Recombinant Nitrosomonas europaea Phenylalanine--tRNA ligase alpha subunit (pheS)

What is the genomic context of pheS in Nitrosomonas europaea?

The pheS gene in Nitrosomonas europaea is located within a conserved gene arrangement between ammonia monooxygenase (amo) and hydroxylamine oxidoreductase (hao) gene clusters. Specifically, it resides in the 15.5-kb intergenic spacer region between these two important ammonia oxidation gene clusters. The genes in this region include threonyl tRNA synthetase (thrS), initiation factor 3 (infC), ribosomal protein L20 (rplT), and phenylalanyl tRNA synthetase α and β subunits (pheS and pheT) . This conserved arrangement is also found in other Nitrosomonas strains such as Nitrosomonas sp. strain ENI-11, suggesting evolutionary importance of this gene organization . The proximity of pheS to these ammonia oxidation genes may indicate functional coordination between protein synthesis and energy metabolism in this specialized bacterium.

How does pheS contribute to the obligate chemolithoautotrophic lifestyle of N. europaea?

As an obligate chemolithoautotroph, N. europaea has highly specialized metabolic requirements and relies on a streamlined set of genes for survival. The pheS gene supports this lifestyle by ensuring accurate protein synthesis necessary for the specialized enzymatic machinery involved in ammonia oxidation and carbon fixation. The genome of N. europaea contains limited genes for catabolism of organic compounds, with a focus instead on genes necessary for biosynthesis, energy generation from ammonia, and CO2 and NH3 assimilation . In this context, pheS helps maintain translational fidelity for the approximately 2,460 protein-coding genes identified in N. europaea, including the critical enzymes for energy metabolism. The conservation of pheS and its genomic location across related Nitrosomonas strains suggests its importance in supporting this specialized metabolic lifestyle.

What key controls should be included in experiments with recombinant pheS?

Robust experimental design with recombinant N. europaea pheS requires thoughtful selection of controls. Positive controls should include a known functional phenylalanine-tRNA ligase, ideally from a related organism with similar biochemical properties. Negative controls should include reaction mixtures lacking either the enzyme, substrate tRNA, or phenylalanine, to establish baseline measurements and identify any non-specific activities. When studying protein-protein interactions or complex formation with pheT (beta subunit), controls should include individual subunits tested separately. For kinetic studies, researchers should include time-course experiments and substrate concentration gradients to establish Michaelis-Menten parameters. Since experimental design decisions significantly impact results, as noted in the literature on contradictory research outcomes , researchers should clearly report all control conditions. This is particularly important when studying enzymes from specialized organisms like N. europaea, where the native cellular environment (including ion concentrations and pH) differs from standard laboratory conditions.

How might pheS contribute to the adaptation of N. europaea to environmental changes?

As a specialized ammonia-oxidizing bacterium, N. europaea must adapt to fluctuating environmental conditions while maintaining its obligate chemolithoautotrophic lifestyle. The pheS gene potentially contributes to this adaptation by ensuring accurate protein synthesis under varying conditions. The genome of N. europaea contains "genes encoding transporters for inorganic ions were plentiful, whereas genes encoding transporters for organic molecules were scant" , indicating a metabolism focused on inorganic substrates. Accurate translation by pheS is critical for maintaining these specialized transport systems and metabolic enzymes. Furthermore, the genomic context of pheS, positioned between ammonia oxidation gene clusters, suggests possible co-regulation with energy metabolism genes in response to environmental changes. The genome also contains numerous insertion sequence elements (85 in eight different families) , which could influence gene expression under stress conditions. Future research could investigate whether pheS expression or activity changes under different environmental conditions, potentially contributing to metabolic adjustments in this highly specialized organism.

What expression systems are suitable for producing recombinant N. europaea pheS?

Several expression systems have been successfully employed for producing recombinant N. europaea pheS, each with distinct advantages depending on research objectives. Based on available commercial products, three main expression systems have been utilized:

For basic enzymatic studies, the E. coli system is typically sufficient and provides adequate protein quantities . For structural studies requiring properly folded protein, yeast or baculovirus systems may be preferable. Some suppliers also offer specialty versions, such as Avi-tag biotinylated protein (CSB-EP744838MLA-B), which enables specific protein immobilization for interaction studies . When designing expression constructs, researchers should consider codon optimization for the selected expression system, as N. europaea has a GC content of 50.7% , which differs from common expression hosts.

What purification strategies are most effective for recombinant N. europaea pheS?

Effective purification of recombinant N. europaea pheS requires a multi-step strategy tailored to the protein's properties and the expression system used. While specific purification protocols for this protein are not detailed in the search results, general approaches can be recommended based on properties of aminoacyl-tRNA synthetases. A common strategy involves affinity chromatography using a fusion tag (His6, GST, or Avi-tag), followed by ion exchange chromatography exploiting the protein's charge properties, and size exclusion chromatography as a polishing step. For co-purification of the complete enzyme with both alpha (pheS) and beta (pheT) subunits, tandem affinity purification may be employed. Quality control should include SDS-PAGE to assess purity, Western blotting for identity confirmation, and activity assays measuring aminoacylation of tRNAPhe. Mass spectrometry can verify integrity and post-translational modifications. Given that N. europaea pheS functions in a specialized bacterium with unique metabolic requirements, buffers containing appropriate ions (particularly Mg2+) should be used throughout purification to maintain enzyme stability and activity. The purification strategy should be adjusted based on the intended application, with structural studies requiring higher purity than basic activity assays.

