Recombinant Clostridium novyi GTPase Era (era)

What is Clostridium novyi GTPase Era and what is its primary function?

GTPase Era is a deeply conserved protein critical for bacterial ribosome assembly. In Clostridium species, including C. novyi, Era functions primarily as a ribosomal assembly factor that intervenes relatively early in small ribosomal subunit biogenesis. Era is essential for the proper shaping of the ribosomal platform, which is a prerequisite for efficient translation . Like other bacterial Era proteins, C. novyi Era likely contains approximately 300-350 amino acids (~35 kDa) and consists of two globular domains: an N-terminal GTPase domain that binds guanosine nucleotides and a C-terminal KH domain that confers RNA-binding activity .

How does the structure of Era affect its function in ribosome assembly?

Era typically consists of two key domains connected by an unstructured linker. The N-terminal GTPase domain functions as a molecular switch triggered by GTP hydrolysis and reset by GDP/GTP exchange. The C-terminal KH domain enables RNA binding and ribosome association . Both domains must maintain their structural integrity for proper Era function. The GTPase domain contains five diagnostic motifs involved in GTP binding and hydrolysis arranged in a characteristic fold where a 6-stranded β-sheet is surrounded by 5 α-helices . Mutations in either domain can lead to severe phenotypes, demonstrating their critical importance for Era function .

What cellular processes are affected by Era activity?

Era impacts multiple cellular processes through its central role in ribosome biogenesis. As a critical nexus of small subunit assembly, Era is subject to sophisticated regulatory mechanisms at transcriptional, post-transcriptional, and post-translational levels . Deficiencies in Era function result in dramatic consequences for protein synthesis and far-reaching pleiotropic effects on organism physiology . In bacteria, Era mutations can cause heat and cold sensitivity, cell filamentation, significant growth delays, and inability to use certain carbon sources . The conserved nature of Era across species suggests that C. novyi Era likely plays similar essential roles.

What expression systems are recommended for producing functional recombinant C. novyi GTPase Era?

For successful expression of recombinant C. novyi GTPase Era, researchers should consider the following methodology:

• Vector selection: Use pET expression vectors with T7 promoter systems for high-yield bacterial expression. Include a His6-tag for purification, preferably at the N-terminus to avoid interference with the C-terminal KH domain functionality.

• Host optimization: E. coli BL21(DE3) strains are recommended, with Rosetta or CodonPlus variants to address potential codon bias issues in C. novyi genes. Alternatively, consider Arctic Express strains for expression at lower temperatures to enhance proper folding.

• Expression conditions: Induce with 0.1-0.5 mM IPTG at reduced temperatures (16-25°C) for 12-18 hours to maximize soluble protein yield while minimizing inclusion body formation. The GTPase activity is sensitive to proper folding, making expression conditions critical .

• Purification approach: Implement a two-step purification using immobilized metal affinity chromatography followed by size exclusion chromatography to ensure high purity and removal of protein aggregates that could interfere with functional assays.

How can researchers accurately measure the GTPase activity of recombinant C. novyi Era?

Measuring GTPase activity requires sensitive methods due to Era's relatively poor intrinsic GTPase activity . Recommended methodological approaches include:

• Malachite green assay: This colorimetric method detects inorganic phosphate released during GTP hydrolysis. Optimize the assay by including potassium ions, which stimulate Era's GTPase activity by an order of magnitude .

• HPLC-based nucleotide quantification: Monitor the conversion of GTP to GDP directly using reversed-phase HPLC with UV detection at 254 nm. Typical reaction conditions should include:

  • 1-5 μM purified recombinant Era protein

  • 50-200 μM GTP substrate

  • Buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2

  • Reaction temperature of 30-37°C

• Fluorescence-based assays: Use BODIPY-FL-GTP or mant-GTP as fluorescent GTP analogs to monitor binding and hydrolysis in real-time through changes in fluorescence intensity or anisotropy.

