Recombinant Mycoplasma mycoides subsp. mycoides SC Ribosome-recycling factor (frr)

What is Mycoplasma mycoides subsp. mycoides SC and why is studying its ribosome-recycling factor important?

Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC) is the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle . As a member of the Mollicutes class, it has a small genome, no cell wall, and reduced metabolic capacities .

The ribosome-recycling factor (frr) catalyzes the fourth step of protein synthesis: disassembly of the post-termination complex of ribosomes, mRNA, and tRNA . The gene encoding RRF is widely distributed in prokaryotes, including Mycoplasma genitalium, which has retained frr despite having only ~500 genes and dispensing with other protein synthesis factors like RF2 and RF3 . This conservation indicates the critical importance of frr for bacterial survival, making it both a fundamental research target and a potential therapeutic avenue.

How are recombinant proteins from M. mycoides SC typically expressed and purified for research purposes?

Expression and purification of recombinant proteins from M. mycoides SC typically involves a multi-step process:

• Selection process: Target proteins are identified through genome sequence analysis using tools like SignalP to identify signal peptide sequences, TMHMM for transmembrane regions, and BLASTP to assess similarity to proteins in other species .

• Cloning methodology: Genes are cloned from M. mycoides SC whole genomic DNA, with careful consideration of TGA codons, which code for tryptophan in Mycoplasma but function as stop codons in standard bacterial expression systems .

• Codon optimization: A two-step PCR process is typically employed for TGA codon mutagenesis, converting Mycoplasma TGA codons to TGG for expression in E. coli .

• Expression system: Recombinant proteins are commonly expressed in E. coli BL21(DE3) with isopropyl 1-thio-β-d-galactopyranoside induction .

• Purification approach: Proteins are typically purified using immobilized metal ion chromatography, often with N-terminal hexahistidine and albumin-binding protein fusion tags (His6ABP) .

For transmembrane proteins, it's common practice to select only the largest extracellular domain to improve solubility and expression efficiency .

What experimental systems are used to study the function of ribosome-recycling factors in Mycoplasma species?

Several experimental approaches have proven valuable for studying ribosome-recycling factors in Mycoplasma and related prokaryotes:

• Temperature-sensitive mutants: Temperature-sensitive bacterial mutants with altered frr genes provide critical insights into in vivo function. When RRF is inactivated at non-permissive temperatures, ribosomes remain on mRNA, scan downstream from termination codons, and re-initiate translation at various sites without canonical initiation codons .

• Growth phase analyses: RRF inactivation shows differential effects depending on growth phase—bacteriostatic in the growing phase but bactericidal during the transition between stationary and growing phases . This provides a valuable experimental window for studying essential gene functions.

• Cryo-electron tomography: This technique has been used to visualize translation dynamics at atomic detail inside Mycoplasma pneumoniae cells, resolving thirteen distinct ribosome states that reflect intermediates during translation . Similar approaches could be adapted to study frr function.

• In vitro reconstitution systems: Though not explicitly mentioned in the search results, purified components can be used to reconstitute the ribosome recycling process in controlled conditions.

How does recombination affect genetic diversity of essential genes in Mycoplasma species?

Recombination plays a measurable but relatively minor role in genetic diversity of Mycoplasma species, with implications for essential genes like frr:

• Recombination frequency: Putative recombination events occur less frequently than vertically inherited mutations in all studied Mycoplasma clades, with ρ/θ ratios (which estimate the relative frequency of recombination versus mutation) ranging from 0 to 0.03 .

• Clade-specific patterns: Different Mycoplasma clades show varying recombination rates, as evidenced by the data below:

• Conservation pressure: Essential genes like frr typically experience strong purifying selection, meaning that recombination events that disrupt function would be selected against. The retention of frr even in minimal genomes like M. genitalium demonstrates its critical importance .

• Methodology for detection: Recombination events can be identified using tools like Gubbins and analyzed through phylogenetic approaches, with each gene scored based on the number of predicted recombination events in each clade .

What approaches are available for visualizing translation dynamics in Mycoplasma species?

Visualization of translation dynamics, including processes involving ribosome-recycling factor, can be achieved through several methodologies:

• Cryo-electron tomography: This technique has been successfully applied to visualize translation dynamics at atomic detail inside Mycoplasma pneumoniae cells . It allows researchers to:

  • Obtain in-cell atomic models of ribosomes

  • Identify distinct extensions of ribosomal proteins

  • Resolve different ribosome states that reflect intermediates during translation

• Classification of ribosomal states: Advanced computational methods can classify ribosomes into distinct states based on conformation and composition, with thirteen different states identified in M. pneumoniae .

