strains with mutated versions

  • Oxidative stress sensitivity tests (H2O2, NaOCl challenge)

  • Spectroscopic analysis to assess [Fe-S] cluster formation

  • In vitro enzyme activity assays

  Research in P. aeruginosa has shown that mutations of the conserved cysteines for [Fe-S] cluster ligation (C115S, C118S, and C206S) completely abolished TtcA function, confirming their essential role in enzyme activity .

• What techniques are available for detecting and quantifying s2C32 modifications in tRNA populations?

  Several complementary approaches are used to analyze s2C32 modifications:

  HPLC-based methods:

  • Nucleoside composition analysis after complete tRNA hydrolysis

  • Characteristic retention time (approximately 10 minutes under standard conditions)

  • UV-visible spectral profile characteristic of s2C32

  Mass spectrometry approaches:

  • LC-MS/MS to identify s2C32 by its mass signature (MH+ = 260.03)

  • RNA mass fingerprinting of oligonucleotide fragments

  Sequencing-based methods:

  • cDNA misincorporation analysis, as thiolated nucleosides cause specific mutation signatures

  • Termination sequencing to identify modification sites

  • Methanethiosulfonate (MTS) chemistry can be used for thiolated RNA enrichment

  Comparative analysis:

  • Comparison between wild-type and ttcA mutant strains

  • Quantitative assessment of modification levels

  Research has shown that combining multiple detection methods provides the most comprehensive characterization of tRNA modification status .

• How does genomic context and evolutionary conservation inform our understanding of ttcA in Pseudomonas species?

  Understanding the genomic context and evolutionary conservation of ttcA provides insights into its function and importance:

  Genomic context in P. syringae pv. tomato DC3000:

  • The ttcA gene is part of the core genome rather than mobile genetic elements

  • May be influenced by genomic plasticity observed in P. syringae

  • Could potentially be subject to duplication events, as observed with other genes in P. syringae pv. tomato DC3000

  Evolutionary conservation:

  • TtcA is conserved across diverse bacterial species, suggesting fundamental importance

  • The TtcA family is divided into two groups, with bacterial TtcA typically belonging to group I (two CXXC motifs)

  • Conservation pattern suggests coevolution with specific tRNA substrates

  Functional implications:

  • Conservation of the unique [Fe-S]-dependent mechanism implies evolutionary advantage

  • The link between TtcA and stress response appears to be conserved across different Pseudomonas species

  • The connection to virulence suggests a potential role in host-pathogen coevolution

  Phylogenetic analysis can reveal potential horizontal gene transfer events and adaptation to specific environmental niches or hosts.

• What role does TtcA play in P. syringae pv. tomato host adaptation and plant pathogenicity?

  TtcA likely contributes to P. syringae pv. tomato host adaptation and pathogenicity through several mechanisms:

  Stress adaptation during infection:

  • Plants produce reactive oxygen species as defense mechanisms

  • TtcA-mediated tRNA modifications may enable efficient translation of stress-response proteins

  • This adaptation would be particularly important during the early stages of infection

  Virulence factor production:

  • Proper translation of key virulence factors may depend on TtcA-modified tRNAs

  • The type III secretion system and effector proteins could be affected by translation efficiency

  • Studies in P. syringae have shown that effectors like AvrE1 and HopM1 are crucial for bacterial growth in planta

  Host range determination:

  • Different P. syringae pathovars show distinct host ranges

  • tRNA modifications could potentially influence adaptation to specific plant hosts

  • Model studies using P. syringae pv. tomato DC3000, which has a wider host range than typical tomato isolates , could reveal connections between TtcA function and host adaptation

  Further research studying ttcA mutants in plant infection models would help elucidate its specific contributions to the pathogenicity of P. syringae pv. tomato.

• How can transcriptomic and proteomic approaches be used to study the impact of ttcA deletion in P. syringae pv. tomato?

  Multi-omics approaches provide comprehensive insights into TtcA function:

  Transcriptomic analysis:

  • RNA-Seq to compare wild-type and ttcA mutant gene expression profiles

  • Temporal dynamic methods for bulk RNA-Seq time series data to capture expression changes during infection

  • Analysis of differential gene expression under stress conditions

  Proteomic analysis:

  • Quantitative proteomics to identify proteins with altered translation efficiency

  • Ribosome profiling to detect translation stalling or efficiency changes

  • Targeted analysis of stress response and virulence factor production

  Integrated analysis:

  • Correlation between transcriptomic and proteomic changes to identify post-transcriptional effects

  • Pathway enrichment analysis to identify affected biological processes

  • Network analysis to understand system-level impacts

  These approaches can reveal which specific molecular pathways are most affected by ttcA deletion, beyond the general phenotypes of stress sensitivity and reduced virulence.

• What complementary genetic systems can be used with ttcA to study tRNA modification networks in Pseudomonas species?

  Several genetic approaches can be combined with ttcA studies:

  TolC-based genetic systems:

  • The tolC gene can serve as a selectable/counter-selectable marker for genetic manipulation

  • This system allows for seamless deletion of genes and precise genomic engineering

  • Can be used to create ttcA deletion strains and subsequent complementation variants

  Recombineering approaches:

  • λ Red/Gam recombination systems adapted for Pseudomonas

  • Can facilitate precise genetic modifications to study ttcA interactions

  • Allows for chromosomal integration of modified ttcA variants

  Multi-gene deletion analysis:

  • Creation of double or triple mutants with other tRNA modification enzymes

  • Analysis of synthetic phenotypes that reveal functional relationships

  • Study of genetic interactions between ttcA and stress response systems

  Reporter systems:

  • Transcriptional and translational fusion reporters to study ttcA regulation

  • Fluorescent protein-based reporters to visualize expression patterns

  • Luciferase-based systems for quantitative and temporal analysis

  These systems allow for detailed genetic dissection of ttcA function and its interactions with other cellular pathways in P. syringae pv. tomato.

• How does the iron-sulfur cluster assembly in TtcA relate to bacterial adaptation to oxidative environments?

  The iron-sulfur cluster in TtcA represents a critical interface between tRNA modification and oxidative stress response:

  Iron-sulfur cluster biosynthesis:

  • TtcA function depends on the iron-sulfur cluster biosynthesis machinery (Isc system)

  • The IscS cysteine desulfurase provides sulfur for both the [Fe-S] cluster and the thiolation reaction

  • The IscU scaffold protein is required for [Fe-S] cluster assembly and transfer to TtcA

  Oxidative stress connections:

  • The [4Fe-4S] cluster in TtcA is oxygen-sensitive and can degrade to [2Fe-2S] or lose activity entirely

  • Environmental oxidative stress can directly impact TtcA function

  • Upregulation of ttcA during oxidative stress may compensate for reduced activity of existing enzyme

  Adaptive significance:

  • The sensitivity of TtcA to oxidation may serve as a sensor for oxidative stress

  • This could represent a mechanism to modulate translation in response to environmental conditions

  • The connection between iron-sulfur proteins and tRNA modification provides multiple layers of regulation

  This relationship highlights how bacteria have evolved intricate connections between basic cellular processes (translation) and environmental adaptation (stress response).