Recombinant Nitrosomonas europaea Glucans biosynthesis protein G (opgG)

What is Recombinant Nitrosomonas europaea Glucans biosynthesis protein G and its structural characteristics?

Recombinant Nitrosomonas europaea Glucans biosynthesis protein G (opgG) is a protein involved in glucan synthesis pathways in the ammonia-oxidizing bacterium Nitrosomonas europaea. The recombinant protein has a UniProt accession number Q82SA9 and consists of 489 amino acids with an expression region covering amino acids 30-518 of the full protein sequence . Its primary structure contains specific functional domains characteristic of glucan synthesis enzymes, and it is typically produced with >85% purity as determined by SDS-PAGE analysis . The complete amino acid sequence includes multiple functional regions that contribute to its catalytic activity and substrate binding capabilities.

What is the genetic context of opgG in Nitrosomonas europaea?

Nitrosomonas europaea's genome consists of a single circular chromosome of 2,812,094 bp, with genes distributed evenly around the genome . The organism contains 2,460 protein-encoding genes, which average 1,011 bp in length . While the search results do not provide the exact genomic location of opgG, Nitrosomonas europaea's genome has been fully sequenced and characterized, allowing researchers to understand the genetic context of opgG relative to other metabolic and structural genes. This bacterium participates in the biogeochemical nitrogen cycle through nitrification, converting ammonia to nitrite, which provides the metabolic context in which opgG functions .

How should recombinant opgG be properly stored and handled in laboratory settings?

For optimal stability and activity of recombinant Nitrosomonas europaea opgG, specific storage conditions are recommended based on the protein's formulation:

• Liquid form: Store at -20°C/-80°C with an expected shelf life of approximately 6 months

• Lyophilized form: Store at -20°C/-80°C with an expected shelf life of approximately 12 months

• Working aliquots: Store at 4°C for up to one week to avoid repeated freeze-thaw cycles

For reconstitution of lyophilized protein, it is recommended to briefly centrifuge the vial before opening and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL . Addition of glycerol to a final concentration of 5-50% is advised for long-term storage, with 50% being the standard recommendation . Repeated freezing and thawing should be avoided as it may lead to protein denaturation and loss of activity.

How does opgG function within Nitrosomonas europaea's adaptive responses to environmental stressors?

Nitrosomonas europaea possesses specific mechanisms to cope with environmental stressors such as low dissolved oxygen (DO) concentrations and high nitrite levels . While the direct role of opgG in these responses isn't explicitly detailed in the available data, as a glucans biosynthesis protein, it likely plays a significant role in cell wall modification under stress conditions. Research shows that N. europaea exhibits distinct responses to DO limitation, including increased mRNA concentrations of ammonia oxidation genes (amoA) and hydroxylamine oxidation genes (hao) during exponential growth phase under low DO conditions . These adaptations suggest a complex regulatory network in which opgG may participate to maintain cell wall integrity under stress.

What is the relationship between opgG and nitrification processes in Nitrosomonas europaea?

Nitrosomonas europaea is a chemolithoautotrophic bacterium that derives its energy and reducing power from ammonia oxidation . The nitrification process involves several key enzymes encoded by genes such as amoA, hao, nirK, and norB . While the specific interaction between opgG and these nitrification enzymes isn't directly addressed in the available data, the cell wall structures that opgG helps synthesize likely provide the cellular architecture necessary for proper functioning of membrane-associated nitrification enzymes. Understanding this relationship would require experimental approaches such as co-immunoprecipitation studies or mutational analyses to determine whether opgG expression affects nitrification rates or enzyme localization.

What expression systems are optimal for producing recombinant Nitrosomonas europaea opgG?

Based on the available data, recombinant Nitrosomonas europaea opgG is effectively produced using a Baculovirus expression system . This system provides several advantages for producing functional bacterial proteins, including proper folding and post-translational modifications. The expression construct typically includes the mature protein sequence (amino acids 30-518), omitting any signal peptides or regulatory regions that might interfere with expression . Researchers should consider the following factors when designing expression systems for opgG:

The choice of tag for purification will be determined during the manufacturing process and should be considered based on the specific experimental requirements .

What analytical techniques are recommended for studying opgG structure and function?

To comprehensively study the structure and function of Nitrosomonas europaea opgG, researchers should employ a combination of biochemical, biophysical, and genetic approaches:

• Structural Analysis:

  • X-ray crystallography to determine three-dimensional structure

  • Circular dichroism spectroscopy to assess secondary structure elements

  • Mass spectrometry for protein characterization and post-translational modifications

• Functional Assays:

  • Enzyme activity assays measuring glucan synthesis

  • Substrate binding studies using isothermal titration calorimetry

  • Site-directed mutagenesis to identify catalytic residues

• Cellular Localization:

  • Immunofluorescence microscopy with anti-opgG antibodies

  • Subcellular fractionation followed by Western blotting

  • Green fluorescent protein (GFP) fusion studies

These approaches should be adapted based on specific research questions and the cellular context of N. europaea, which is an obligate chemolithoautotroph with specific growth requirements .

