Recombinant Nitrosomonas europaea Elongation factor G (fusA), partial

What is Nitrosomonas europaea and where is the fusA gene located in its genome?

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. Its genome consists of a single circular chromosome of 2,812,094 bp with approximately 47% of genes transcribed from one strand and 53% from the complementary strand .

The fusA gene encoding elongation factor G is located within a conserved gene cluster that includes tRNA genes, several ribosomal genes, elongation factor Tu (tufB), and the transcription anti-termination gene nusG. This 23.1-kb spanning region is positioned near one of the ammonia monooxygenase (amo) and hydroxylamine oxidoreductase (hao) gene clusters that are essential for ammonia oxidation . Comparative genomic studies between N. europaea and other Nitrosomonas strains show conservation of this arrangement despite significant genomic rearrangements in other regions.

What is the function of elongation factor G in Nitrosomonas europaea?

While the search results don't specifically detail the function of fusA in N. europaea, elongation factor G typically plays a critical role in bacterial protein synthesis. In N. europaea, it functions during the translocation step of protein synthesis, catalyzing the movement of the ribosome along mRNA and the translocation of tRNAs from the A-site to the P-site.

The fusA gene is part of the essential cellular machinery supporting the chemolithoautotrophic lifestyle of N. europaea, where energy generation depends on ammonia oxidation and carbon acquisition relies primarily on CO2 fixation . This protein is particularly important for expression of metabolic enzymes involved in ammonia oxidation, energy generation, and CO2 fixation pathways.

How does the genetic organization of fusA in N. europaea compare to other bacteria?

The fusA gene in N. europaea is positioned within a conserved genomic region that includes tRNA genes, ribosomal genes, and elongation factor Tu (tufB). This pattern of organization is similar to what is observed in other bacteria, reflecting the fundamental importance of protein synthesis machinery.

Interestingly, comparative genomic studies between N. europaea ATCC 19718 and Nitrosomonas sp. strain ENI-11 reveal that while certain gene clusters (including those containing fusA) show remarkable conservation in proximity and organization, other genomic regions display significant rearrangements . For example, while both genomes maintain two amo/hao gene clusters in similar arrangements, the spanning regions between these clusters differ dramatically (87 kb in ENI-11 versus 1,300 kb in N. europaea) . This suggests that even conserved housekeeping genes like fusA maintain stable positional relationships despite broader genomic reorganization.

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

For successful expression of recombinant N. europaea fusA, researchers should consider the following methodological approach:

• Expression system selection: E. coli BL21(DE3) is generally recommended due to its reduced protease activity and compatibility with T7 promoter-based expression vectors.

• Codon optimization: N. europaea has a different codon usage pattern than E. coli. Analyzing the fusA sequence for rare codons and potentially optimizing the sequence is advisable, especially considering N. europaea's GC content.

• Expression conditions:

  • Temperature: Lower temperatures (16-25°C) often improve soluble protein yield

  • Induction: 0.1-0.5 mM IPTG for T7-based systems

  • Media: Rich media (e.g., LB) supplemented with glucose to prevent leaky expression

  • Duration: 4-16 hours post-induction depending on temperature

• Protein extraction: Due to the large size of fusA (typically 70-80 kDa), gentle cell lysis methods like sonication with cooling intervals are recommended to prevent protein aggregation.

The methodological approach would parallel techniques used for sequencing and analyzing other N. europaea genes, such as those used in whole-genome shotgun strategies described in the search results .

What purification strategy is most effective for recombinant N. europaea fusA?

Based on properties of elongation factors and the specific characteristics of N. europaea proteins, the following purification strategy is recommended:

• Affinity tag selection:

  • N-terminal 6×His tag generally performs well for fusA purification

  • Alternative: Strep-tag II or FLAG tag if antibody detection is important

• Purification buffer considerations:

  • Base buffer: 50 mM Tris-HCl pH 7.5-8.0

  • Salt: 100-300 mM NaCl

  • Reducing agent: 1-5 mM DTT or 2-10 mM β-mercaptoethanol

  • Stabilizers: 5-10% glycerol and 0.1 mM EDTA

  • Protease inhibitors: PMSF (1 mM) or commercial cocktail

• Purification protocol:

• Quality control:

  • SDS-PAGE to verify purity

  • Western blot with anti-His antibodies

  • Mass spectrometry to confirm identity

This approach incorporates similar methodological principles to those used for isolating and characterizing other N. europaea proteins, such as the nitrite reductase purification described in search result .

