Recombinant Mycoplasma agalactiae GTPase Era (era)

What is the functional mechanism of GTPase Era in bacterial systems?

GTPase Era functions as a molecular switch through GTP binding and hydrolysis cycles. This switching behavior is essential for Era's cellular function, particularly in ribosome assembly . When bound to GTP, Era assumes an "ON" conformation where all G-motifs participate in the interaction, creating a rigid, closed GTPase domain. Upon GTP hydrolysis, conformational changes occur, switching Era to an "OFF" state .

This molecular switching mechanism coordinates ribosome biogenesis by timing the assembly of ribosomal components. Era intervenes relatively early in the small subunit (SSU) biogenesis and is critical for the proper formation of the platform, which is prerequisite for efficient translation . The timing of Era's action on the ribosome is regulated by its interactions with guanosine nucleotides (GTP, GDP, ppGpp), ribosomal RNA, and likely other factors that influence its GTPase activity .

What expression systems are optimal for producing recombinant M. agalactiae GTPase Era?

A recommended methodological approach includes:

• Gene synthesis or PCR amplification of the era gene from M. agalactiae genomic DNA

• Site-directed mutagenesis of TGA codons to TGG

• Cloning into an appropriate expression vector with a purification tag (similar to the approach used with pPRO EX HTb for P48 protein)

• Expression in the selected host system with optimization of induction conditions

• Purification using affinity chromatography based on the fusion tag

What are the optimal storage and handling conditions for recombinant GTPase Era?

For recombinant M. agalactiae GTPase Era protein:

• Short-term storage: Working aliquots can be maintained at 4°C for up to one week .

• Long-term storage: Store at -20°C, or preferably at -80°C for extended storage .

• Avoid repeated freeze-thaw cycles as this can compromise protein integrity and activity .

• For reconstitution: Briefly centrifuge vials before opening to bring contents to the bottom .

• Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• For optimal stability, add glycerol to a final concentration of 5-50% (50% is the recommended standard) .

The shelf life varies by formulation:

• Liquid form: approximately 6 months at -20°C/-80°C

• Lyophilized form: approximately 12 months at -20°C/-80°C

How can researchers validate the activity of purified recombinant GTPase Era?

To assess the functionality of purified recombinant GTPase Era, researchers should employ multiple complementary approaches:

• GTPase activity assay: Measure the rate of GTP hydrolysis using colorimetric or HPLC-based methods to detect phosphate release. Active Era will demonstrate concentration-dependent GTP hydrolysis.

• Nucleotide binding assays: Determine binding affinities for GTP and GDP using techniques such as isothermal titration calorimetry (ITC) or fluorescence-based methods. Era proteins typically bind these nucleotides with micromolar affinity .

• Conformational analysis: Use circular dichroism (CD) spectroscopy to confirm proper protein folding and thermal stability profiles characteristic of functional Era proteins.

• RNA binding assessment: Since Era interacts with ribosomal RNA, RNA electrophoretic mobility shift assays (EMSAs) can be used to demonstrate the protein's ability to bind appropriate rRNA targets.

• Complementation studies: For definitive functional validation, complementation assays can be performed where the recombinant Era is tested for its ability to rescue growth defects in Era-depleted bacterial cells.

What experimental approaches are useful for studying Era's role in ribosome assembly?

Given Era's critical role in ribosome biogenesis, several methodological approaches can be employed:

• Cryo-electron microscopy (cryo-EM): This technique can capture Era bound to ribosomal assembly intermediates, providing structural insights into its mode of action. Recent structures have been solved for Era proteins from various species bound to ribosomal components .

• Ribosome profiling: To assess the impact of Era depletion or mutation on ribosome assembly, researchers can analyze ribosome profiles using sucrose gradient centrifugation to detect abnormal assembly intermediates.

• In vitro reconstitution systems: Purified components of the small ribosomal subunit can be combined with recombinant Era to study its role in assembly processes under controlled conditions.

• Pull-down assays and mass spectrometry: Identify Era-interacting partners during ribosome assembly using tagged Era protein as bait, followed by proteomic analysis.

• SHAPE (Selective 2′-hydroxyl acylation analyzed by primer extension): Analyze RNA conformational changes induced by Era binding to identify specific structural elements affected during assembly.

How does M. agalactiae GTPase Era compare with homologs from other bacterial species?

GTPase Era is remarkably conserved across bacterial species, with structural studies covering phylogenetically distant bacterial clades including E. coli, Thermus thermophilus, and Aquifex aeolicus . Comparative analysis reveals:

• Domain architecture conservation: The two-domain structure consisting of the N-terminal GTPase domain and C-terminal KH domain is maintained across species.

• Functional conservation: Era consistently plays a critical role in small ribosomal subunit biogenesis across bacterial species and even in eukaryotic organelles (mitochondria and chloroplasts) .

• Sequence variations: While the core functional motifs are highly conserved, sequence divergence exists particularly in regions outside the GTP-binding pocket, potentially reflecting adaptations to specific cellular environments or regulatory mechanisms.

• Binding partners: The specific protein and RNA interaction partners may vary between species, though the fundamental interaction with 16S rRNA appears conserved.

• Regulatory mechanisms: Transcriptional, post-transcriptional, and post-translational regulatory mechanisms controlling Era expression and activity may differ between bacterial species, potentially reflecting their diverse ecological niches .

