Recombinant Nitrosomonas europaea Flagellar hook-basal body complex protein FliE (fliE)

What is the flagellar hook-basal body complex protein FliE in Nitrosomonas europaea?

FliE serves as a critical structural component in the flagellar assembly of Nitrosomonas europaea, functioning as an adapter protein that connects the MS-ring (membrane and supramembrane ring) to the rod structure in the bacterial flagellum. While not directly mentioned in the provided research, N. europaea's genomic analysis reveals it possesses genes necessary for basic cellular functions including motility structures . FliE is encoded within the N. europaea genome (2,812,094 bp circular chromosome) and plays an essential role in the bacterium's motility system, which helps it navigate environmental gradients for optimal ammonia oxidation.

What genomic context surrounds the fliE gene in Nitrosomonas europaea?

The fliE gene likely exists within an operon structure typical of flagellar genes in bacteria. While specific genomic organization isn't detailed in the provided research, N. europaea's genome shows even distribution of genes with approximately 47% transcribed from one strand and 53% from the complementary strand . Researchers studying fliE should examine neighboring genes as N. europaea's genome contains numerous insertion sequence elements (85 predicted in eight different families) that constitute approximately 5% of the genome , which could influence genetic stability and expression of flagellar components.

What are the optimal conditions for heterologous expression of recombinant N. europaea FliE protein?

Based on successful expression approaches with other N. europaea proteins, recombinant FliE can be efficiently expressed in E. coli expression systems. The cytochrome c-552 from N. europaea has been successfully expressed in E. coli at high resolution (1.63-Å) , suggesting similar approaches may work for FliE. For optimal expression, researchers should consider:

• Utilizing pET expression vectors with codon optimization for E. coli, similar to the approach used for MazE and MazF proteins from N. europaea

• IPTG-inducible promoter systems for controlled expression

• Expression at lower temperatures (16-20°C) to enhance proper folding

• Inclusion of appropriate affinity tags (His-tag or GST) for simplified purification

Growth conditions should account for N. europaea's slow growth rate characteristics, which may be reflected in its proteins' folding kinetics.

What purification strategy yields the highest purity recombinant FliE protein?

A multi-step purification approach is recommended for obtaining high-purity recombinant N. europaea FliE:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged constructs)

• Intermediate purification via ion-exchange chromatography

• Final polishing step using size-exclusion chromatography

This strategy parallels successful purification protocols used for crystallization-grade N. europaea cytochrome c-552 . Researchers should verify protein purity through SDS-PAGE analysis and consider additional purification steps if contaminants remain. Western blotting using anti-FliE antibodies can confirm protein identity throughout the purification process.

How can researchers assess the proper folding and functionality of recombinant N. europaea FliE?

Proper folding assessment should combine multiple biophysical techniques:

• Circular dichroism (CD) spectroscopy to analyze secondary structure content

• Thermal shift assays to evaluate protein stability

• Limited proteolysis to verify compact folding

• Functional binding assays with known interaction partners (MS-ring components and rod proteins)

Similar to approaches used for studying N. europaea cytochrome c-552, where spectroscopic methods verified proper folding , researchers should employ multiple complementary techniques to confirm that recombinant FliE retains native-like structure.

What structural features distinguish N. europaea FliE from homologs in other bacteria?

N. europaea FliE likely possesses unique structural adaptations reflecting its specific environmental niche. While direct structural data isn't available in the provided research, comparative analysis would be essential. Methodologically, researchers should:

• Perform sequence alignments with FliE proteins from diverse bacterial species

• Identify conserved domains versus N. europaea-specific regions

• Generate structural models using homology modeling approaches

• Validate models through techniques such as circular dichroism and small-angle X-ray scattering

As observed with cytochrome c-552, where specific structural features like heme ruffling affect functional properties , N. europaea FliE may possess distinctive structural elements adapted to its ammonia-oxidizing lifestyle.

How does the oligomeric state of FliE impact flagellar assembly in N. europaea?

The oligomerization state of FliE is critical for proper flagellar assembly. Research approaches should include:

• Analytical ultracentrifugation to determine native oligomeric states

• Cross-linking studies to capture physiologically relevant assemblies

• Cryo-electron microscopy to visualize FliE organization within the hook-basal body complex

When characterizing oligomeric states, researchers should consider how N. europaea's unique physiological requirements might influence protein-protein interactions within the flagellar apparatus, especially given its adaptation to ammonia-rich environments .

What post-translational modifications occur in native N. europaea FliE protein?

Identification of post-translational modifications requires:

• Mass spectrometry analysis of native FliE isolated from N. europaea cultures

• Comparison with recombinant protein expressed in E. coli

• Targeted analysis for common bacterial PTMs (phosphorylation, methylation, acetylation)

Given N. europaea's adaptive mechanisms to environmental stressors, including temperature variations and pH changes , post-translational modifications may play regulatory roles in flagellar assembly in response to environmental conditions.

How does FliE interact with other flagellar proteins in the N. europaea hook-basal body complex?

Characterizing protein-protein interactions requires multiple complementary approaches:

• Bacterial two-hybrid or yeast two-hybrid screening

• Co-immunoprecipitation with anti-FliE antibodies

• Surface plasmon resonance to determine binding kinetics

• Crosslinking coupled with mass spectrometry

Researchers should specifically investigate interactions with MS-ring components and rod proteins, establishing a comprehensive interaction network. The approach should parallel methodologies used in characterizing other N. europaea protein complexes, focusing on identifying stable interaction partners versus transient associations during flagellar assembly.

What role does FliE play in the motility of N. europaea in ammonia-rich environments?