• Polysome mapping: Three-dimensional mapping of translating ribosomes reveals how they arrange to form polysomes, with evidence that ribosomal protein L9 mediates local coordination .

• Translation animation: By capturing various intermediates, researchers can create animations of translation elongation, demonstrating the process at an atomic level of detail .

• Antibiotic perturbation studies: Antibiotics can be used to reshape the translation landscape inside cells, providing insights into the mechanisms of translation and potentially ribosome recycling .

What methodologies are most effective for studying surface proteins in M. mycoides SC, and how might these be applied to ribosome-associated proteins?

A systematic approach to characterize the surface proteome of M. mycoides SC has been developed with potential applications for studying ribosome-associated proteins like frr:

• Proteome selection strategy: Initially, the complete proteome is analyzed with SignalP to identify signal peptide sequences, followed by TMHMM and BLASTP analysis to identify transmembrane regions and assess similarity to proteins in other species .

• High-throughput expression platform: A multiplex approach allows expression of numerous proteins simultaneously:

  • Codon optimization through a two-step PCR mutagenesis process

  • Primer-based introduction of biotin for solid-phase restriction

  • Religation into appropriate expression vectors like pAff8c

  • Expression in E. coli BL21(DE3) with appropriate induction

• Multiplex analysis system: The Luminex suspension array technology enables simultaneous analysis of antibody binding to multiple individual proteins in minute sample volumes, allowing detection and clear signal separation between positive and negative sera .

• Validation through multiple approaches: Protein-specific signals are confirmed through inhibition experiments and compared with Western blot results .

• Longitudinal monitoring capabilities: The system allows tracking of IgG, IgM, and IgA responses over time, as demonstrated in a proof-of-concept study with 116 sera from eight animals in a CBPP vaccine study .

For ribosome-associated proteins like frr, this platform could be adapted to study:

• Protein-protein interactions with ribosomal components

• Conformational changes during the ribosome recycling process

• Effects of mutations on binding affinity and function

• Cross-reactivity with antibodies to identify conserved epitopes

How can homologous recombination be used to modify the frr gene in M. mycoides SC?

Homologous recombination represents an important genome editing tool for Mycoplasma species, with specific considerations for targeting essential genes like frr:

• Plasmid delivery systems: OriC plasmids have been developed to examine gene function in mycoplasmas, mesoplasmas, and spiroplasmas, providing vehicles for delivering homologous recombination constructs .

• Essential gene modification strategy: Since frr is likely essential, complete knockout would be lethal. Alternative approaches include:

  • Creating temperature-sensitive mutations similar to those used in E. coli studies

  • Introducing subtle mutations that affect function without completely eliminating it

  • Using inducible promoters to control expression levels

  • Implementing complementation strategies with wild-type copies at alternative genomic locations

• Recombination frequency considerations: The relatively low frequency of natural recombination in Mycoplasma species (ρ/θ ratios of 0-0.03) suggests that engineered recombination systems may require optimization for efficiency.

• Selection strategies: Appropriate selection markers must be chosen, with consideration for the limited metabolic capabilities of Mycoplasma species.

• Verification methodology: Successful recombination must be verified through:

  • PCR and sequencing to confirm the desired modification

  • Phenotypic assays to assess impact on growth and protein synthesis

  • For temperature-sensitive mutations, comparative growth at permissive and non-permissive temperatures

What challenges exist in developing CRISPR/Cas systems for editing the frr gene in M. mycoides SC?

CRISPR/Cas systems represent emerging tools for Mycoplasma genome editing, but face several challenges when targeting essential genes like frr:

• Delivery challenges: The lack of established transformation methods for many Mycoplasma species complicates the introduction of CRISPR/Cas components .

• Essential gene targeting: Since frr is likely essential for growth, complete knockout would be lethal, necessitating more sophisticated approaches:

  • Conditional knockdown strategies

  • Precise editing to introduce specific mutations

  • Complementation systems to maintain cell viability

• Genome peculiarities: Several features of Mycoplasma genomes present challenges:

  • Alternative genetic code (TGA coding for tryptophan)

  • Low GC content affecting guide RNA design

  • Reduced genome lacking some repair mechanisms that typically work alongside CRISPR systems

• Limited genetic toolkit: The paucity of genetic tools to manipulate mycoplasma genomes has impeded studies of virulence factors and essential genes , requiring customization of CRISPR systems for these organisms.

• Validation strategies: For essential genes like frr, distinguishing between unsuccessful editing and lethal modifications requires careful experimental design and appropriate controls.

How do ribosome-recycling factors from different Mycoplasma species compare, and what does this reveal about their evolution?