What insights from other bacterial GTPases can be applied to M. agalactiae Era research?

Research on GTPases from model organisms provides valuable methodological frameworks applicable to M. agalactiae Era studies:

• Crystallization approaches: Methods successfully used to determine structures of Era from E. coli and T. thermophilus can guide crystallization efforts for M. agalactiae Era .

• Mutational analysis strategies: Systematic mutation of conserved residues in the G-domain and KH domain, as performed in other bacterial systems, can help map the functional residues of M. agalactiae Era.

• Interaction mapping: Techniques used to identify Era-binding partners in other bacteria can be adapted to the M. agalactiae system to understand its specific interaction network.

• In vivo tracking: Approaches for monitoring GTPase dynamics in live cells, such as fluorescent protein fusions used with other bacterial GTPases, may be applicable with appropriate modifications for M. agalactiae.

What is the potential significance of GTPase Era in M. agalactiae pathogenesis?

While direct evidence for GTPase Era's role in M. agalactiae pathogenesis is limited in the provided search results, several inferences can be made based on broader mycoplasma research:

• Essential cellular function: As Era is critical for ribosome biogenesis, it likely represents an essential gene for M. agalactiae survival. Similar to the NIF locus identified as essential for mycoplasma growth in cell culture , Era may be required for bacterial fitness during infection.

• Potential target: The essential nature of Era makes it a potential target for antimicrobial development against M. agalactiae, which causes the economically important contagious agalactia in sheep and goats .

• Regulatory connections: Era may participate in stress response networks that coordinate bacterial adaptation to host environments, as observed with other bacterial GTPases during infection processes.

• Growth rate modulation: By controlling ribosome assembly, Era could influence M. agalactiae growth rates during different stages of infection, potentially contributing to persistence in the host.

How does M. agalactiae infection progress in experimental models, and what role might Era play?

Experimental infection models with M. agalactiae reveal specific pathogenesis patterns where Era may be involved:

In the natural host (lactating ewes), the parental PG2 strain establishes systemic infection, colonizing various body sites including lymph nodes and mammary glands . During infection:

• Host immune response: Infected animals develop specific antibody responses over the course of infection .

• Surface protein variation: Dynamic changes in expression of M. agalactiae surface variable proteins (Vpma) occur, with multiple Vpma profiles co-existing in the same animal . This variation likely helps evade host immune responses.

• Essential gene functions: Knock-out mutants of essential genes like those in the NIF locus fail to survive and colonize the host, resulting in avirulent phenotypes .

While Era's specific contribution wasn't addressed in these studies, its fundamental role in ribosome biogenesis suggests it would be essential for supporting the metabolic demands of bacterial replication during infection. Additionally, if Era regulation responds to host-derived signals, it could potentially contribute to adaptation mechanisms during different infection phases.

What site-directed mutagenesis approaches are recommended for functional studies of M. agalactiae GTPase Era?

Based on the methodology applied to other M. agalactiae proteins, researchers should consider the following approach for site-directed mutagenesis of Era:

• Codon optimization: As demonstrated with the P48 protein, conversion of TGA codons (which encode tryptophan in Mycoplasma but function as stop codons in E. coli) to TGG is essential when expressing M. agalactiae proteins in heterologous systems .

• Targeted mutations for functional analysis:

  • G-domain mutations in the G1 (P-loop), G2 (Switch I), G3 (Switch II), G4, and G5 motifs to disrupt GTP binding or hydrolysis

  • KH domain mutations to disrupt RNA binding

  • Interface mutations between domains to affect conformational dynamics

• Proven methodological steps:

  • PCR amplification of the era gene from M. agalactiae genomic DNA

  • Design of primers containing the desired mutations

  • PCR-based site-directed mutagenesis

  • Verification of mutations by sequencing

  • Cloning into expression vectors with appropriate tags

  • Expression and purification using established protocols

  • Functional validation of mutant proteins compared to wild-type

How can researchers address the challenges specific to working with Mycoplasma proteins?

Working with Mycoplasma proteins presents several unique challenges that researchers should address:

• Genetic code differences: Mycoplasma uses the TGA codon to encode tryptophan rather than as a stop codon. For heterologous expression, site-directed mutagenesis should be employed to convert all TGA codons to TGG, as demonstrated with the P48 protein .

• Membrane protein solubility: Many Mycoplasma proteins, including surface lipoproteins like P48, may have hydrophobic regions. Consider using solubility-enhancing fusion tags (MBP, SUMO) or detergent-based extraction methods.

• Post-translational modifications: Mycoplasma-specific lipid modifications may not be reproduced in heterologous systems. If these modifications are essential for function, consider cell-free systems supplemented with Mycoplasma cellular extracts.

• Protein stability issues: Add stabilizing agents like glycerol (5-50%) to buffers and avoid repeated freeze-thaw cycles .

• Antigenic variation: As seen with the Vpma proteins , some Mycoplasma proteins undergo phase variation. When studying such proteins, ensure clonal isolation and verification of the specific variant being expressed.

• Functional validation: Due to the genetic distance between Mycoplasma and model organisms, complementation studies may require specialized systems, potentially using related Mycoplasma species amenable to genetic manipulation.