Recombinant Burkholderia ambifaria Ribosome-recycling factor (frr)

What is the ribosome-recycling factor (frr) and what is its function in bacterial protein synthesis?

Ribosome-recycling factor (RRF), encoded by the frr gene, is an essential bacterial protein required for the disassembly of post-termination ribosomal complexes. RRF works in conjunction with elongation factor G (EF-G) to release 70S ribosomes from mRNA after translation termination, allowing ribosomal subunits to be recycled for subsequent rounds of protein synthesis .

Methodology for characterizing RRF function includes:

• Conditional RRF depletion systems

• Ribosome profiling to map ribosome positions genome-wide

• In vitro reconstitution assays with purified components

• Reporter assays to measure translation efficiency

What is Burkholderia ambifaria and why is it significant for research on bacterial translation?

Burkholderia ambifaria is a gram-negative bacterium belonging to the Burkholderia cepacia complex (BCC) . This organism has remarkable ecological versatility, exhibiting a dual lifestyle that makes it particularly interesting for research:

• As a plant-associated bacterium, it colonizes the rhizosphere of many crop plants including maize and functions as a biocontrol agent against plant pathogens. B. ambifaria produces antifungal compounds like pyrrolnitrin and demonstrates capabilities for phosphate solubilization, indole-3-acetic acid (IAA) production, and siderophore synthesis .

• As an opportunistic human pathogen, it can cause respiratory infections in immunocompromised individuals, particularly those with cystic fibrosis .

This dual ecological role makes B. ambifaria an excellent model for studying how translational machinery, including RRF, adapts to function across diverse environments. Understanding the molecular basis of this adaptability could provide insights into bacterial evolution and host-microbe interactions at the translational level.

How is the frr gene typically identified and cloned from B. ambifaria?

The identification and cloning of the frr gene from B. ambifaria typically involves the following methodological steps:

• Genomic sequence analysis: Start with bioinformatics approaches to identify the putative frr gene in B. ambifaria genome databases using homology to known frr sequences from related bacteria. The gene is typically located in conserved genomic contexts - in P. aeruginosa, for example, frr is positioned downstream of the genes for ribosomal protein S2 (rpsB), elongation factor Ts (tsf), and UMP kinase (pyrH) .

• PCR amplification: Design primers based on the identified sequence to amplify the complete frr gene and its regulatory elements from B. ambifaria genomic DNA.

• Functional complementation: Clone the PCR product into an appropriate expression vector and test its functionality by complementation of a temperature-sensitive frr mutant of E. coli, similar to the approach used for P. aeruginosa frr .

• Verification and characterization:

  • Sequence the cloned gene to confirm its identity

  • Analyze the promoter and regulatory regions

  • Compare sequence conservation with RRF from other bacteria

  • Assess cross-species functionality in heterologous systems

This approach not only isolates the gene but also confirms its functionality, providing the foundation for further studies of B. ambifaria RRF.

What expression systems are most effective for producing recombinant B. ambifaria RRF?

For efficient production of recombinant B. ambifaria RRF, several expression systems can be considered, each with specific advantages for different research applications:

• E. coli-based expression systems:

  • pET vector systems with T7 promoter in BL21(DE3) strains offer high-yield expression

  • Addition of affinity tags (6xHis, as used in other recombinant proteins ) facilitates purification

  • Expression optimization parameters include:

    • Induction temperature (typically 16-25°C to enhance proper folding)

    • IPTG concentration (0.1-1.0 mM)

    • Culture media composition (LB, TB, or minimal media depending on downstream applications)

    • Co-expression with chaperones if folding issues occur

• Native host expression:

  • Expression in Burkholderia species may preserve native folding and potential post-translational modifications

  • Inducible promoter systems adapted for Burkholderia should be used

  • Lower yields but potentially higher specific activity

• Cell-free protein synthesis:

  • Allows rapid production without cellular constraints

  • Useful for proteins that may be toxic when overexpressed in living cells

  • Permits incorporation of modified amino acids for structural studies

For functional studies requiring active RRF, expression conditions should be optimized to maintain proper protein folding, with activity verified through in vitro ribosome recycling assays using both homologous and heterologous ribosome systems .