While the search results don't provide direct comparisons of frr across different Mycoplasma species, several inferences can be made:

• Evolutionary conservation: The frr gene is widely distributed in prokaryotes, including minimalist genomes like Mycoplasma genitalium . This suggests strong selective pressure to maintain this gene throughout evolution.

• Functional importance: Even the minimal genome of M. genitalium has retained frr while dispensing with other protein synthesis factors such as RF2 and RF3 . This indicates that the ribosome-recycling step may be more essential than some aspects of termination.

• Comparative analysis approach: Researchers can employ several methods to compare frr across species:

  • Phylogenetic analysis to trace evolutionary relationships

  • Structural comparison to identify conserved domains

  • Functional complementation to test interchangeability

• Recombination impact assessment: Using tools like those described in search result , researchers can determine whether frr genes show evidence of recombination events or primarily evolve through vertical inheritance.

• Species-specific adaptations: Comparing frr sequences across Mycoplasma species that infect different hosts could reveal adaptations related to specific ecological niches or host interactions.

What experimental designs are most appropriate for studying temperature-sensitive frr mutants in Mycoplasma species?

Based on studies with E. coli temperature-sensitive frr mutants , several experimental designs would be valuable for Mycoplasma research:

• Mutant generation strategy:

  • Site-directed mutagenesis based on known temperature-sensitive mutations in E. coli

  • Random mutagenesis followed by temperature-based selection

  • Homologous recombination to introduce mutations into the native locus

• Phenotypic characterization:

  • Growth curves at permissive and non-permissive temperatures

  • Microscopy to observe cellular morphology changes

  • Protein synthesis assays to measure translation efficiency

• Molecular analysis of translation events:

  • Analysis of re-initiation events downstream from termination codons

  • Measurement of ribosome occupancy on mRNA

  • Determination of whether re-initiation occurs in all reading frames

• Growth phase considerations:

  • Comparison of bacteriostatic effects in growing phase versus bactericidal effects during transition from stationary to growing phase

  • Time-course experiments to track changes after temperature shift

• Rescue experiments:

  • Complementation with wild-type frr gene

  • Expression of frr from related species to test functional conservation

  • Chemical rescue approaches if applicable

How can suspension array technology be optimized for studying multiple aspects of frr function simultaneously?

The magnetic bead-based suspension array technology described for surface protein analysis could be adapted for comprehensive frr functional studies:

• Multiplex binding partner analysis:

  • Coupling of recombinant frr to uniquely identifiable beads

  • Simultaneous testing of interactions with multiple ribosomal components

  • Quantification of binding affinities under various conditions

• Mutant screening platform:

  • Creation of bead sets with different frr variants

  • Parallel assessment of functional changes resulting from mutations

  • Identification of critical residues for specific interactions

• Conformational analysis:

  • Development of conformation-specific antibodies

  • Detection of structural changes in response to binding partners or conditions

  • Monitoring of frr states during the ribosome recycling process

• Inhibitor screening:

  • Evaluation of compounds that disrupt frr-ribosome interactions

  • Quantification of inhibition constants for multiple compounds simultaneously

  • Structure-activity relationship studies for inhibitor optimization

• Optimization parameters:

  • Sample volume minimization (demonstrated capability with minute volumes)

  • Signal-to-noise ratio enhancement (20-fold mean signal separation demonstrated between positive and negative samples)

  • Cross-validation with alternative techniques like Western blot

What are the most promising approaches for visualizing frr-ribosome interactions at atomic resolution in Mycoplasma species?

Visualization of frr-ribosome interactions at atomic resolution can be achieved through several cutting-edge approaches:

• Cryo-electron tomography: This technique has successfully visualized translation dynamics in M. pneumoniae at atomic detail and could be applied to capture frr-ribosome interactions by:

  • Targeting cells at specific stages of translation termination

  • Utilizing classification approaches to identify frr-bound ribosome states

  • Comparing wild-type cells with frr mutants

• Sub-tomogram analysis: This computational approach allows researchers to:

  • Obtain in-cell atomic models for the Mycoplasma ribosome

  • Resolve distinct conformational states

  • Identify the position and orientation of frr during the recycling process

• Translation state classification: Advanced classification methods have identified thirteen ribosome states in M. pneumoniae . Similar approaches could focus specifically on the recycling step to:

  • Characterize conformational changes during frr binding

  • Track movements of ribosomal subunits during recycling

  • Identify additional factors that participate in the process

• Animation of recycling process: By capturing multiple intermediates, researchers could create animated visualizations of the ribosome recycling process at atomic detail .

• Combined methodological approach: Integration of structural data with biochemical and genetic approaches would provide comprehensive understanding of frr function in the cellular context.