Recombinant Nitrosomonas europaea Probable tRNA threonylcarbamoyladenosine biosynthesis protein Gcp (gcp)

What is Nitrosomonas europaea and what is its genomic context?

Nitrosomonas europaea (ATCC 19718) is a gram-negative obligate chemolithoautotroph that derives all its energy and reductant for growth from the oxidation of ammonia to nitrite. It plays a crucial role in the biogeochemical nitrogen cycle through the process of nitrification. The genome of N. europaea consists of a single circular chromosome of 2,812,094 base pairs with approximately 50.7% G+C content. The genome encodes a total of 2,460 protein-encoding genes, averaging 1,011 bp in length, with intergenic regions averaging 117 bp .

To study N. europaea effectively, researchers should first obtain pure cultures from established repositories such as ATCC. The organism requires minimal media containing ammonia as an energy source and mineral salts for growth. Due to its obligate chemolithoautotrophic nature, establishing proper growth conditions with controlled ammonia concentrations (typically 25-50 mM) and appropriate aeration is essential for experiments involving protein expression and functional analysis.

How is the Gcp protein characterized within N. europaea genomic organization?

The Gcp protein in N. europaea is characterized as a probable tRNA threonylcarbamoyladenosine biosynthesis protein. Based on genomic organization studies of N. europaea, genes involved in tRNA modification and processing are often found in conserved clusters. The gcp gene has been identified in proximity to other genes involved in translation machinery, including thrS (encoding threonyl tRNA synthetase), infC (initiation factor 3), rplT (ribosomal protein L20), and genes encoding phenylalanyl tRNA synthetase subunits .

For experimental characterization, researchers should employ comparative genomic approaches combined with transcriptional analysis to understand the regulatory context of the gcp gene. Methods such as RT-qPCR and RNA-seq can reveal co-expression patterns with functionally related genes, while ChIP-seq can identify potential transcription factors regulating the expression of gcp.

What is the role of tRNA threonylcarbamoyladenosine biosynthesis in bacterial physiology?

tRNA threonylcarbamoyladenosine biosynthesis is a critical process for proper protein translation and cellular function. This modification occurs at position 37 of tRNAs and is one of the few universally conserved tRNA modifications . The threonylcarbamoyladenosine modification enhances translational fidelity by stabilizing codon-anticodon interactions, preventing frameshifting, and ensuring proper decoding of ANN codons.

In bacteria like N. europaea, which has limited capacity for catabolizing organic compounds but possesses plentiful genes for inorganic ion transporters , maintaining translational accuracy is particularly important for optimizing cellular resources. Research approaches to study the physiological impact include creating knockout or conditional mutants of gcp, followed by phenotypic characterization and ribosome profiling to assess translational fidelity under various growth conditions.

What are the optimal expression systems for producing recombinant N. europaea Gcp protein?

The optimal expression system for producing recombinant N. europaea Gcp protein should consider the protein's properties and research objectives. For high-yield production, E. coli-based expression systems (BL21(DE3), Rosetta, or Arctic Express strains) are recommended, particularly when using pET-series vectors with T7 promoter control.

A recommended methodology includes:

• Gene synthesis or PCR amplification of the gcp gene from N. europaea genomic DNA

• Cloning into an expression vector with an appropriate affinity tag (His6, GST, or MBP)

• Transformation into the selected E. coli strain

• Expression optimization by testing various:

  • Induction temperatures (18°C, 25°C, 30°C, 37°C)

  • IPTG concentrations (0.1-1.0 mM)

  • Expression durations (4-24 hours)

For difficult-to-express proteins, consider codon optimization for E. coli, fusion with solubility-enhancing tags, or expression in cell-free systems. Researchers working with N. europaea proteins should be aware that its moderately high G+C content (50.7%) might necessitate codon optimization for efficient expression in some systems.

What purification strategies yield the highest purity and activity of recombinant Gcp?

A multi-step purification approach is recommended to achieve high purity and activity of recombinant Gcp protein:

Table 1: Recommended Purification Protocol for Recombinant N. europaea Gcp

For optimal activity preservation, all buffers should include 1 mM DTT or 2 mM β-mercaptoethanol and protease inhibitors during initial steps. Considering that N. europaea proteins may be sensitive to salinity changes , careful optimization of salt concentrations during purification is advised.

Activity assays should be performed after each purification step to monitor the retention of enzymatic function. These assays typically involve measuring the protein's ability to catalyze tRNA modification using radiolabeled substrates or mass spectrometry-based approaches.

How can researchers verify the proper folding and activity of recombinant Gcp?

Verification of proper folding and activity of recombinant Gcp involves both structural and functional analyses:

• Structural verification methods:

  • Circular Dichroism (CD) spectroscopy to assess secondary structure content

  • Thermal shift assays to evaluate protein stability

  • Limited proteolysis to probe for folded domains

  • Dynamic Light Scattering (DLS) to check for aggregation

• Functional activity assays:

  • In vitro tRNA modification assays using purified tRNA substrates

  • Mass spectrometry to detect threonylcarbamoyladenosine formation

  • Complementation assays in gcp-deficient bacterial strains

For N. europaea Gcp specifically, researchers should consider performing activity assays under conditions that mimic the bacterium's natural environment, including appropriate pH (around 7.0-7.5) and metal cofactors. Since N. europaea possesses numerous genes for iron acquisition , testing the effect of iron availability on Gcp activity would be informative.

