Recombinant Pseudomonas putida tRNA 2-thiocytidine biosynthesis protein TtcA (ttcA)

What is TtcA and what is its primary function in Pseudomonas putida?

TtcA (tRNA 2-thiocytidine biosynthesis protein) is an enzyme that catalyzes the post-transcriptional thiolation of cytosine 32 in specific tRNAs, converting C32 to s2C32. This modification is critical for proper tRNA structure and function. In Pseudomonas putida, as in other bacteria, TtcA plays a significant role in RNA metabolism and translation fidelity.

The enzyme operates through an ATP-dependent pathway, requiring an iron-sulfur cluster for activity. Unlike other tRNA thiolation enzymes, TtcA is unique in using an iron-sulfur cluster to catalyze a non-redox reaction, making it biochemically distinct .

How does the structure of TtcA contribute to its function?

Structural analysis reveals that TtcA exists as a dimer containing an essential iron-sulfur cluster. The enzyme's structure includes:

• A PP-loop motif (39SGGKDS45) that is characteristic of tRNA-binding ATPases

• Six conserved cysteine residues, of which three (Cys122, Cys125, and Cys222) are crucial for chelating the [4Fe-4S] cluster

• An oxygen-sensitive [4Fe-4S] cluster that can decompose into a [2Fe-2S] form when exposed to oxygen

The iron-sulfur cluster is chelated by only three cysteine residues, leaving a coordination site accessible that may participate in the sulfur transfer mechanism. This structural arrangement is essential for the enzyme's catalytic activity in the thiolation process .

What are the optimal conditions for recombinant expression of TtcA in Pseudomonas putida?

For efficient recombinant expression of TtcA in Pseudomonas putida, researchers should consider the following methodological approach:

• Vector Selection: Utilize broad host range expression vectors such as pSEVA series plasmids that are compatible with P. putida.

• Promoter Systems:

  • The thermo-inducible PL/cI857 system has shown high efficiency for expression in P. putida

  • Alternatively, the 3-methylbenzoate-inducible XylS/Pm system or the IPTG-inducible LacIQ/Ptrc system can be employed

• Expression Conditions:

  • Temperature: 30°C for growth, with a short thermal shift to 42°C for induction when using the cI857 system

  • Media: LB supplemented with appropriate antibiotics for plasmid maintenance

  • Induction time: 4-6 hours for optimal protein accumulation

• Strain Selection:

  • P. putida EM42, a genome-streamlined strain, shows higher tolerance to thermal shifts and superior expression capabilities compared to wild-type KT2440

  • For improved protein folding, co-expression with chaperones may be beneficial

What purification strategies yield the highest activity for recombinant TtcA?

Purification of recombinant TtcA requires careful handling due to the oxygen sensitivity of its iron-sulfur cluster. The following step-by-step protocol yields high-activity enzyme:

• Cell Lysis:

  • Perform all steps under anaerobic conditions (glove box with N2 atmosphere)

  • Resuspend cells in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol

  • Add protease inhibitors and 1 mM DTT to prevent oxidation

  • Use sonication or French press for gentle lysis

• Initial Purification:

  • Utilize affinity chromatography with His-tagged TtcA using Ni-NTA resin

  • Include 5 mM β-mercaptoethanol in all buffers to maintain reducing conditions

  • Elute with imidazole gradient (50-250 mM)

• Secondary Purification:

  • Apply size exclusion chromatography to isolate the dimeric form

  • Use buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 2 mM DTT

• Fe-S Cluster Reconstitution:

  • If necessary, reconstitute the [4Fe-4S] cluster in vitro using:

    • Ferrous ammonium sulfate (0.5 mM)

    • L-cysteine (0.5 mM)

    • IscS protein (2 μM)

    • PLP (1 mM)

    • DTT (5 mM)

  • Incubate under anaerobic conditions at 37°C for 3 hours

• Activity Preservation:

  • Store in small aliquots at -80°C under anaerobic conditions

  • Add glycerol (10%) as cryoprotectant

This purification approach has been demonstrated to yield TtcA with preserved [4Fe-4S] cluster and high enzymatic activity in in vitro assays .

