Recombinant Nitrosomonas europaea UPF0102 protein NE0719 (NE0719)

What is Nitrosomonas europaea UPF0102 protein NE0719?

NE0719 is an uncharacterized protein belonging to the UPF0102 family found in Nitrosomonas europaea strain ATCC 19718. It is a small protein of 118 amino acids encoded by a gene located at position 774690-775046 on the negative strand of the N. europaea chromosome . The protein belongs to a family of conserved bacterial proteins with unknown function. As part of the UPF0102 family, it contains conserved domains whose specific functions remain unelucidated, making it a target for fundamental research on bacterial protein functions.

What is the genomic context of the NE0719 gene in N. europaea?

The NE0719 gene is situated within a genetic neighborhood that provides potential clues to its function. According to the genome annotation, NE0719 is positioned near genes encoding:

• NE0718: A hypothetical protein (located at position 773820-774359)

• NE0720: An ABC transporter with fused permease and ATPase domains (located at position 775153-776940)

This genomic arrangement suggests that NE0719 might be functionally related to membrane transport processes or might be co-regulated with these neighboring genes. Researchers can use this contextual information to guide hypotheses about NE0719 function.

What are the properties of the host organism, Nitrosomonas europaea?

Nitrosomonas europaea is a Gram-negative obligate chemolithoautotroph that derives all its energy and reductant for growth from the oxidation of ammonia to nitrite . Key characteristics include:

• Habitat: Found in soil, sewage, freshwater, building surfaces, and monuments, especially in areas with high levels of nitrogen compounds

• Metabolism: Aerobic metabolism, with the ability to "burn" ammonia with oxygen

• Growth conditions: Prefers pH 6.0-9.0 (optimal: slightly basic), temperature 20-30°C

• Growth rate: Slow cell division (takes several days) due to large ammonia requirements

• Genome: Single circular chromosome of 2,812,094 bp with 2,460 protein-coding genes

• Environmental significance: Critical in nitrification processes and wastewater treatment

What expression systems are available for recombinant NE0719 production?

The recombinant NE0719 protein can be produced using several expression systems:

According to available product information, recombinant NE0719 is commercially available with various tags (His, GST, Flag, MBP) and multiple specifications (20μg/100μg/1mg) . Researchers can select the most appropriate system based on their specific experimental requirements.

What computational approaches can predict the function of NE0719?

Since NE0719 is an uncharacterized protein, computational approaches offer valuable starting points for functional characterization:

• Structural prediction and modeling:

  • Homology modeling based on solved structures of related UPF0102 family proteins

  • Ab initio structure prediction using platforms like AlphaFold

  • Analysis of conserved domains and potential binding sites

• Genomic context analysis:

  • Investigation of operonic structures and co-expression patterns

  • Phylogenetic profiling to identify co-evolving genes

  • The proximity to ABC transporter genes (NE0720) suggests potential involvement in transport functions

• Protein-protein interaction prediction:

  • Based on the genomic location near an ABC transporter with fused permease and ATPase domains, NE0719 might function as an accessory protein in transport processes

  • Network analysis based on genomic context and co-expression data

What experimental approaches are optimal for characterizing NE0719 function?

Given the uncharacterized nature of NE0719, a multi-faceted experimental approach is recommended:

• Knockout/knockdown studies:

  • CRISPR-Cas9 or homologous recombination-based gene deletion

  • Analysis of phenotypic changes, particularly under different ammonia oxidation conditions

  • Metabolomic analysis comparing wild-type vs. NE0719 mutants

• Transcriptomic analysis:

  • RNA-seq under various growth conditions to determine when NE0719 is expressed

  • Similar to approaches used in studying N. europaea's response to oxygen limitation

  • Co-expression network analysis to identify functionally related genes

• Protein interaction studies:

  • Pull-down assays using tagged recombinant NE0719

  • Bacterial two-hybrid screening

  • Crosslinking mass spectrometry to identify interaction partners

• Biochemical characterization:

  • Enzymatic activity assays based on predicted functions

  • Substrate binding assays

  • Structural studies (X-ray crystallography, cryo-EM)

How can genetic engineering be applied to study NE0719 function in vivo?

