Recombinant Nitrosomonas europaea tRNA 2-thiocytidine biosynthesis protein TtcA (ttcA)

What is the recommended culture medium for growing Nitrosomonas europaea for TtcA studies?

The standard ATCC #2265 medium is commonly used for N. europaea cultivation, but researchers should be aware of spontaneous precipitation issues reported in this medium. A modified formulation has been developed to prevent precipitation problems while maintaining optimal growth conditions.

When preparing culture medium, consider the following components:

• Ammonium source (typically (NH₄)₂SO₄)

• Appropriate buffer system (pH 7.5-8.0)

• Essential trace elements

• No organic carbon sources (for chemolithoautotrophic growth)

For accurate cell concentration determination during cultivation, fluorescent in situ hybridization (FISH) followed by flow cytometry quantification is recommended. The Nsm156 probe (5′-TATTAGCACATCTTTCGAT-3′) labeled with 6-carboxyfluorescein (6-FAM) fluorophore is specific for N. europaea .

Growth in chemostats allows for more controlled studies of TtcA expression under various conditions. A typical pre-inoculum should be prepared 10-15 days before each culture to ensure healthy starting cultures .

What cloning strategy should be used for recombinant expression of Nitrosomonas europaea TtcA?

Based on established protocols for TtcA from other organisms, the following strategy is recommended:

• Amplify the ttcA gene from Nitrosomonas europaea genomic DNA using PCR with high-fidelity polymerase (e.g., Pwo DNA polymerase)

• Design primers with appropriate restriction sites (e.g., NdeI at 5' end and HindIII at 3' end)

• Clone the PCR product into an expression vector (e.g., pT7-7 for E. coli expression systems)

• Add an N-terminal hexahistidine tag to facilitate purification

• Confirm the sequence to ensure no errors were introduced during PCR

For expression, transform the construct into E. coli BL21(DE3) strain, which is widely used for recombinant protein production .

How can I verify the activity of recombinant TtcA protein from Nitrosomonas europaea?

TtcA activity can be verified through the following in vitro assay:

• Prepare a reaction mixture containing:

  • 100 mM Tris-HCl buffer (pH 8.0)

  • 200 mM NaCl

  • 4 mM ATP

  • 5 mM MgCl₂

  • tRNA bulk (1 mg/ml)

  • 500 μM L-cysteine

  • 3 mM DTT

  • 1.5 μM IscS (E. coli cysteine desulfurase)

  • 2-40 μM purified TtcA enzyme

• Incubate the reaction mixture at 37°C for 2 hours under anaerobic conditions

• Digest the tRNAs and analyze by HPLC for s²C₃₂ modification

• Confirm s²C₃₂ presence by:

  • Retention time comparison (approximately 10 min in standard conditions)

  • UV-visible spectral analysis

  • Mass spectrometry (expected m/z ratio for protonated pseudo-molecular ion: 260.03)

Complementation assays in ttcA-deficient E. coli strains can also verify in vivo activity .

What are the key steps for purifying recombinant Nitrosomonas europaea TtcA protein?

For successful purification of recombinant N. europaea TtcA:

• Express TtcA with an N-terminal hexahistidine tag in E. coli BL21(DE3)

• Grow cells under the appropriate induction conditions

• Harvest cells and disrupt by sonication or French press

• Clarify lysate by centrifugation

• Purify using immobilized metal affinity chromatography (IMAC)

• Consider further purification using ion exchange or size exclusion chromatography if needed

• Assess purity by SDS-PAGE

For iron-sulfur cluster reconstitution in apoTtcA:

• Perform all steps under strictly anaerobic conditions (e.g., in a glove box with <2 ppm O₂)

• Treat the purified apoprotein with 5 mM DTT for 30 min

• Add 6-fold molar excess of ferrous ammonium sulfate

• Add 10-fold molar excess of L-cysteine

• Add catalytic amount of E. coli cysteine desulfurase (IscS)

• Remove excess Fe and cysteine using gel filtration

How do oxygen limitation conditions affect TtcA expression and function in Nitrosomonas europaea?

Oxygen limitation significantly impacts the gene expression profile of Nitrosomonas europaea, potentially affecting TtcA expression and function. Research investigating N. europaea under oxygen-limited conditions reveals:

• Under oxygen limitation (0.5 mg/L DO vs. 2.0 mg/L DO):

  • Growth yield is reduced

  • Ammonia-to-nitrite conversion becomes non-stoichiometric

  • Cells become more susceptible to environmental stressors

• Transcriptional changes under oxygen limitation include:

  • Upregulation of both heme-copper-containing cytochrome c oxidases

  • Significant increase in B-type heme-copper oxidase (proposed to function as a nitric oxide reductase)

  • Altered expression of genes involved in energy metabolism

What is the role of conserved cysteine residues in TtcA function and iron-sulfur cluster binding?