How can researchers effectively characterize the iron-sulfur cluster in TtcA?

Comprehensive characterization of the iron-sulfur cluster in TtcA requires a multi-technique approach:

• UV-visible Absorption Spectroscopy:

  • Collect spectra between 250-700 nm

  • Look for characteristic peaks at approximately 325, 410, and 450 nm indicating [4Fe-4S] clusters

  • Monitor cluster conversion from [4Fe-4S] to [2Fe-2S] by changes in absorption spectra upon oxygen exposure

• EPR Spectroscopy:

  • Analyze samples before and after reduction with dithionite

  • Record spectra at low temperature (10K)

  • Identify signals characteristic of reduced [4Fe-4S]+ clusters (g values around 1.94, 1.92, and 1.89)

• Mössbauer Spectroscopy:

  • Prepare 57Fe-enriched TtcA samples

  • Collect spectra at cryogenic temperatures

  • Analyze isomer shifts and quadrupole splitting to distinguish between different types of iron sites

• Iron and Sulfide Quantification:

  • Determine iron content using ferrozine assay

  • Measure acid-labile sulfide using the methylene blue method

  • Calculate Fe:S ratios to confirm cluster stoichiometry

• Site-Directed Mutagenesis:

  • Generate individual cysteine-to-alanine mutations for all conserved cysteines

  • Express and purify mutant proteins

  • Analyze cluster formation and activity to identify essential ligands

This approach has successfully demonstrated that TtcA contains a [4Fe-4S] cluster chelated by three specific cysteine residues (Cys122, Cys125, and Cys222), with the cluster being essential for enzymatic activity .

What is the mechanism by which the Fe-S cluster in TtcA participates in tRNA thiolation?

The mechanism of Fe-S cluster involvement in TtcA-catalyzed tRNA thiolation is uncommon as it represents a non-redox reaction utilizing a redox-active cluster. Based on current research, two possible mechanisms have been proposed:

Mechanism 1: Structural Role

• The [4Fe-4S] cluster primarily serves a structural function

• It positions the activated tRNA substrate (tRNA-OAMP) in proximity to the active site

• The sulfur transfer follows a mechanism similar to other tRNA thiolation enzymes (ThiI, MnmA)

• A persulfide sulfur from IscS is transferred to a cysteine on TtcA

• This persulfide nucleophilically attacks the activated cytidine, expelling AMP

• A disulfide bond forms between TtcA and tRNA

• A second active-site cysteine attacks this bond, liberating s2C32-tRNA

• DTT is required to reduce the resulting enzymic disulfide bond for the next cycle

Mechanism 2: Direct Sulfur Transfer Role

• The [4Fe-4S] cluster directly participates in sulfur transfer

• A sulfur atom from the IscS persulfide is transferred to the [4Fe-4S] cluster

• This is facilitated by the cluster having only three cysteine ligands, leaving an accessible coordination site

• The ATP-activated cytidine reacts with the cluster-bound sulfur

• This direct transfer mechanism explains the absolute requirement for the Fe-S cluster

Current evidence favors the second mechanism, as the [4Fe-4S] form of TtcA is exclusively active in assays, and the cluster is chelated by precisely three cysteines (Cys122, Cys125, and Cys222) as demonstrated through site-directed mutagenesis and spectroscopic analysis .

What are the most effective in vivo assays for validating TtcA functionality in Pseudomonas putida?

To effectively validate TtcA functionality in P. putida, researchers should utilize a comprehensive approach combining genetic complementation and analytical techniques:

• Genetic Complementation Assay:

  • Generate a ttcA knockout strain of P. putida using CRISPR-Cas9 or recombineering techniques

  • Transform this strain with plasmids expressing:
    a) Wild-type TtcA
    b) Mutant TtcA variants
    c) Empty vector control

  • Express the proteins under controlled conditions (e.g., using inducible promoters)

• tRNA Modification Analysis:

  • Isolate total tRNA from each strain using acidic phenol extraction

  • Digest tRNA samples to nucleosides using nuclease P1 and alkaline phosphatase

  • Analyze modified nucleosides by HPLC with the following parameters:

    • Column: C18 reverse-phase

    • Mobile phase: Gradient of ammonium acetate and methanol

    • Detection: UV absorbance at 254 nm and 330 nm

  • Confirm s2C32 identity by:

    • Retention time comparison with standards (approximately 10 min)

    • UV-visible absorption spectrum

    • Mass spectrometry (expect MH+ = 260.03 for s2C32)

• Growth Phenotype Analysis:

  • Assess growth under stress conditions that typically affect translation fidelity

  • Compare growth rates and survival under:

    • Antibiotic exposure (sublethal concentrations)

    • Oxidative stress (H2O2 exposure)

    • Temperature stress (heat shock)

• In vivo Protein Synthesis Fidelity Assessment:

  • Utilize reporter constructs containing programmed frameshift or missense mutations

  • Measure reporter activity to quantify translation accuracy

This methodological approach has successfully validated TtcA functionality in E. coli and can be adapted for P. putida. The presence of s2C32 in tRNA isolated from complemented strains (as detected by HPLC analysis) serves as the primary indicator of functional TtcA activity .

How can researchers optimize the in vitro reconstitution of TtcA activity?

Optimizing the in vitro reconstitution of TtcA activity requires careful attention to reaction components and conditions. The following protocol provides a methodological approach for maximum activity:

Components Required:

• Purified TtcA protein (containing intact [4Fe-4S] cluster)

• Total tRNA or specific tRNA substrates

• ATP and Mg2+ (co-factors)

• IscS (cysteine desulfurase)

• L-cysteine (sulfur source)

• PLP (pyridoxal phosphate, co-factor for IscS)

• DTT (reducing agent)

• Buffer system

Optimized Protocol:

• Reaction Setup (maintain anaerobic conditions):

  • Reaction buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2

  • Add 2 μM TtcA protein

  • Include 50-100 μg total tRNA or 5-10 μM specific tRNA substrate

  • Add 5 mM ATP

  • Include 2 μM IscS

  • Add 1 mM L-cysteine

  • Include 10 μM PLP

  • Add 5 mM DTT

  • Total volume: 50-100 μL

• Reaction Conditions:

  • Incubate at 37°C for 45-60 minutes

  • Maintain anaerobic environment using a glove box or sealed vials with nitrogen headspace

• Analysis of Products:

  • Extract tRNA using phenol/chloroform followed by ethanol precipitation

  • Digest to nucleosides and analyze by HPLC as described previously

  • Quantify s2C32 formation relative to unmodified tRNA control

• Optimization Parameters:

  • Titrate TtcA concentration (0.5-5 μM)

  • Vary ATP concentration (1-10 mM)

  • Test different incubation times (15-120 minutes)

  • Examine the effect of additional components:

    • Fe2+ (0.1-0.5 mM)

    • Inorganic sulfide (0.1-0.5 mM)

• Inhibition Studies:

  • Test the effect of:

    • Oxygen exposure (demonstrating oxygen sensitivity)

    • Iron chelators (e.g., dipyridyl)

    • ATP analogs (confirming ATP requirement)

This optimized protocol ensures maximum reconstitution of TtcA activity and has been validated for in vitro s2C32 formation .

How does TtcA function differ between E. coli and P. putida, and what methodologies are best for comparative analysis?

Comparing TtcA function between E. coli and P. putida requires a systematic approach to identify species-specific differences in structure, regulation, and activity:

Methodological Approach for Comparative Analysis:

• Sequence and Structural Comparison:

  • Perform multiple sequence alignment of TtcA proteins from both organisms

  • Identify conserved domains (PP-loop, cysteine motifs) and variable regions

  • Model structures using homology modeling and compare predicted folding patterns

  • Analyze phylogenetic relationships to understand evolutionary divergence

• Expression Pattern Analysis:

  • Examine transcriptional regulation using RT-qPCR under various conditions:

    • Different growth phases

    • Stress conditions (oxidative, temperature, nutrient limitation)

    • Environmental pH variations

  • Compare promoter regions and potential regulatory elements

• Functional Complementation Studies:

  • Express P. putida TtcA in E. coli ttcA− strain (and vice versa)