• Control experiments: Include measurements with inactive Era mutants (modifications in G1 or G2 motifs) as negative controls. The K21R mutation in the G1 motif, known to be lethal in E. coli Era, can serve as a reference for inactive protein .

What methodologies are effective for studying Era-RNA interactions in C. novyi?

RNA-binding studies are crucial for understanding Era function, as the KH domain interacts specifically with ribosomal RNA. Recommended approaches include:

• Electrophoretic mobility shift assays (EMSA): Use labeled RNA fragments corresponding to the 3' end of 16S rRNA to detect complex formation with recombinant Era. Compare binding in the presence of different nucleotides (GTP, GDP, non-hydrolyzable GTP analogs) to assess how nucleotide binding affects RNA interaction.

• Surface plasmon resonance (SPR): Quantitatively determine binding kinetics by immobilizing either the RNA or the protein on a sensor chip. This approach can reveal on/off rates and affinity constants under various conditions.

• RNA footprinting: Employ chemical or enzymatic probing methods to identify specific nucleotides protected by Era binding. This approach can map the exact interaction sites on the RNA.

• Microscale thermophoresis (MST): Use this solution-based technique to measure binding affinities with minimal sample consumption, allowing for rapid screening of different RNA constructs or Era mutants.

• Cryo-EM studies: For structural characterization of Era-ribosome complexes, cryo-electron microscopy can provide insights into binding modes similar to those recently revealed for human ERAL1 on nascent mitochondrial small subunits .

How does Era coordinate with other C. novyi proteins during ribosome biogenesis?

While specific information about C. novyi Era interactors is limited, comparative analysis with other bacterial systems suggests several methodological approaches to investigate these interactions:

• Bacterial two-hybrid screening: Identify potential protein interactors by screening C. novyi genomic libraries against Era as bait. Focus particular attention on proteins encoded by genes that show syntenic association with era, as these may be functionally related .

• Co-immunoprecipitation coupled with mass spectrometry: Use antibodies against tagged recombinant Era to pull down protein complexes from C. novyi lysates, followed by mass spectrometric identification of binding partners.

• Proximity labeling approaches: Express Era fused to BioID or APEX2 enzymes in C. novyi to biotinylate proximal proteins, which can then be isolated and identified by mass spectrometry.

• Genetic approaches: Create conditional Era depletion strains in C. novyi and identify suppressor mutations that rescue growth defects, potentially revealing functional interactions.

• Ribosome profile analysis: Compare ribosome assembly intermediates in wild-type and Era-depleted conditions using sucrose gradient centrifugation and RNA-seq to identify specific assembly steps affected by Era depletion.

Based on studies in other bacteria, potential interactors may include factors involved in DNA replication (dnaG), recombination (recO), transcription (rpoD), translation (glyQS), and protein folding (dnaJ) .

What is the relationship between Era function and C. novyi virulence?

The relationship between Era and virulence in C. novyi represents an important research direction, particularly given the role of C. novyi alpha-toxin (TcnA) in modifying small GTPases . To investigate this relationship, consider these methodological approaches:

• Conditional knockdown systems: Develop tetracycline-inducible or CRISPRi-based systems to modulate Era expression levels in C. novyi and assess impacts on:

  • Alpha-toxin production and secretion

  • Bacterial growth rates under different conditions

  • Sporulation efficiency

  • Antibiotic susceptibility profiles

• Toxin activity assays: Compare TcnA activity in wild-type and Era-depleted conditions using cell rounding assays with HEp-2 cells, which have been established for studying TcnA effects .

• Phosphoproteome analysis: Apply mass spectrometry-based phosphoproteomics to detect changes in GTPase-dependent signaling pathways when Era function is altered, similar to approaches used to study TcnA effects .

• Animal infection models: Use conditional Era expression strains in appropriate animal models to assess whether Era modulation affects C. novyi virulence in vivo.

• Transcriptome analysis: Perform RNA-seq comparing wild-type and Era-depleted conditions to identify virulence factors whose expression may be affected by Era dysfunction.

What crystallization strategies are most promising for structural studies of recombinant C. novyi Era?