How can researchers verify the functionality of recombinant N. europaea fusA?

Verifying the functionality of recombinant fusA requires assessing its ability to perform its catalytic role in protein synthesis. Researchers should implement the following multi-step verification process:

• In vitro translation assay:

  • Components: ribosomes, mRNA template, aminoacyl-tRNAs, initiation factors, and elongation factor Tu

  • Replace native EF-G with recombinant N. europaea fusA

  • Measure peptide synthesis rates using radiolabeled amino acids or fluorescent reporters

• GTPase activity assay:

  • Monitor GTP hydrolysis using colorimetric phosphate detection methods

  • Compare kinetic parameters (Km, kcat) with those of well-characterized EF-G proteins

  • Test activity under various conditions (temperature, pH, salt concentration)

• Ribosome binding studies:

  • Fluorescence anisotropy to measure binding affinity to ribosomes

  • Filter binding assays with radiolabeled GTP

  • Surface plasmon resonance (SPR) for real-time binding kinetics

• Complementation testing:

  • Use temperature-sensitive E. coli fusA mutants

  • Transform with plasmid expressing N. europaea fusA

  • Test for growth restoration at non-permissive temperatures

This approach integrates molecular biology techniques similar to those used for characterizing other functional proteins in N. europaea, such as the nitric oxide reductase activity measurements described in search result .

How does the structure of N. europaea fusA compare to those from other bacteria, and what are the implications for function?

Though specific structural information for N. europaea fusA isn't provided in the search results, comparative structural analysis would likely reveal important insights:

The fusA protein typically consists of five domains (I-V):

• Domain I contains the GTP-binding site

• Domains II-V are involved in interactions with the ribosome

Researchers investigating structural differences should focus on:

• Sequence alignment analysis: Comparing N. europaea fusA with well-characterized homologs from model organisms (E. coli, T. thermophilus)

• Homology modeling: Using solved crystal structures of bacterial EF-G as templates

• Key functional regions to analyze:

  • GTP-binding pocket (G-domain)

  • Switch regions involved in conformational changes

  • Ribosome-interaction surfaces

• Potential adaptations: N. europaea's chemolithoautotrophic lifestyle and specific temperature preferences may have driven adaptive changes in fusA structure that optimize protein synthesis under these conditions.

As with comparative genomic studies described in search result , structural analysis could reveal conserved features alongside organism-specific adaptations that reflect N. europaea's ecological niche.

What role might fusA play in the stress response of N. europaea to environmental challenges?

N. europaea faces unique environmental stresses as an ammonia-oxidizing bacterium, including nitrite toxicity, oxygen limitation, and oxidative stress. The role of fusA in these stress responses may include:

• Translational regulation under stress:

  • Selective translation of stress-responsive genes

  • Altered translational accuracy during stress conditions

  • Specialized ribosomes with differential affinity for fusA variants

• Response to nitrite stress:

  • N. europaea can experience self-inhibition from the nitrite it produces

  • The search results indicate that nitrite reductase (NirK) deficiency reduces nitrite tolerance

  • fusA may be involved in translating proteins needed for nitrite tolerance

• Oxygen limitation response:

  • N. europaea can grow anaerobically with nitrite as an electron acceptor

  • Transition to anaerobic metabolism likely requires significant translational reprogramming

  • fusA activity may be regulated during this transition

• Integration with regulatory systems:

  • Potential interaction with transcription factors like Fnr

  • The search results mention an Fnr protein containing four conserved cysteine residues involved in ligation of a [4Fe-4S] cluster

  • This oxygen-responsive regulator may indirectly influence fusA activity

This question connects to findings about N. europaea's adaptability to different growth conditions as described in search results and .

How can site-directed mutagenesis of N. europaea fusA inform our understanding of elongation factor evolution in specialized bacterial niches?