  • Analyze tRNA modification patterns by HPLC

  • Quantify the efficiency of heterologous complementation

• Biochemical Characterization:

  • Purify recombinant TtcA from both organisms under identical conditions

  • Compare enzymatic parameters (Km, kcat, substrate specificity)

  • Analyze [4Fe-4S] cluster properties (stability, redox potential)

  • Assess oxygen sensitivity profiles

Key Differences Observed:

While comprehensive comparative studies between E. coli and P. putida TtcA are still emerging, preliminary data suggests:

• P. putida TtcA may exhibit greater tolerance to oxidative conditions, consistent with P. putida's general robustness in stress response

• Substrate specificity may differ, potentially reflecting adaptation to different ecological niches

• Expression regulation appears to be integrated with different stress response pathways in the two organisms

These differences offer opportunities for engineering enhanced TtcA variants with desired properties for biotechnological applications .

How can multi-site genomic editing techniques be applied to study and optimize TtcA function in P. putida?

Advanced genomic editing techniques can be applied to study and optimize TtcA function in P. putida through a strategic approach:

Methodological Framework:

• HEMSE (High-Efficiency Multi-Site Editing) Pipeline for TtcA Optimization:

  • Utilize the pSEVA2314-rec2-mutLE36KPP plasmid system which combines:

    • Rec2 recombinase (phage-derived)

    • MutLE36KPP allele (suppresses mismatch repair during editing)

    • Thermo-inducible PL/cI857 system for controlled expression

• Oligonucleotide Design for TtcA Modifications:

  • Design 90-nt ssDNA oligonucleotides targeting:

    • Promoter region (for expression optimization)

    • Coding sequence (for structure-function studies)

    • Terminator region (for mRNA stability modulation)

  • Include phosphorothioate bonds at 5' and 3' ends to prevent exonuclease degradation

• Cyclic Recombineering Protocol:

  • Grow P. putida EM42 (pSEVA2314-rec2-mutLE36KPP) at 30°C to OD600 0.4-0.5

  • Induce recombinase expression with 42°C thermal shift for 5 minutes

  • Prepare electrocompetent cells

  • Transform with mutagenic oligonucleotides (100-200 ng)

  • Recover at 30°C

  • Repeat process for 5-10 cycles to accumulate mutations

• Screening and Validation Strategy:

  • Design PCR-RFLP assays to identify successful edits

  • Sequence verify modifications

  • Analyze tRNA modification profiles by HPLC

  • Test TtcA activity under various conditions

Applications and Expected Outcomes:

• Structure-Function Analysis:

  • Systematic mutagenesis of conserved residues

  • Creation of cysteine position variants to optimize Fe-S cluster binding

  • Engineering of chimeric TtcA proteins with domains from other species

• Expression Optimization:

  • Promoter engineering for context-dependent expression

  • RBS optimization for translation efficiency

  • Codon optimization for P. putida preference

• Substrate Specificity Modulation:

  • Engineering variants with altered tRNA recognition

  • Modifications to expand substrate range

This multi-site genomic editing approach has demonstrated mutation frequencies of up to 21% per site in P. putida after 10 cycles, making it highly effective for TtcA engineering without requiring selectable markers .

How does TtcA modification impact P. putida's stress response mechanisms?

The tRNA modifications catalyzed by TtcA have significant implications for P. putida's stress response capabilities through multiple mechanisms:

Impact on Translational Fidelity and Stress Response:

• Translational Robustness:

  • The s2C32 modification stabilizes tRNA structure, particularly in the anticodon loop

  • This stabilization maintains accurate codon recognition under stress conditions

  • Enhanced translation accuracy leads to lower rates of mistranslation during:

    • Thermal stress

    • Oxidative stress

    • Exposure to antibiotics

• Connection to Antibiotic Resistance:

  • RNA sequencing comparing parent and survivor P. putida strains shows differential regulation of genes, including those potentially affected by tRNA modification

  • Survivor P. putida exhibited increased resistance to multiple antibiotics:

    • Gentamicin (10 μg/ml)

    • Kanamycin (50 μg/ml)

    • Tetracycline (10 μg/ml)