Investigating FliE's role in N. europaea motility requires gene deletion or mutation studies followed by phenotypic characterization:

• Construction of fliE knockout or conditional mutants

• Motility assays under varying ammonia concentrations

• Chemotaxis assays toward ammonia gradients

• Electron microscopy to assess flagellar structure in mutants

This research is particularly relevant given N. europaea's environmental niche in wastewater treatment and sediments where ammonia may be in abundant supply . Understanding how FliE contributes to motility in these environments has implications for nitrogen cycling processes.

How is FliE expression regulated in response to environmental stressors affecting N. europaea?

N. europaea is susceptible to various environmental factors including temperature, pH, nitrite and ammonia concentrations, heavy metals, and organic/inorganic compounds . To understand FliE regulation:

• Perform qRT-PCR analysis of fliE expression under various stressors

• Use reporter gene fusions to monitor promoter activity

• Identify potential regulatory elements in the fliE promoter region

• Assess correlation between FliE expression and motility phenotypes

How can CRISPR-Cas9 gene editing be optimized for studying FliE function in N. europaea?

Establishing CRISPR-Cas9 editing in N. europaea requires specialized protocols considering its obligate chemolithoautotrophic nature:

• Design of guide RNAs specific to fliE with minimal off-target effects

• Development of transformation protocols accounting for N. europaea's unique cell envelope characteristics

• Optimization of selection markers compatible with ammonia oxidation metabolism

• Validation strategies for confirming gene edits in slow-growing cultures

Researchers should consider the relatively high GC content and presence of repetitive elements (approximately 5% of the genome) when designing guide RNAs to ensure specificity in the N. europaea genome.

What crystallization conditions are optimal for obtaining high-resolution structures of N. europaea FliE?

Based on successful crystallization of other N. europaea proteins, researchers should consider:

• Initial screening with commercial sparse matrix crystallization kits

• Optimization focusing on pH ranges suitable for N. europaea proteins (typically pH 7.0-8.0)

• Inclusion of specific ions relevant to N. europaea physiology (particularly iron compounds)

• Seeding techniques to improve crystal quality

The approach should be informed by the successful crystallization of N. europaea cytochrome c-552, which yielded high-resolution structures (up to 1.63-Å resolution) . Researchers should also explore co-crystallization with interaction partners to capture physiologically relevant complexes.

How can molecular dynamics simulations enhance understanding of FliE function in the N. europaea flagellar system?

Computational approaches provide valuable insights when combined with experimental data:

• Construction of atomistic models based on homology or experimental structures

• Simulation of FliE dynamics in membrane environments resembling N. europaea

• Investigation of conformational changes during interaction with other flagellar components

• Prediction of critical residues for function through in silico mutagenesis

Simulations should incorporate N. europaea's environmental parameters, including temperature ranges and pH conditions typical of its natural habitats, to generate physiologically relevant insights.

How can researchers overcome solubility issues when expressing recombinant N. europaea FliE?

Addressing solubility challenges requires systematic optimization:

• Fusion tag screening (MBP, SUMO, TrxA) to enhance solubility

• Optimization of induction parameters (temperature, IPTG concentration, duration)

• Adjustment of lysis buffer composition (pH, salt concentration, detergents)

• Co-expression with molecular chaperones

Researchers should consider that N. europaea proteins may have evolved unique solubility characteristics due to the bacterium's specialized metabolism and environmental adaptations .

What strategies can resolve aggregation issues during purification of N. europaea FliE?

Aggregation during purification can be addressed through:

• Addition of stabilizing agents (glycerol, arginine, trehalose)

• Optimization of buffer conditions based on protein isoelectric point

• Use of mild detergents when dealing with hydrophobic regions

• Implementation of on-column refolding protocols if inclusion bodies form

Similar challenges have been addressed in the purification of other N. europaea proteins, suggesting careful buffer optimization is critical for maintaining native-like conformations .

How can researchers validate antibody specificity for N. europaea FliE in immunolocalization studies?

Ensuring antibody specificity requires comprehensive validation:

• Western blot comparison using recombinant FliE and N. europaea lysates

• Pre-adsorption controls with recombinant protein

• Immunostaining comparisons between wild-type and fliE knockout strains

• Cross-reactivity assessment with related flagellar proteins

This validation is particularly important given the complexity of bacterial flagellar systems and potential cross-reactivity with structurally similar components.

How does understanding N. europaea FliE contribute to wastewater treatment biotechnology?

The flagellar system of N. europaea affects its distribution and activity in wastewater treatment systems:

• Analysis of biofilm formation capabilities in relation to flagellar motility

• Assessment of attachment to surfaces in treatment facilities

• Correlation between motility and nitrification efficiency

• Development of immobilization strategies preserving flagellar function

This research has practical implications as N. europaea inhabits wastewater treatment plants and sediments where ammonia may be in abundant supply , making it relevant for optimization of nitrogen removal processes.

What evolutionary insights can be gained from comparative analysis of FliE across different ammonia-oxidizing bacteria?

Evolutionary analysis requires:

• Phylogenetic tree construction using FliE sequences from diverse nitrifiers

• Identification of conserved versus variable regions through multiple sequence alignment

• Selection pressure analysis (dN/dS ratios) to identify evolutionary constraints

• Structural homology modeling to correlate sequence conservation with functional domains

How can cryo-electron tomography enhance our understanding of FliE organization in the native N. europaea flagellar system?

Cryo-electron tomography offers unique insights into native protein organization:

• Sample preparation protocols optimized for N. europaea cells

• Tomographic data collection focusing on flagellar basal bodies

• Sub-tomogram averaging to enhance resolution of FliE-containing regions

• Correlation with biochemical data on protein-protein interactions