How can researchers assess the functional activity of recombinant N. europaea pheS?

To assess the functional activity of recombinant N. europaea pheS, researchers should employ a combination of biochemical and biophysical approaches. Since pheS encodes the alpha subunit of phenylalanine-tRNA ligase, functional assays should measure its aminoacylation activity—the attachment of phenylalanine to tRNAPhe. The standard aminoacylation assay involves incubating the enzyme with phenylalanine, ATP, and tRNAPhe, followed by quantification of charged tRNAPhe using either radioactive [14C/3H]-phenylalanine incorporation or more modern non-radioactive methods such as thin-layer chromatography or HPLC. When performing these assays, researchers should note that the alpha subunit (pheS) typically requires the beta subunit (pheT) for full activity, so reconstitution of the complete enzyme may be necessary. Kinetic parameters (KM, kcat) should be determined for all substrates (phenylalanine, ATP, and tRNAPhe) under varying conditions (pH, temperature, ion concentrations) to establish optimal reaction conditions. Given that N. europaea has a specialized metabolism, comparing its pheS activity parameters with those from other bacteria may provide insights into potential adaptations. Additionally, thermal shift assays can assess protein stability, while isothermal titration calorimetry can measure substrate binding affinities.

What bioinformatic approaches can provide insights into N. europaea pheS structure and function?

Bioinformatic approaches offer valuable insights into N. europaea pheS structure and function without requiring extensive laboratory work. Sequence analysis should begin with multiple sequence alignment of pheS from N. europaea with homologs from related nitrifying bacteria and model organisms to identify conserved catalytic residues and unique sequence features. Phylogenetic analysis can place N. europaea pheS in evolutionary context, potentially revealing adaptation patterns related to its specialized metabolism. Structural prediction using homology modeling based on crystallized phenylalanine-tRNA ligases can generate hypotheses about substrate binding pockets and subunit interactions. Researchers should pay particular attention to the interface between pheS and pheT subunits, as this interaction is critical for enzyme function. Genomic context analysis already reveals interesting patterns, with pheS positioned between ammonia oxidation gene clusters in a conserved arrangement . This positioning may suggest co-regulation patterns that can be further explored through promoter analysis and transcriptome data mining. Network analysis incorporating protein-protein interaction predictions may identify potential regulatory partners beyond the expected pheT beta subunit. These bioinformatic approaches generate testable hypotheses that can guide subsequent experimental work with recombinant pheS.

What genomic insights from N. europaea can inform pheS research?

The complete genome sequence of N. europaea provides valuable context for pheS research. With a single circular chromosome of 2,812,094 bp and a GC content of 50.7%, the genome contains 2,460 protein-encoding genes with an average length of 1,011 bp . The proximity of pheS to ammonia oxidation genes offers important insights for researchers. Located in a 15.5-kb intergenic spacer between amo and hao gene clusters, pheS is part of a conserved gene arrangement that includes thrS, infC, rplT, and pheT , suggesting potential co-regulation or functional coordination. This genomic context should inform experimental design, particularly when studying transcriptional regulation or protein-protein interactions. The genome also features 85 insertion sequence elements in eight different families, constituting approximately 5% of the genome , which may influence gene expression patterns. Researchers studying pheS should consider whether these mobile genetic elements affect pheS expression under different conditions. Additionally, the comparative genomic analysis showing conservation of pheS positioning in related Nitrosomonas strains despite larger genomic rearrangements suggests strong evolutionary pressure to maintain this arrangement, which may relate to functional importance in this specialized bacterium.

How can researchers integrate pheS studies with broader understanding of N. europaea metabolism?

To integrate pheS studies with the broader understanding of N. europaea metabolism, researchers should consider the enzyme's role within the context of this bacterium's specialized chemolithoautotrophic lifestyle. N. europaea derives all its energy from ammonia oxidation and must fix carbon dioxide for growth . In this metabolic framework, protein synthesis supported by pheS is essential for maintaining the enzymatic machinery required for energy generation and carbon fixation. Researchers should investigate potential regulatory links between pheS expression/activity and the bacterium's central metabolic processes. The genomic proximity of pheS to ammonia oxidation genes suggests possible co-regulation , which could be explored through transcriptomic studies under varying ammonium concentrations or carbon availability. Metabolic flux analysis incorporating isotope labeling could reveal how amino acid metabolism, including phenylalanine, connects to central carbon and nitrogen metabolism in this specialized organism. Additionally, researchers should consider how the high number of transporters for inorganic ions but limited transporters for organic molecules might affect cellular conditions relevant to pheS function. This integration would contribute to understanding how protein synthesis is coordinated with energy metabolism in this environmentally important nitrifying bacterium.

