Recombinant Nitrosomonas europaea GTPase Der (der)

What is Nitrosomonas europaea GTPase Der and what is its fundamental role in bacterial physiology?

GTPase Der (also known as EngA or YphC in some bacteria) is a translational GTPase found in Nitrosomonas europaea, a gram-negative obligate chemolithoautotroph. Der belongs to a family of proteins whose GTPase activity is stimulated by the large ribosomal subunit . GTPase Der is nearly universally conserved across bacterial species, suggesting an essential role in basic cellular functions.

Functionally, Der is implicated in ribosome biogenesis and assembly. Unlike other translational GTPases such as EF-Tu and EF-G that are directly involved in the elongation phase of translation, Der appears to play a regulatory role in ribosome maturation. It contains characteristic domains including:

• N-terminal GTPase domain

• Central GTPase domain

• C-terminal KH-domain-like region

These structural features enable Der to interact with the ribosome during assembly, potentially facilitating the association of ribosomal proteins or rRNA processing.

How can researchers accurately identify and classify GTPase Der within the translational GTPase family?

Translational GTPases can be identified and classified using a combination of methods:

Hidden Markov Model (HMM) Approach:

• Create subfamily-specific HMM profiles from well-conserved trGTPases

• Compute phylogenetic trees based on the GTPase domain

• Use iterative HMMSEARCH at increasing sensitivity levels

As detailed in Chain et al. (2007), this methodology was used to classify nine subfamilies of translational GTPases across 191 bacterial genomes . GTPase Der classification can be confirmed through phylogenetic analysis by:

• Retrieving sequences through BLAST searches using known Der sequences

• Aligning GTPase domains (full-length sequence alignment is often unreliable)

• Constructing phylogenetic trees to validate subfamily placement

The protein contains specific domains that distinguish it from other GTPases including the unique two-GTPase domain structure and C-terminal KH-domain, which can be identified through domain analysis tools .

What expression systems are recommended for producing functional recombinant Nitrosomonas europaea GTPase Der?

For successful expression of functional recombinant N. europaea GTPase Der, consider the following methodological approaches:

Expression System Selection:

• E. coli BL21(DE3) or derivative strains are commonly used for recombinant expression

• pET-based vectors with T7 promoter systems have demonstrated successful expression of Der proteins

• Consider codon optimization if expression levels are low (particularly important for N. europaea genes which may have codon usage bias different from E. coli)

Expression Conditions:

Protein Solubility Considerations:
To enhance solubility of recombinant Der, researchers have successfully employed fusion partners such as MBP (maltose-binding protein) or SUMO tags, which can later be removed through specific protease cleavage sites.

What are the optimal purification strategies for obtaining high-purity, active recombinant Nitrosomonas europaea GTPase Der?

Purification of N. europaea GTPase Der requires careful consideration of protein characteristics and activity requirements:

Multi-step Purification Protocol:

• Initial Capture:

  • Immobilized metal affinity chromatography (IMAC) using His-tagged constructs

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1-5 mM MgCl2 (critical for GTPase stability)

  • Imidazole gradient: 20-250 mM for washing and elution

• Intermediate Purification:

  • Ion exchange chromatography (typically Q-Sepharose)

  • Buffer: 20 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT

  • NaCl gradient: 50-500 mM for elution

• Polishing Step:

  • Size exclusion chromatography (Superdex 200)

  • Buffer: 20 mM HEPES pH 7.5, 150 mM KCl, 5 mM MgCl2, 1 mM DTT, 10% glycerol

Activity Preservation Considerations:

• Include 1-5 mM MgCl2 in all buffers to maintain nucleotide binding capability

• Add 5-10% glycerol to prevent protein aggregation

• Consider adding 0.1 mM GDP or GTP in storage buffers to stabilize protein conformation

• Flash-freeze aliquots in liquid nitrogen and store at -80°C for long-term stability

Purified protein should reach ≥85% purity as verified by SDS-PAGE , with expected yield of 2-5 mg per liter of bacterial culture. Activity assays should be performed promptly to confirm functional integrity.

How does the genetic context of the der gene in Nitrosomonas europaea compare to other bacterial species, and what are the implications for its function?

The genomic context of the der gene in N. europaea provides important insights into its functional relationships:

In N. europaea ATCC 19718, the der gene is positioned in a genomic region containing several critical genes involved in translation and transcription processes. This arrangement is similar to that found in other bacteria but with notable differences:

Comparative Genomic Organization:

As reported in Chain et al. (2003), the genome of N. europaea contains genes for ribosome-associated functions including elongation factors G (fusA) and Tu (tufB), and transcription anti-termination gene nusG . The der gene is likely part of this functional gene cluster.