Multiple genetic engineering approaches have been successfully applied to N. europaea and can be adapted for NE0719 studies:

• Reporter gene fusions:

  • Transcriptional fusions with GFP (similar to those used with mbla and clpB promoters in N. europaea)

  • Translational fusions to study protein localization

• Gene complementation:

  • Introduction of intact NE0719 in trans to knockout mutants, as demonstrated with norCBQD gene cluster

  • Heterologous expression in related bacteria

• Transformation protocols:

  • Plasmid-based transformation with promoters such as amoC P1

  • The ColEI replication origin has been shown to function in N. europaea

• Biosensor development:

  • If NE0719 responds to specific environmental conditions, its promoter could be used to develop biosensors similar to those created for chloroform detection

What role might NE0719 play in N. europaea's unique metabolism?

Based on N. europaea's specialized metabolism, several hypotheses about NE0719 function can be proposed:

• Ammonia oxidation pathway:

  • N. europaea's energy metabolism revolves around ammonia oxidation via ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO)

  • NE0719 might serve as an accessory protein in these pathways, particularly under stress conditions

• Iron acquisition:

  • N. europaea dedicates over 20 genes to iron receptors

  • The genomic context of NE0719 should be examined for potential involvement in iron metabolism

• Stress response:

  • Similar to stress-responsive genes identified in N. europaea (such as those responding to chloroform oxidation)

  • NE0719 expression patterns under various stressors could be analyzed

• Carbon fixation:

  • As an autotroph, N. europaea fixes CO₂ for carbon needs

  • NE0719 might participate in regulatory processes related to balancing energy generation and carbon fixation

How does protein NE0719 compare to other UPF0102 family proteins across bacterial species?

Comparative analysis of UPF0102 family proteins can provide valuable insights:

• Sequence conservation analysis:

  • Multiple sequence alignment of UPF0102 proteins from diverse bacteria

  • Identification of highly conserved residues potentially crucial for function

  • NE0719 shares sequence similarity with UPF0102 proteins from other bacterial species, but its exact relationship requires detailed phylogenetic analysis

• Phylogenetic distribution:

  • Analysis of UPF0102 protein presence across bacterial phyla

  • Correlation with metabolic capabilities and ecological niches

• Structural comparison:

  • Comparative modeling of UPF0102 proteins from different species

  • Identification of conserved structural features versus species-specific adaptations

• Genomic context conservation:

  • Analysis of gene neighborhoods across species

  • Identification of consistently co-localized genes suggesting functional relationships

What are the optimal conditions for expressing recombinant NE0719?

Based on the general properties of N. europaea proteins and recombinant protein production approaches:

• Expression system selection:

  • E. coli BL21(DE3) is commonly used for initial expression trials

  • Five expression systems have been developed for recombinant proteins from N. europaea, including E. coli, yeast, baculovirus-infected insect cells, mammalian cells, and cell-free E. coli systems

• Expression optimization:

  • Temperature: Lower temperatures (16-25°C) often improve solubility

  • Induction: Optimize IPTG concentration for E. coli systems

  • Media: Enriched media for higher cell density

  • Consider codon optimization for the expression host

• Protein solubility enhancement:

  • Fusion tags: His, GST, Flag, and MBP tags have been used successfully

  • Solubility enhancers: SUMO or thioredoxin fusions

  • Lysis conditions: Optimize buffer composition

• Purification strategy:

  • Affinity chromatography based on selected tag

  • Size exclusion chromatography for final polishing

  • Quality control via SDS-PAGE and mass spectrometry

What analytical techniques are most informative for characterizing NE0719?

Multiple analytical approaches can provide complementary information about NE0719:

• Structural analysis:

  • X-ray crystallography for high-resolution structure

  • NMR spectroscopy for solution structure and dynamics

  • Circular dichroism for secondary structure content

  • Thermal shift assays for stability assessment

• Functional analysis:

  • Enzymatic activity assays (if enzymatic function is predicted)

  • Binding assays using isothermal titration calorimetry or surface plasmon resonance

  • Mass spectrometry for post-translational modifications

• Interaction studies:

  • Pull-down assays using tagged NE0719

  • Crosslinking followed by mass spectrometry

  • Yeast two-hybrid or bacterial two-hybrid screening

• Localization studies:

  • Immunofluorescence microscopy with anti-NE0719 antibodies

  • Subcellular fractionation followed by Western blotting

  • GFP fusion localization in live cells

How can researchers design knockout experiments for NE0719 in N. europaea?