Conserved cysteine residues play a critical role in TtcA function, particularly in coordinating the [4Fe-4S] cluster essential for enzymatic activity. Based on studies of TtcA from other organisms:

• TtcA contains six conserved cysteine residues, but only three are involved in coordinating the [4Fe-4S] cluster

• Site-directed mutagenesis studies with cysteine-to-alanine substitutions reveal:

• The unique three-cysteine coordination of the [4Fe-4S] cluster, rather than the typical four-cysteine coordination, may be functionally significant

• This atypical coordination may create an open coordination site that is important for substrate binding or catalysis

These findings suggest that TtcA utilizes a specialized Fe-S cluster arrangement for its non-redox thiolation reaction .

How does environmental stress from TiO₂ nanoparticles affect TtcA expression in Nitrosomonas europaea?

Exposure to TiO₂ nanoparticles (NPs) significantly impacts N. europaea at both physiological and transcriptional levels, which may affect TtcA expression and function:

• Initial exposure to 50 mg/L TiO₂ NPs causes:

  • Inhibited cell growth

  • Compromised membrane integrity

  • Reduced nitritation rate

  • Decreased ammonia monooxygenase activity

• After 40 days of chronic exposure, N. europaea demonstrates adaptation with:

  • Recovery of metabolic activities

  • Remission of membrane distortion

  • Adjusted gene expression profiles

• Transcriptional changes during adaptation include:

  • Upregulation of aminoacyl-tRNA biosynthesis genes (gatAB)

  • Increased expression of amino acid biosynthesis genes (gcvTH1, argC, leuA)

  • Enhanced ribosomal protein biogenesis (rpsBU)

  • Stimulation of RNA translation metabolism

• Membrane metabolism regulations observed:

  • Upregulation of acriflavin resistance (NE0669)

  • Increased membrane efflux/fusion protein expression (NE0373, NE0668, NE0670)

  • Enhanced major facilitator transporter (MFC) (NE2454) gene expression

• Low dissolved oxygen (0.5 mg/L) exacerbates TiO₂ NP toxicity, requiring longer adaptation periods

These changes in aminoacyl-tRNA and RNA translation processes likely affect TtcA expression and activity, as TtcA is directly involved in tRNA modification .

What spectroscopic techniques should be used to characterize the iron-sulfur cluster in recombinant Nitrosomonas europaea TtcA?

Comprehensive characterization of the iron-sulfur cluster in TtcA requires multiple complementary spectroscopic techniques:

• UV-visible absorption spectroscopy:

  • Useful for initial confirmation of iron-sulfur cluster presence

  • Characteristic absorption peaks at approximately 320, 410, and 420 nm

  • Can monitor cluster stability under different conditions

• Electron Paramagnetic Resonance (EPR) spectroscopy:

  • Essential for determining the redox state of the cluster

  • [4Fe-4S] clusters typically show signals at g = 2.04, 1.94, and 1.89 when in the reduced state

  • Sample preparation requires anaerobic conditions and appropriate reductants

• Mössbauer spectroscopy:

  • Provides definitive information about the type and oxidation state of iron atoms

  • Requires preparation with ⁵⁷Fe-enriched samples

  • Preparation protocol:

    • Use ⁵⁷Fe-enriched FeCl₃ reduced in situ with 4 mM DTT

    • Perform reconstitution under strictly anaerobic conditions

    • Collect data at multiple temperatures (e.g., 4.2K and 100K)

• Complementary techniques:

  • Iron and sulfide content analysis to confirm the 4:4 stoichiometry

  • Circular dichroism spectroscopy to assess changes in protein structure

  • Resonance Raman spectroscopy for additional cluster characterization

These techniques, combined with site-directed mutagenesis of cysteine residues, provide comprehensive characterization of the iron-sulfur cluster in TtcA .

What research framework should be used when designing experiments for Nitrosomonas europaea TtcA studies?

When designing experiments for N. europaea TtcA studies, researchers should follow the PIONER framework:

Additional self-assessment questions should address:

• Feasibility: Are resources, expertise, and timeframe sufficient?

• Interest: Is the research question relevant to the scientific community?

• Ethics: Have all appropriate oversight processes been engaged?

This framework ensures that TtcA research questions are clearly defined, novel, and relevant to the field .

How can the oxygen sensitivity of TtcA's iron-sulfur cluster be managed during recombinant protein studies?

The [4Fe-4S] cluster in TtcA is oxygen-sensitive, presenting challenges for recombinant protein studies. Recommended approaches include:

• Expression considerations:

  • Use anaerobic expression systems when possible

  • Express protein at lower temperatures (16-18°C) to improve proper folding

  • Consider co-expression with iron-sulfur cluster assembly proteins (ISC system)

• Purification under anaerobic conditions:

  • Conduct all purification steps in an anaerobic chamber with <2 ppm O₂

  • Include reducing agents (e.g., 3-5 mM DTT) in all buffers

  • Use oxygen-scavenging systems in buffers (glucose/glucose oxidase/catalase)

  • Perform rapid purification to minimize exposure time

• Reconstitution of iron-sulfur clusters:

  • Chemical reconstitution using ferrous iron, cysteine, and IscS enzyme

  • Strict anaerobic conditions during reconstitution

  • Buffer optimization to stabilize reconstituted clusters

• Storage and handling:

  • Store protein under argon or nitrogen atmosphere

  • Flash-freeze samples in liquid nitrogen for long-term storage

  • Include glycerol (10-20%) as a cryoprotectant

  • Thaw samples under anaerobic conditions immediately before use

These approaches help maintain the integrity of TtcA's iron-sulfur cluster during experimental procedures .