What potential biotechnological applications exist for N. europaea pheS?

N. europaea pheS offers several promising biotechnological applications beyond basic research. As an aminoacyl-tRNA synthetase from an organism with specialized metabolism, it may have unique properties advantageous for certain applications. One potential application is as a selection marker in bacterial genetics, exploiting the toxicity of certain phenylalanine analogs in cells with functional pheS. Mutant forms of pheS could be developed that confer resistance to these analogs, providing a counterselection system for genetic manipulation of bacteria, particularly those where traditional antibiotic selection is challenging. Another application lies in synthetic biology, where orthogonal aminoacyl-tRNA synthetase/tRNA pairs are used to incorporate non-canonical amino acids into proteins. The unique properties of N. europaea pheS might make it suitable for engineering such systems, expanding the toolkit for protein engineering. In environmental biotechnology, understanding pheS function could contribute to optimizing nitrification processes in wastewater treatment, where Nitrosomonas species play crucial roles. Finally, given that N. europaea participates in the biogeochemical nitrogen cycle , its pheS could be explored as a potential biomarker for monitoring nitrification in environmental samples, providing insights into microbial community function in different ecosystems.

How might protein engineering of pheS enhance our understanding of N. europaea metabolism?

Protein engineering of N. europaea pheS offers a powerful approach to probe the specialized metabolism of this ammonia-oxidizing bacterium. Site-directed mutagenesis targeting catalytic residues and substrate binding sites could generate variants with altered specificity or activity, providing insights into structure-function relationships. Creating chimeric proteins by swapping domains between N. europaea pheS and homologs from other bacteria might reveal adaptations specific to ammonia-oxidizing metabolism. Researchers could engineer inducible or repressible pheS variants to control protein synthesis in vivo, allowing investigation of how translation rates affect ammonia oxidation and carbon fixation. Given the genomic proximity of pheS to ammonia oxidation genes , introducing reporter fusions could elucidate potential co-regulation mechanisms. More advanced approaches include directed evolution to identify mutations that enhance activity under conditions mimicking environmental stresses faced by N. europaea. Complementation studies using engineered pheS variants in model organisms could assess functional conservation and specialization. These protein engineering approaches would not only enhance understanding of aminoacyl-tRNA synthetase biology but also provide insights into how translation is integrated with the specialized energy metabolism of this environmentally important bacterium.

What role might pheS play in adaptation of N. europaea to environmental stressors?

The potential role of pheS in N. europaea's adaptation to environmental stressors represents an important research frontier. As an obligate chemolithoautotroph residing in dynamic environments, N. europaea must maintain protein synthesis under varying conditions. The pheS gene, encoding a critical enzyme for translation, may contribute to stress adaptation through several mechanisms. Under oxidative stress, which is particularly relevant for ammonia-oxidizing bacteria generating reactive nitrogen species, maintaining accurate phenylalanine incorporation is essential as phenylalanine residues are often targets of oxidative damage. The genomic context of pheS, positioned between ammonia oxidation gene clusters , suggests possible co-regulation with energy metabolism genes during stress responses. Researchers should investigate whether pheS expression or activity changes under stressors like ammonia limitation, pH fluctuations, or heavy metal exposure. Comparative analysis with other Nitrosomonas strains could reveal whether pheS sequence variations correlate with environmental adaptations, particularly given the conservation of genomic arrangement despite larger genomic rearrangements between strains . The specialized lifestyle of N. europaea, with its limited number of genes for organic compound catabolism but numerous transporters for inorganic ions , suggests a streamlined metabolism where accurate protein synthesis by pheS may be particularly crucial during stress adaptation.

What are the most promising future research directions for N. europaea pheS studies?

Future research on N. europaea pheS should pursue several promising directions to enhance understanding of this protein's role in ammonia-oxidizing bacteria. Structural studies using X-ray crystallography or cryo-electron microscopy would reveal the detailed architecture of the enzyme, particularly in complex with the beta subunit (pheT) and substrates. This structural information could explain any unique adaptations to N. europaea's specialized metabolism. Systems biology approaches integrating transcriptomics, proteomics, and metabolomics could elucidate how pheS expression and activity coordinate with ammonia oxidation pathways under varying environmental conditions. Given the conserved genomic positioning of pheS between ammonia oxidation gene clusters across Nitrosomonas strains , chromatin immunoprecipitation sequencing (ChIP-seq) could identify shared regulatory elements. In vivo studies using gene editing, now possible in previously challenging bacterial systems, could assess the phenotypic effects of pheS mutations. Comparative studies across multiple ammonia-oxidizing bacteria would reveal evolutionary patterns in aminoacyl-tRNA synthetase adaptation to chemolithoautotrophic metabolism. Finally, exploring the potential biotechnological applications of N. europaea pheS, particularly in environmental monitoring, synthetic biology, and nitrogen cycle engineering, represents an exciting frontier. These multidisciplinary approaches would significantly advance understanding of both fundamental aminoacyl-tRNA synthetase biology and specialized bacterial metabolism.