How does the structure of N. europaea Gcp compare to homologs in other bacteria?

While specific structural data for N. europaea Gcp is limited in the provided search results, comparative analysis with known Gcp structures from other bacteria can provide valuable insights.

Gcp proteins belong to a conserved family present across all domains of life. They typically contain a metallopotease-like domain and require metal ions (often zinc) for catalytic activity. Structural comparison can be conducted using:

• Homology modeling based on crystal structures of Gcp homologs

• Multiple sequence alignment to identify conserved catalytic residues

• Conservation mapping onto structural models to predict functional sites

• Molecular dynamics simulations to assess structural stability

Based on sequencing data, the N. europaea genome encodes numerous metal ion transporters, particularly for iron acquisition , suggesting that metal availability may be important for Gcp function in this organism. Researchers should investigate whether iron or other metals serve as cofactors for the N. europaea Gcp, which would represent an adaptation to its specialized ecological niche.

What experimental approaches can resolve the catalytic mechanism of N. europaea Gcp?

Resolving the catalytic mechanism of N. europaea Gcp requires a combination of biochemical, biophysical, and structural approaches:

• Site-directed mutagenesis of predicted catalytic residues combined with activity assays to identify essential amino acids

• Enzyme kinetics studies with various substrates (tRNA, threonine, carbamoyl donors) to determine:

  • Km and Vmax values

  • Reaction order

  • pH and temperature optima

  • Cofactor requirements

• Structural studies including:

  • X-ray crystallography of Gcp alone and in complex with substrates

  • Cryo-EM analysis of Gcp-tRNA complexes

  • NMR for dynamics analysis

• Reaction intermediate trapping using:

  • Rapid quench-flow techniques

  • Modified substrates

  • Mass spectrometry analysis

Given that N. europaea has adapted to a chemolithoautotrophic lifestyle with specific metabolic pathways , investigating whether its Gcp has unique catalytic properties compared to homologs from heterotrophic bacteria would provide valuable insights into potential adaptation of this enzyme to N. europaea's specialized metabolism.

How does Gcp interact with the cellular tRNA pool and other components of the translation machinery?

Understanding Gcp interactions with cellular components requires studies that capture both direct physical interactions and functional relationships:

• Protein-tRNA interaction studies:

  • RNA electrophoretic mobility shift assays (EMSA)

  • Filter binding assays

  • Surface plasmon resonance

  • CLIP-seq (crosslinking immunoprecipitation-sequencing) to identify tRNA targets in vivo

• Protein-protein interaction studies:

  • Co-immunoprecipitation followed by mass spectrometry

  • Bacterial two-hybrid screening

  • Proximity labeling (BioID or APEX)

  • Crosslinking mass spectrometry

• Integration with translation machinery:

  • Ribosome profiling to assess translation effects

  • Polysome fractionation to determine association with active ribosomes

  • In vitro translation assays with modified and unmodified tRNAs

Given that N. europaea's genome contains genes encoding threonyl tRNA synthetase (thrS) and other components of the translation machinery in proximity to gcp , researchers should investigate potential functional coupling between these proteins, which might reflect co-evolution of the tRNA modification and aminoacylation systems in this specialized bacterium.

How does salinity stress affect the expression and function of Gcp in N. europaea?

A comprehensive approach to study salinity effects on Gcp would include:

• Expression analysis:

  • RT-qPCR and Western blotting to quantify gcp mRNA and protein levels under various salinity conditions

  • Promoter-reporter fusions to monitor transcriptional regulation

  • Proteomics to place Gcp regulation in the context of global salinity response

• Functional analysis:

  • tRNA modification profiling by mass spectrometry to assess changes in threonylcarbamoyladenosine levels

  • Translation efficiency measurements using reporter constructs

  • Metabolic labeling to measure protein synthesis rates

N. europaea exhibits specific adaptations to salinity stress, including changes in transporters and carbon metabolism enzymes . Since tRNA modifications can regulate translation efficiency under stress conditions, researchers should investigate whether Gcp-mediated tRNA modification serves as a regulatory mechanism during osmotic adaptation in this ammonia-oxidizing bacterium.

What is the relationship between ammonia oxidation pathways and tRNA modification in N. europaea?

The relationship between ammonia oxidation (N. europaea's primary energy source) and tRNA modification represents an intriguing intersection of energy metabolism and translational regulation:

• Metabolic coupling analysis:

  • Measure Gcp activity and tRNA modification levels under different ammonia oxidation rates

  • Assess the effects of ammonia oxidation inhibitors on tRNA modification

  • Investigate potential regulatory links between nitrogen and carbon metabolism and tRNA modification

• Transcriptional co-regulation:

  • Analyze promoter elements of gcp and ammonia oxidation genes

  • Perform ChIP-seq to identify common transcription factors

  • Use global transcriptomics to establish co-expression networks

The search results indicate that N. europaea's ammonia oxidation machinery (including ammonia monooxygenase) can be affected by environmental stressors, with one subunit (ammonia monooxygenase subunit B) showing reduced abundance under salinity stress . Researchers should investigate whether similar regulatory mechanisms affect Gcp expression and whether tRNA modification status influences the translation of mRNAs encoding proteins involved in ammonia oxidation.