What strategies can address the challenges of studying TtcA in the context of Nitrosomonas europaea's slow growth?

N. europaea is a slow-growing organism, which presents challenges for studying TtcA expression and function. Effective strategies include:

• Optimized culture conditions:

  • Use modified ATCC #2265 medium to prevent precipitation issues

  • Maintain optimal pH (7.5-8.0) and temperature (28-30°C)

  • Prepare pre-inoculum 10-15 days before experiments

  • Monitor ammonia consumption as growth indicator

• Chemostat cultivation:

  • Enables steady-state growth conditions

  • Allows precise control of dissolved oxygen levels

  • Facilitates reproducible sampling for transcriptomic analysis

  • Permits long-term studies under defined conditions

• Heterologous expression systems:

  • Express N. europaea TtcA in faster-growing hosts (E. coli)

  • Use codon optimization for improved expression

  • Create fusion proteins to enhance stability if needed

• Molecular and analytical techniques:

  • Use FISH combined with flow cytometry for accurate cell counting

  • Employ qRT-PCR for sensitive detection of ttcA transcript levels

  • Consider microarray analysis for global gene expression patterns

  • Use sensitive analytical methods for detecting tRNA modifications

• Complementation approaches:

  • Test N. europaea TtcA function in ttcA-deficient E. coli strains

  • Compare activity with TtcA from other organisms

These approaches can overcome the inherent challenges of studying slowly growing nitrifying bacteria .

How can the effects of environmental stressors on TtcA function be systematically evaluated?

To systematically evaluate how environmental stressors affect TtcA function in N. europaea:

• Integration of data:

  • Correlate transcriptomic changes with enzymatic activity

  • Compare different stressors to identify common response pathways

  • Use statistical modeling to determine significant factors affecting TtcA function

This systematic approach provides comprehensive understanding of how environmental factors influence TtcA expression and activity in N. europaea .

What are the most promising areas for future research on Nitrosomonas europaea TtcA?

Future research on N. europaea TtcA should focus on:

• Structure-function relationships:

  • Crystal structure determination of N. europaea TtcA

  • Comparative analysis with TtcA from other organisms

  • Detailed mechanism of the non-redox thiolation reaction

  • Role of the unique three-cysteine coordination of the [4Fe-4S] cluster

• Physiological significance:

  • Impact of tRNA thiolation on N. europaea fitness under environmental stress

  • Role of TtcA in adaptation to oxygen limitation

  • Connection between TtcA activity and nitrogen metabolism

  • Differential expression of TtcA under various growth conditions

• Ecological and evolutionary aspects:

  • Comparative analysis of TtcA across different ammonia-oxidizing bacteria

  • Evolution of tRNA modification systems in nitrifiers

  • Ecological significance of tRNA modifications in environmental adaptation

• Biotechnological applications:

  • Engineered TtcA variants with enhanced stability or altered specificity

  • Development of TtcA-based biosensors for environmental monitoring

  • Integration of TtcA studies with genome-scale metabolic models of N. europaea

These research directions will contribute to a comprehensive understanding of TtcA's role in N. europaea physiology and ecology .

How might genome-scale metabolic modeling incorporate TtcA function in Nitrosomonas europaea?

Incorporating TtcA function into genome-scale metabolic models of N. europaea requires:

This approach would make the genome-scale model more comprehensive and provide insights into the role of TtcA in N. europaea metabolism .

What methodological advances are needed to better understand TtcA's role in microbial communities?

Advancing our understanding of TtcA's role in microbial communities requires:

• Community-level analytical techniques:

  • Development of methods to track tRNA modifications in complex communities

  • Antibody-based or aptamer-based detection of TtcA in environmental samples

  • Single-cell approaches to monitor TtcA expression in individual bacteria

  • Meta-transcriptomic analyses targeting ttcA genes in environmental samples

• Functional assays:

  • High-throughput methods to detect s²C₃₂ modifications in environmental samples

  • Development of activity-based probes for TtcA function

  • Reporter systems to monitor TtcA activity in vivo

  • Correlation of TtcA activity with community metabolic functions

• Systems biology approaches:

  • Integration of TtcA function into community-level metabolic models

  • Network analysis to identify interactions between TtcA and other cellular processes

  • Machine learning approaches to predict TtcA activity from environmental parameters

  • Multi-scale modeling connecting molecular mechanisms to community dynamics

• Environmental monitoring innovations:

  • Biosensors for detecting changes in tRNA modification patterns

  • Field-deployable methods for monitoring TtcA expression

  • Long-term studies correlating TtcA activity with ecosystem function

  • Community composition analysis tools that include functional genes like ttcA