Genetic manipulation of N. europaea requires specialized approaches:

• Gene knockout strategies:

  • Homologous recombination with antibiotic resistance cassettes

  • Similar approaches to those used for norB gene disruption in N. europaea

  • CRISPR-Cas9 systems adapted for N. europaea

• Verification methods:

  • PCR confirmation of gene disruption

  • RT-PCR to confirm absence of transcript

  • Western blotting to confirm absence of protein

• Phenotypic analysis:

  • Growth rate assessment under various conditions

  • Ammonia oxidation capacity measurements

  • Metabolomic profiling

  • Transcriptomic response to knockout

• Complementation:

  • Reintroduction of intact NE0719 gene in trans

  • Use of inducible promoters for controlled expression

What is the potential relevance of NE0719 to environmental biotechnology?

Given N. europaea's environmental importance, understanding NE0719 could have significant applications:

• Wastewater treatment:

  • N. europaea is crucial in nitrification processes in wastewater treatment

  • If NE0719 affects ammonia oxidation efficiency, it could be a target for optimization

• Bioremediation:

  • N. europaea can degrade benzene and halogenated organic compounds

  • NE0719 might play a role in these degradation pathways

• Biosensor development:

  • Similar to GFP reporter systems developed for sensing chloroform and H₂O₂

  • NE0719 promoter could potentially be used if responsive to specific environmental conditions

• Climate change mitigation:

  • N. europaea contributes to N₂O production, a potent greenhouse gas

  • Understanding if NE0719 influences N₂O production could have climate implications

How might functional characterization of NE0719 contribute to understanding bacterial UPF0102 proteins?

Characterizing NE0719 would advance understanding of the entire UPF0102 protein family:

• Functional annotation transfer:

  • Functions discovered for NE0719 could be extrapolated to homologs in other bacteria

  • Creation of testable hypotheses for related proteins

• Evolutionary insights:

  • Understanding of selective pressures on UPF0102 proteins

  • Identification of conserved functional motifs

• Bacterial physiology insights:

  • UPF0102 proteins may represent novel aspects of bacterial metabolism or regulation

  • Potential discovery of new regulatory mechanisms or metabolic pathways

• Structural biology advancements:

  • Addition of a new protein structure to databases

  • Improvement of structure prediction algorithms through new structural data

How can researchers overcome the slow growth of N. europaea when studying NE0719?

N. europaea's slow growth presents challenges for research:

• Culture optimization:

  • High ammonia concentrations to support growth

  • Optimized mineral medium composition

  • Carefully controlled pH (6.0-9.0) and temperature (20-30°C)

• Alternative approaches:

  • Heterologous expression of NE0719 in faster-growing hosts

  • In vitro studies with purified recombinant NE0719

  • Computational predictions to guide focused experiments

• Enhanced growth strategies:

  • Engineering N. europaea to express Vitreoscilla hemoglobin (VHb) has been shown to increase ammonia conversion by ~30%

  • Similar approaches could be applied to create research strains with faster growth

• High-throughput methodologies:

  • Miniaturized culture systems

  • Automation of sampling and analysis

  • Sensitive detection methods requiring less biomass

What are the key considerations for ensuring reproducibility in NE0719 research?

Ensuring reproducible results with this challenging organism requires:

• Standardized protocols:

  • Detailed documentation of growth conditions

  • Consistent medium preparation

  • Standardized recombinant protein expression conditions

• Quality control measures:

  • Regular verification of strain identity

  • Monitoring of culture purity

  • Protein quality assessment (e.g., SDS-PAGE, mass spectrometry)

• Data validation approaches:

  • Multiple biological and technical replicates

  • Independent verification of key findings

  • Use of appropriate statistical methods

• Reporting standards:

  • Complete methods description including exact strain designations

  • Sharing of materials and protocols

  • Data deposition in public repositories