How can researchers effectively generate and characterize opgG mutants?

Creating and characterizing opgG mutants is essential for understanding its function in Nitrosomonas europaea. Based on standard molecular biology approaches, researchers should consider:

• Mutation Strategy Selection:

  • Site-directed mutagenesis for specific amino acid changes

  • Deletion mutagenesis to remove functional domains

  • Random mutagenesis to identify critical regions

• Genetic Tools:

  • Homologous recombination for chromosomal integration

  • CRISPR-Cas9 system for precise genome editing

  • Complementation studies to confirm phenotype specificity

• Phenotypic Characterization:

  • Growth rate analysis under various environmental conditions

  • Cell wall composition analysis

  • Stress response assays (oxygen limitation, nitrite exposure)

  • Microscopy to assess morphological changes

When designing mutagenesis experiments, researchers should pay particular attention to the conserved domains identified in the protein sequence and consider the genomic context of opgG within N. europaea's single circular chromosome .

How does opgG interact with other proteins in Nitrosomonas europaea's metabolic network?

Understanding the protein-protein interactions of opgG requires an integrative approach combining genomic, proteomic, and functional analyses. Nitrosomonas europaea has a relatively compact genome with 2,460 protein-encoding genes , which facilitates comprehensive interaction studies. Potential research approaches include:

• Computational Predictions:

  • Genomic context analysis to identify functionally related genes

  • Protein domain interaction predictions

  • Structural modeling of potential interaction interfaces

• Experimental Validation:

  • Co-immunoprecipitation followed by mass spectrometry

  • Bacterial two-hybrid assays

  • Cross-linking studies to capture transient interactions

• Functional Validation:

  • Co-expression analysis under different growth conditions

  • Mutational studies of predicted interaction partners

  • Physiological assays measuring cellular responses

Special attention should be given to potential interactions with proteins involved in ammonia oxidation and nitrite reduction pathways, as these are central to N. europaea's metabolism and environmental adaptations .

What is the evolutionary significance of opgG across different ammonia-oxidizing bacteria?

Comparative genomic analyses can provide insights into the evolutionary conservation and specialization of opgG across different ammonia-oxidizing bacteria. Although specific details about opgG conservation aren't provided in the search results, the comparative genomics section in the genome study of N. europaea provides a framework for such analyses . Research approaches should include:

• Phylogenetic Analysis:

  • Sequence alignment of opgG homologs across bacterial species

  • Construction of phylogenetic trees to trace evolutionary history

  • Identification of conserved and variable regions

• Functional Domain Analysis:

  • Mapping of conserved domains across different species

  • Identification of species-specific adaptations

  • Correlation with ecological niches and metabolic capabilities

• Genomic Context Comparison:

  • Analysis of gene neighborhoods across species

  • Identification of synteny and genome rearrangements

  • Correlation with metabolic specializations

These approaches would help determine whether opgG has undergone adaptive evolution in N. europaea and related bacteria in response to specific environmental pressures or metabolic specializations.

What are the promising applications of recombinant opgG in biotechnology and environmental studies?

Based on the understanding of Nitrosomonas europaea's role in the nitrogen cycle and the structural function of opgG, several promising research directions emerge:

• Bioremediation Enhancement:

  • Engineering opgG to improve N. europaea's resilience in wastewater treatment

  • Developing opgG-based biosensors for monitoring nitrification efficiency

  • Creating optimized strains for specific environmental conditions

• Structural Biology Advances:

  • Determining the high-resolution structure of opgG

  • Mapping the catalytic mechanism for glucan synthesis

  • Engineering modified versions with enhanced stability or activity

• Environmental Monitoring Tools:

  • Developing antibodies against opgG for monitoring ammonia-oxidizing bacterial populations

  • Creating diagnostic tools for assessing nitrification potential in soil and water

Research in these areas would build upon the understanding that N. europaea participates in important environmental processes including nitrogen cycling and has potential for bioremediation of sites contaminated with chlorinated aliphatic hydrocarbons .

What techniques are emerging for studying opgG regulation under different environmental conditions?

To understand how opgG expression and activity are regulated under different environmental conditions, researchers should consider emerging high-throughput and single-cell techniques:

• Transcriptomics Approaches:

  • RNA-seq to capture global expression patterns in response to environmental changes

  • Ribosome profiling to assess translational regulation

  • Single-cell RNA-seq to capture cell-to-cell variability

• Protein-Level Analyses:

  • Proteomics to measure opgG abundance under different conditions

  • Phosphoproteomics to identify regulatory post-translational modifications

  • Activity-based protein profiling to assess functional state

• Real-time Monitoring:

  • Reporter gene fusions for in vivo expression monitoring

  • Microfluidic systems for controlled environmental perturbations

  • Time-lapse microscopy to visualize dynamic responses

These approaches would build upon the existing knowledge that N. europaea has specific mechanisms to cope with environmental stressors such as low dissolved oxygen and high nitrite concentrations , and would help elucidate how opgG contributes to these adaptive responses.