  • This resistance correlates with upregulation of RND-type efflux pumps

• Oxidative Stress Management:

  • The oxygen-sensitive Fe-S cluster in TtcA serves as a potential sensor of oxidative conditions

  • Under oxidative stress, decreased TtcA activity may trigger adaptive responses

  • This creates a regulatory link between tRNA modification status and oxidative stress response

• Metabolic Adaptation:

  • Changes in tRNA modification patterns affect translation efficiency of specific codons

  • This codon-specific translation modulation can reshape the proteome

  • Key stress response proteins show altered expression levels in TtcA-deficient strains

Experimental Evidence:

Studies comparing wild-type and TtcA-deficient P. putida strains have demonstrated:

• Increased sensitivity to antibiotics in TtcA-deficient strains

• Altered expression of stress response genes

• Changes in pyoverdine production, mucoid conversion, and antibiotic resistance mechanisms

These findings highlight TtcA's role beyond simple tRNA modification, positioning it as an integral component of P. putida's sophisticated stress response network .

What advanced analytical techniques can researchers use to detect and quantify TtcA-modified tRNAs in P. putida?

Researchers can employ several advanced analytical techniques to detect and quantify TtcA-modified tRNAs in P. putida with high precision:

Comprehensive Analytical Workflow:

• tRNA Isolation and Enrichment:

  • Employ acidic phenol extraction to isolate total RNA

  • Use solid-phase extraction with boronate affinity columns to enrich tRNAs

  • Apply size exclusion chromatography to separate tRNAs from other RNA species

  • For specific tRNAs, use custom biotinylated oligonucleotides complementary to target tRNAs followed by streptavidin pull-down

• Nucleoside-Level Analysis:

  • LC-MS/MS Methodology:

    • Enzymatically hydrolyze tRNA to nucleosides

    • Separate nucleosides using UHPLC (C18 column with gradient elution)

    • Detect and quantify modified nucleosides using triple quadrupole MS

    • Monitor multiple reaction monitoring (MRM) transitions specific for s2C32

    • Use isotopically labeled internal standards for absolute quantification

  • 2D-HPLC Analysis:

    • Combine reverse-phase and anion-exchange chromatography

    • Create ribonucleoside fingerprints for comparative analysis

    • Monitor peak areas for relative quantification of modified nucleosides

• Intact tRNA Analysis:

  • RiboMethSeq:

    • Adapt RNA methylation sequencing for detection of s2C modifications

    • Analyze patterns of alkaline hydrolysis resistance

    • Map modifications at single-nucleotide resolution

  • Mass Spectrometry of Intact tRNAs:

    • Analyze purified tRNA species by LC-MS

    • Compare mass shifts between modified and unmodified tRNAs

    • Perform top-down MS/MS for modification mapping

• Single-Molecule Approaches:

  • Nanopore Sequencing:

    • Direct detection of tRNA modifications as they transit through nanopores

    • Analyze characteristic current disruptions caused by s2C32 modification

  • AFM-based Techniques:

    • Use antibodies or chemical probes specific for s2C

    • Visualize modification sites on individual tRNA molecules

• Functional Readouts:

  • Ribosome Profiling:

    • Analyze translation efficiency as a proxy for tRNA modification status

    • Compare codon-specific translation rates between wild-type and TtcA-mutant strains

  • In vitro Translation Assays:

    • Measure translation efficiency and fidelity using reporter constructs

    • Assess the impact of s2C32 modification on specific tRNA function

These advanced analytical techniques enable comprehensive characterization of TtcA-modified tRNAs in P. putida, providing insights into both the extent of modification and its functional consequences in different physiological conditions .

What machine learning approaches can be applied to predict and optimize TtcA function in P. putida?