Site-directed mutagenesis of N. europaea fusA provides a powerful approach to understanding how this essential protein has adapted to support the specialized chemolithoautotrophic lifestyle of this organism:

• Key residues for mutational analysis:

  • GTP-binding pocket residues

  • Residues at the interface with ribosomes

  • Switch regions that undergo conformational changes during GTP hydrolysis

  • Unique residues conserved among ammonia-oxidizing bacteria but divergent from other bacteria

• Functional characterization approaches:

  • In vitro translation efficiency with mutant variants

  • GTPase activity measurements

  • Ribosome binding affinity

  • Thermostability analysis

• Evolutionary context analysis:

  • Comparing effects of mutations in conserved vs. variable regions

  • Relating functional changes to phylogenetic patterns

  • Identifying selection pressures specific to ammonia-oxidizing bacteria

• Potential applications: Understanding fusA adaptations could provide insights into:

  • Metabolic specialization mechanisms in chemolithoautotrophs

  • Evolution of core cellular machinery in specialized niches

  • Protein synthesis optimization under energy-limited conditions

This approach connects to the comparative genomics findings in search result , which highlight both conservation and divergence in the genomic organization of essential genes in Nitrosomonas species.

What are common challenges in expressing soluble recombinant N. europaea fusA and how can they be addressed?

Researchers often encounter several challenges when expressing recombinant N. europaea fusA:

• Protein aggregation and inclusion body formation:

  • Solution: Lower induction temperature (16-20°C)

  • Alternative: Co-express with chaperones (GroEL/ES, DnaK/J)

  • Consider fusion partners (SUMO, MBP, thioredoxin) to enhance solubility

• Low expression levels:

  • Evaluate codon optimization for expression host

  • Test different promoter systems (T7, tac, arabinose-inducible)

  • Optimize induction conditions (inducer concentration, OD at induction)

  • Screen multiple E. coli strains (BL21, Rosetta, Arctic Express)

• Protein degradation:

  • Include protease inhibitors during purification

  • Express in protease-deficient strains

  • Reduce induction time

  • Maintain samples at 4°C throughout processing

• Purification issues:

  • Test multiple affinity tags (His, GST, Strep-tag II)

  • Optimize buffer conditions (pH, salt concentration, additives)

  • Consider on-column refolding if inclusion bodies are unavoidable

This methodological troubleshooting approach is relevant to researchers working with challenging proteins from specialized bacteria like N. europaea, similar to the careful isolation approaches described for other N. europaea proteins in the search results .

How can researchers optimize activity assays for recombinant N. europaea fusA?

Optimizing activity assays for recombinant N. europaea fusA requires careful consideration of multiple factors:

• GTPase activity measurement:

  • Malachite green assay: Optimize protein and GTP concentrations

  • NADH-coupled assay: Monitor interference from sample components

  • Radioactive [γ-32P]GTP assay: Ensure accurate quenching and background subtraction

• Translation activity assessment:

  • Ribosome source selection: Test both homologous and heterologous ribosomes

  • mRNA template optimization: Use templates with various secondary structures

  • Reaction conditions: Titrate Mg2+ (5-15 mM) and K+ (50-200 mM) concentrations

  • Detection method: Compare radioactive, fluorescent, and luminescent reporters

• Environmental parameter optimization:

• Controls and validation:

  • Positive control: E. coli fusA for comparison

  • Negative control: GTPase-deficient mutant (e.g., H91A)

  • Validation: Correlation between GTPase activity and translation efficiency

This approach to assay optimization aligns with the methodological rigor shown in the activity assays for nitrite reductase and nitric oxide reductase described in search results and .

What strategies can overcome challenges in crystallizing N. europaea fusA for structural studies?

Crystallizing elongation factors like fusA can be challenging due to their size, flexibility, and multiple conformational states. Researchers should consider these strategies:

• Protein sample preparation optimization:

  • Extreme purity (>95% by SDS-PAGE)

  • Concentration screening (5-20 mg/ml)

  • Buffer optimization through thermal shift assays

  • Limited proteolysis to identify stable domains

  • Dynamic light scattering to assess monodispersity

• Construct design approaches:

  • Full-length protein with removable affinity tags

  • Domain truncations based on sequence analysis

  • Surface entropy reduction mutations (Lys/Glu → Ala)

  • Stabilized conformations through nucleotide analogs (GTPγS, GDPNP)

• Crystallization condition screening:

  • Initial broad screening (500-1000 conditions)

  • Optimization of promising conditions:

    • pH fine-screening (±0.2 units)

    • Precipitant concentration gradients

    • Additive screening

    • Seeding techniques (micro- and macro-seeding)

• Alternative approaches if crystallization fails:

  • Cryo-electron microscopy

  • Small-angle X-ray scattering (SAXS)

  • Hydrogen-deuterium exchange mass spectrometry

  • NMR of individual domains

This systematic approach to structural studies follows principles similar to those used in the careful biochemical characterization of other N. europaea proteins described in the search results.