How does carbon metabolism in N. europaea influence Gcp function and tRNA modification?

N. europaea, as an autotroph, has a distinctive carbon metabolism centered on CO2 fixation. The search results reveal specific adaptations in carbon metabolism under salinity stress, including increased abundance of carbonic anhydrase and enzymes of the Pentose Phosphate Pathway (PPP) .

To investigate the relationship between carbon metabolism and Gcp function:

• Metabolic manipulation experiments:

  • Culture N. europaea under different CO2 concentrations and analyze Gcp expression and activity

  • Use stable isotope labeling (13C) to track carbon flow through central metabolism and potential incorporation into tRNA modifications

  • Inhibit specific steps of carbon metabolism and assess impacts on tRNA modification

• Integration with stress responses:

  • Analyze tRNA modification status during carbon limitation

  • Investigate whether carbon metabolism intermediates regulate Gcp activity

  • Assess translation of carbon metabolism enzymes in Gcp-deficient strains

Table 2: Carbon Metabolism Enzymes with Differential Expression Under Salinity Stress in N. europaea

This pattern of upregulation in PPP enzymes suggests a shift toward generating NADPH under salinity stress, which could affect the redox environment in which Gcp functions. Researchers should investigate whether these metabolic shifts indirectly influence tRNA modification through changes in cellular energetics or redox state.

What are the most effective genetic manipulation techniques for studying Gcp function in N. europaea?

• Gene knockout/knockdown strategies:

  • Homologous recombination for gene replacement (efficiency may be low)

  • CRISPR-Cas9 system adapted for N. europaea

  • Antisense RNA for conditional knockdown

  • Inducible degradation tags for regulated protein depletion

• Complementation and expression systems:

  • Integration of complementation constructs at neutral genomic loci

  • Development of N. europaea-specific expression vectors with appropriate promoters

  • Heterologous expression in related bacteria followed by functional testing

• Reporter systems:

  • Translational fusions to track Gcp localization

  • Transcriptional fusions to monitor gcp expression

  • Fluorescent tRNA reporters to assess modification status in vivo

When designing genetic manipulation experiments, researchers should consider N. europaea's specific genomic features, including its GC content (50.7%) and the presence of repetitive elements that constitute approximately 5% of the genome, including 85 predicted insertion sequence elements in eight different families .

How can advanced mass spectrometry techniques be applied to study tRNA modifications in N. europaea?

Mass spectrometry represents a powerful approach for studying tRNA modifications, including threonylcarbamoyladenosine:

• Sample preparation protocols:

  • Total tRNA isolation from N. europaea cultures using acidic phenol extraction

  • Specific tRNA purification by oligonucleotide-directed RNase H cleavage

  • Enzymatic digestion to nucleosides using nuclease P1 and bacterial alkaline phosphatase

• Analysis techniques:

  • Liquid chromatography-mass spectrometry (LC-MS/MS) for nucleoside quantification

  • High-resolution mass spectrometry for accurate mass determination

  • Tandem mass spectrometry for structural characterization

  • Multiple reaction monitoring (MRM) for targeted quantification

• Comparative analysis frameworks:

  • Profiling tRNA modifications under different growth conditions

  • Comparing wild-type and gcp mutant strains

  • Temporal analysis during different growth phases

The search results indicate that N. europaea undergoes significant proteomic changes in response to environmental stressors . Researchers should apply these mass spectrometry techniques to investigate whether tRNA modification patterns also change under these conditions, potentially representing an additional layer of stress response regulation.

What computational approaches can predict the impact of Gcp-mediated tRNA modifications on the N. europaea proteome?

Computational approaches can provide insights into the functional implications of tRNA modifications across the proteome:

• Codon usage analysis:

  • Calculate codon adaptation indices for all N. europaea genes

  • Identify genes enriched in codons that rely on threonylcarbamoyladenosine-modified tRNAs

  • Compare codon usage in highly expressed genes versus average genes

• Translation efficiency prediction:

  • Develop models correlating tRNA modification status with translation rates

  • Predict mRNAs most sensitive to changes in modification status

  • Simulate the effects of reduced modification on proteome composition

• Evolutionary analysis:

  • Compare tRNA gene sequences and anticodon modifications across related bacteria

  • Identify selection pressures on tRNA modification systems

  • Correlate tRNA modification systems with ecological niches

• Systems biology integration:

  • Create network models linking tRNA modification to cellular processes

  • Predict metabolic flux changes resulting from altered translation efficiency

  • Identify potential regulatory feedback loops

Given N. europaea's specialized metabolism and ecological niche, researchers should investigate whether its codon usage and tRNA modification systems have co-evolved to optimize translation of genes critical for ammonia oxidation and autotrophic growth, which would represent an adaptation at the level of translation regulation.