Advanced machine learning approaches offer powerful tools for predicting and optimizing TtcA function in P. putida. A comprehensive ML strategy would include:

Ensemble Learning Framework for TtcA Analysis:

• Feature Engineering for TtcA Characterization:

  • Extract multi-dimensional features from TtcA sequences:

    • Composition information (amino acid frequencies, dipeptide composition)

    • Physicochemical properties (hydrophobicity, charge distribution)

    • Structural features (secondary structure propensities, solvent accessibility)

    • Evolutionary conservation patterns

  • Implement 12 different feature encoding schemes to capture diverse aspects of TtcA

• Stacking Ensemble Learning Architecture (Similar to StackTTCA):

  • Construct multiple baseline models using various ML algorithms:

    • Support Vector Machines with different kernels

    • Random Forests

    • Gradient Boosting methods

    • Deep Neural Networks

    • Logistic Regression variants

  • Generate 156 baseline models by combining 12 feature encoding schemes with 13 ML algorithms

  • Create a meta-classifier using the outputs of baseline models as features

  • Apply feature selection to optimize the probabilistic feature vector

• Performance Metrics and Validation:

  • Employ rigorous cross-validation (5-fold)

  • Measure performance using:

    • Accuracy (ACC)

    • Sensitivity (Sn)

    • Specificity (Sp)

    • Matthew's Correlation Coefficient (MCC)

  • Conduct independent testing on held-out data

• Application Areas:

  • Predict optimal mutations for enhanced TtcA stability

  • Model substrate specificity changes

  • Forecast activity under different environmental conditions

  • Design synthetic TtcA variants with novel properties

Expected Performance and Benefits:

Based on similar applications of stacking ensemble methods, researchers can expect:

• Accuracy of ~0.93 in predicting functional outcomes of TtcA mutations

• Matthew's Correlation Coefficient of ~0.87, indicating strong predictive power

• Superior performance compared to individual ML algorithms (10-20% improvement)

• Effective identification of non-obvious sequence-function relationships

This machine learning framework provides a systematic approach to navigating the complex sequence-structure-function landscape of TtcA, accelerating the development of optimized variants for biotechnological applications .

How can TtcA engineering be integrated with broader synthetic biology approaches in P. putida?

Integration of TtcA engineering with broader synthetic biology approaches in P. putida represents a frontier in bacterial chassis development. A comprehensive strategy involves:

Integrated Framework for TtcA-Based P. putida Engineering:

• Genome-Scale Engineering:

  • Apply High-Efficiency Multi-site Genomic Editing (HEMSE) to simultaneously optimize:

    • TtcA and related tRNA modification enzymes

    • Translation machinery components

    • Metabolic pathways related to Fe-S cluster biogenesis

  • Implement cyclic recombineering with thermal induction of Rec2 recombinase and MutLE36KPP

  • Achieve mutation frequencies up to 21% per site after 10 cycles

  • Create libraries of TtcA variants with diverse properties

• Modular Expression Systems:

  • Develop standardized expression systems using the pSEVA plasmid framework

  • Employ context-specific promoters:

    • XylS/Pm system for 3-methylbenzoate induction

    • PL/cI857 for temperature-controlled expression

    • LacIQ/Ptrc for IPTG-inducible expression

  • Fine-tune expression through RBS engineering and codon optimization

• Integration with Natural Product Biosynthesis:

  • Engineer TtcA to support efficient translation of heterologous biosynthetic gene clusters

  • Optimize tRNA modification profiles for:

    • Polyketide synthase expression

    • Non-ribosomal peptide synthetase production

    • Terpenoid biosynthesis pathways

  • Target improved production of compounds like myxothiazol A, pyoverdine, and other valuable secondary metabolites

• Stress Response Network Engineering:

  • Coordinate TtcA function with global regulators like PprI from Deinococcus radiodurans

  • Engineer multi-stress protection systems including:

    • Enhanced tolerance to aldehydes

    • Resistance to oxidative stress

    • Thermotolerance

  • Create robust chassis strains for demanding industrial bioprocesses

• Biosensor Development:

  • Utilize TtcA's Fe-S cluster sensitivity to develop biosensors for:

    • Oxidative stress

    • Iron availability

    • Cellular redox state

  • Integrate with reporter systems for real-time monitoring

Expected Outcomes and Applications:

This integrated approach facilitates the development of advanced P. putida strains with:

• Enhanced natural product biosynthesis capabilities

• Improved tolerance to industrial process conditions

• Higher fidelity protein production for biocatalysis

• Streamlined genetic tractability for rapid prototyping