The genomic proximity of der to these essential translation factors suggests coordinated expression and functional relationships in ribosome biogenesis pathways, potentially influenced by the unique metabolic constraints of N. europaea as an obligate chemolithoautotroph.

What methodologies are most effective for studying the in vivo interactions of GTPase Der with the ribosome in Nitrosomonas europaea?

Investigating the in vivo interactions of GTPase Der with ribosomes in N. europaea presents unique challenges due to the slow growth rate and specialized metabolism of this organism. The following methodological approaches can be employed:

Genetic Manipulation Approaches:

• Gene Disruption/Replacement:

  • Integration of antibiotic resistance gene cassettes via homologous recombination

  • Conjugation-based plasmid transfer systems as demonstrated for nirK mutations

  • Construction of conditional mutants when complete deletion is lethal

• Fluorescent Tagging:

  • GFP fusion constructs similar to those described by Gvakharia et al. (2009)

  • Promoter-reporter fusions to study der gene expression under different conditions

Biochemical Interaction Analysis:

• Ribosome Profiling:

  • Sucrose gradient centrifugation of cell lysates to separate ribosomal fractions

  • Western blot analysis of fractions using Der-specific antibodies

  • RNA analysis to identify associated ribosomal species

• In vivo Crosslinking:

  • Formaldehyde or UV-based crosslinking to capture transient interactions

  • Immunoprecipitation with Der-specific antibodies

  • Mass spectrometry analysis of crosslinked complexes

Specialized Adaptation for N. europaea:

• Custom media formulations supporting optimal growth (25 mM (NH4)2SO4 as nitrogen source)

• Extended incubation periods (several days) to account for slow growth rates

• Temperature control at 30°C in shaken batch cultures (175 rpm)

Because N. europaea has a doubling time of several days, experiments must be designed with longer timeframes than typical bacterial studies.

How can structural characterization of Nitrosomonas europaea GTPase Der inform our understanding of its molecular mechanism?

Structural characterization of N. europaea GTPase Der can provide critical insights into its functional mechanisms. Based on structural analysis approaches for related GTPases, researchers should consider:

Structural Determination Methods:

• X-ray Crystallography:

  • Crystallization screening with nucleotide analogs (GDP, GTP, non-hydrolyzable GTP analogs)

  • Co-crystallization with ribosomal components or RNA fragments

  • Resolution targets of 2.5Å or better to resolve domain interactions

• Cryo-electron Microscopy:

  • Sample preparation with ribosomes to capture interaction complexes

  • Classification analysis to identify different conformational states

  • Focus on structural transitions upon GTP binding/hydrolysis

Key Structural Features to Analyze:

• The dual GTPase domains and their nucleotide-binding states

• The C-terminal KH-domain and its RNA interaction surfaces

• Conformational changes between GTP- and GDP-bound states

• Interaction interfaces with ribosomal components

Molecular Dynamics Simulations:
Computational approaches can complement experimental structural data by:

• Modeling nucleotide-dependent conformational changes

• Predicting interaction surfaces with ribosomal components

• Calculating energetics of domain movements during GTPase cycles

Understanding the structural basis of Der function would inform its specific role in ribosome biogenesis, which appears to be essential across bacterial species .

What is the relationship between GTPase Der function and the unique metabolic characteristics of Nitrosomonas europaea as an ammonia-oxidizing bacterium?

The function of GTPase Der in N. europaea must be considered within the context of this organism's unique metabolism as an obligate chemolithoautotroph:

Metabolic Context:
N. europaea derives all its energy from ammonia oxidation and must fix CO2 for carbon acquisition . This demanding metabolic lifestyle creates unique cellular conditions that may influence Der function:

• Energy Limitations:

  • The energetic constraints of chemolithoautotrophy may require precise regulation of ribosome assembly

  • Der may play a critical role in balancing translation capacity with available energy resources

• Growth Rate Adaptation:

  • Cell division in N. europaea can take several days due to the limited energy available from ammonia oxidation

  • Der activity might be adapted to coordinate ribosome assembly with this slow growth rate

Experimental Approaches to Investigate This Relationship:

• Transcriptome analysis under varying ammonia concentrations to monitor der expression

• Proteomics studies comparing Der protein levels across growth phases

• Metabolic flux analysis to correlate translation machinery components with energy availability

Comparison with Other Bacterial Systems:

Given that N. europaea must carefully balance energy expenditure, GTPase Der likely plays a crucial role in optimizing ribosome assembly according to the energy available from ammonia oxidation, potentially with regulatory mechanisms unique to this metabolic specialist.