How can recombinant N. europaea fusA be used to study the molecular mechanisms of energy-efficient translation in chemolithoautotrophs?

Chemolithoautotrophs like N. europaea face unique energy constraints, as they derive energy solely from inorganic compounds like ammonia . Recombinant fusA provides a powerful tool to investigate energy-efficient translation:

• Comparative kinetic analysis:

  • Measure GTP hydrolysis rates and efficiency compared to heterotrophic bacteria

  • Quantify the number of GTP molecules consumed per amino acid incorporated

  • Compare translation rates under varying energy availability conditions

• Energy conservation mechanisms:

  • Investigate potential specialized interactions with ribosomes

  • Examine coupling between GTP hydrolysis and mechanical movement

  • Test for differential regulation under energy-limited conditions

• Experimental approaches:

  • Single-molecule FRET to track conformational changes

  • Reconstituted translation systems with defined components

  • Ribosome profiling with recombinant fusA variants

  • Cryo-EM structures of N. europaea fusA bound to ribosomes in different states

• Integration with cellular energetics:

  • Correlate translation rates with cellular ATP/GTP levels

  • Model energy allocation between protein synthesis and ammonia oxidation

  • Compare fusA activity in aerobic versus anaerobic conditions

This research direction connects directly to N. europaea's chemolithoautotrophic lifestyle described in search result , where energy constraints shape cellular processes.

What insights might comparative analysis of fusA across different ammonia-oxidizing bacteria provide about adaptation to specialized metabolic niches?

Comparative analysis of fusA across ammonia-oxidizing bacteria (AOB) could reveal important adaptations to their specialized metabolic lifestyle:

• Evolutionary analysis framework:

  • Phylogenetic comparison of fusA sequences from diverse AOB

  • Identification of positive selection signatures in specific lineages

  • Correlation with metabolic capabilities and environmental niches

• Structural and functional comparison:

  • Residue conservation patterns in catalytic sites versus peripheral regions

  • Differences in kinetic parameters across AOB fusA homologs

  • Temperature and pH optima variations reflecting habitat preferences

• Key comparison groups:

  • Beta-proteobacterial AOB (Nitrosomonas, Nitrosospira)

  • Gamma-proteobacterial AOB (Nitrosococcus)

  • Comparison with non-AOB relatives

  • Extremophilic versus mesophilic AOB

• Potential adaptations to investigate:

  • Efficiency of GTP utilization

  • Stability under varying nitrite concentrations

  • Response to oxygen limitation

  • Coordination with ammonia oxidation pathways

This comparative approach mirrors the genomic comparison between N. europaea and Nitrosomonas sp. strain ENI-11 described in search result , which revealed both conservation and divergence in key genomic regions.

How might CRISPR-Cas9 genome editing of fusA in N. europaea advance our understanding of protein synthesis regulation in ammonia oxidizers?

CRISPR-Cas9 genome editing of the fusA gene in N. europaea presents exciting opportunities for understanding protein synthesis regulation in this specialized bacterium:

• Precision engineering approaches:

  • Single amino acid substitutions in key functional regions

  • Domain swaps with fusA from heterotrophic bacteria

  • Promoter modifications to alter expression levels

  • Introduction of regulatory elements for controlled expression

• Phenotypic analyses of fusA mutants:

  • Growth rates under varying ammonia and oxygen concentrations

  • Protein synthesis rates using metabolic labeling

  • Stress tolerance (nitrite, pH, temperature extremes)

  • Ammonia oxidation efficiency measurements

• Genome-wide effects of fusA modifications:

  • Ribosome profiling to assess translation landscape changes

  • RNA-seq to identify compensatory transcriptional responses

  • Proteomics to evaluate altered protein abundances

  • Metabolomics to detect shifts in central metabolism

• Integration with existing knowledge:

  • Connection to nitrite tolerance mechanisms described in search result

  • Relationship with anaerobic metabolism outlined in search result

  • Potential coordination with regulatory systems like Fnr mentioned in search result

This cutting-edge approach would build upon the genetic techniques described in the search results, such as those used for gene disruption and complementation in N. europaea .