Crystallization of GTPases presents specific challenges due to their conformational flexibility. Methodological recommendations include:

• Protein preparation: Express and purify Era in both nucleotide-free and nucleotide-bound states (with GTP, GDP, or non-hydrolyzable analogs like GMPPNP). Use size exclusion chromatography as the final purification step to ensure monodispersity.

• Truncation and mutation approaches: Create both full-length constructs and domain-specific constructs (separate GTPase domain and KH domain) to increase crystallization probabilities. Consider mutations in switch regions to reduce conformational heterogeneity.

• Crystallization screening: Employ a diverse initial screen including:

  • PEG-based conditions (PEG 3350, 4000, and 8000) at varying concentrations (10-25%)

  • Salt-based conditions (ammonium sulfate, sodium malonate)

  • pH ranges from 5.5 to 8.5

  • Various additives (particularly K+ ions, which affect GTPase activity)

• Co-crystallization approaches: Attempt crystallization with:

  • Nucleotide analogs (GMPPNP, GDP-AlF4)

  • RNA fragments corresponding to the 3' end of 16S rRNA

  • Potential protein binding partners

• Crystal optimization: Use seeding techniques and additive screens to improve crystal quality. Consider dehydration or annealing protocols to enhance diffraction quality.

• Alternative structural approaches: If crystallization proves challenging, consider cryo-EM for Era-ribosome complexes or NMR for individual domains.

How can CRISPR-Cas9 technology be applied to study Era function in C. novyi?

CRISPR-Cas9 technologies offer powerful approaches for studying Era function in C. novyi. Recommended methodological strategies include:

• Conditional expression systems: As Era is likely essential based on studies in other bacteria , design a two-plasmid system:

  • First plasmid: Inducible expression of wild-type Era

  • Second plasmid: CRISPR-Cas9 targeting the endogenous era gene

• Domain-specific mutagenesis: Create precise mutations in key functional regions:

  • G1 motif: Target conserved lysine residues essential for GTP binding

  • Switch regions: Create mutations analogous to the K21R mutation that causes lethality in E. coli

  • KH domain: Target conserved residues in the RNA-binding surface

• Protocol optimization:

  • Electroporation parameters: 1.5-2.0 kV, 200-400 Ω, 25 μF

  • Recovery media: Brain Heart Infusion supplemented with hemin and vitamin K

  • Selection strategy: Use appropriate antibiotics for each plasmid and inducers for conditional expression

• Phenotypic analysis: Assess the effects of Era depletion or mutation on:

  • Growth kinetics under various conditions

  • Cell morphology by phase-contrast and electron microscopy

  • Ribosome profiles using sucrose gradient analysis

  • Protein synthesis rates using pulse-chase experiments

  • Toxin production using immunoblotting or activity assays

What methods are most effective for studying the impact of Era on C. novyi stress response?

Era likely plays a role in stress responses, as mutations in other bacterial species result in heat and cold sensitivity . To investigate this aspect, consider:

• Stress exposure experiments: Challenge C. novyi strains with conditional Era expression with various stressors:

  • Temperature stress (heat shock, cold shock)

  • Oxidative stress (H2O2, paraquat)

  • Nutritional stress (amino acid starvation, carbon limitation)

  • Antibiotic stress (sublethal doses of translation inhibitors)

• Quantitative analysis methods:

  • Growth curve analysis under stress conditions using automated plate readers

  • Survival rate determination by colony forming unit (CFU) counts after stress exposure

  • Measurement of stress-responsive gene expression using RT-qPCR

  • Proteome analysis using 2D gel electrophoresis or LC-MS/MS

• Guanosine nucleotide measurements: Quantify intracellular (p)ppGpp levels, as these alarmones interact with Era and regulate its activity . Use HPLC or mass spectrometry-based methods for nucleotide quantification.

• Ribosome assembly monitoring: Assess ribosome profiles under stress conditions using sucrose gradient centrifugation to determine how Era depletion affects ribosome assembly during stress.