What analytical methods are used to characterize recombinant B. ambifaria RRF?

Comprehensive characterization of recombinant B. ambifaria RRF requires a combination of structural, functional, and biochemical approaches:

• Structural characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure elements

  • X-ray crystallography or cryo-electron microscopy for high-resolution structural determination

  • Thermal shift assays to evaluate stability under different conditions

  • Size exclusion chromatography to confirm monomeric state

• Functional analysis:

  • In vitro ribosome recycling assays using:

    • Purified B. ambifaria ribosomes

    • Heterologous ribosome systems (e.g., E. coli) to assess cross-species activity

  • Polysome disassembly assays measuring release of monosomes from polysomes

  • GTPase activation assays to measure stimulation of EF-G GTPase activity

• Interaction studies:

  • Surface plasmon resonance (SPR) to determine binding kinetics with ribosomes

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of binding

  • Pull-down assays to identify interaction partners

• Biochemical properties:

  • Optimal pH and temperature for activity

  • Effects of ionic strength on function

  • Stability in different buffer conditions

  • Mass spectrometry to confirm protein integrity and any modifications

These analytical approaches provide complementary data for understanding both the structure-function relationships of B. ambifaria RRF and its potential adaptations for the organism's dual lifestyle.

How does the structure and function of B. ambifaria RRF compare to RRF from other bacterial species?

Comparative analysis of B. ambifaria RRF with RRF proteins from other bacteria can reveal species-specific adaptations that may contribute to its ecological versatility:

• Structural comparisons:

  • While specific B. ambifaria RRF structural data is not yet widely available, comparative analysis with other bacterial RRFs would likely reveal conservation of the characteristic L-shaped conformation with a tRNA-mimicking domain

  • Variations in surface residues may reflect adaptation to different ribosomal binding environments

  • Key differences to investigate include:

    • Interface regions between domains

    • Residues that interact directly with the ribosome

    • Flexible regions that undergo conformational changes during recycling

• Functional cross-species activity:

  • P. aeruginosa RRF has been shown to function with E. coli ribosomes, releasing monosomes from polysomes in heterologous assays

  • Testing whether B. ambifaria RRF shows similar cross-species functionality would provide insights into the conservation of the recycling mechanism

  • Variations in activity across temperature and pH ranges may reveal adaptations to B. ambifaria's diverse habitats

• Sequence-function relationships:

  • Molecular phylogenetic analysis can identify residues under selective pressure

  • Site-directed mutagenesis of these residues can establish their functional significance

  • Chimeric proteins containing domains from different bacterial RRFs can help map species-specific functional regions

This comparative approach can reveal how evolutionary pressures from B. ambifaria's dual lifestyle as both plant-beneficial and human-pathogenic have shaped its translational machinery.

What role does RRF play in translational coupling in B. ambifaria operons?

Translational coupling in bacterial operons has been hypothesized to involve ribosome recycling mechanisms, but recent research in E. coli suggests this may not be the primary mechanism . Investigating this question in B. ambifaria would provide valuable comparative data:

• Experimental approaches to investigate coupling mechanisms:

  • Ribosome profiling before and after RRF depletion to examine ribosome density at intergenic regions

  • Analysis of the ratio of ribosome density on neighboring genes in polycistronic transcripts

  • Construction of reporter systems with different intergenic regions between coupled genes

  • Comparison of coupling efficiency in operons with overlapping versus separated stop/start codons

• Expected outcomes based on E. coli data:

  • In E. coli, RRF depletion did not significantly affect the ratio of ribosome density on neighboring genes in polycistronic transcripts, suggesting re-initiation is not a major coupling mechanism

  • If B. ambifaria shows different patterns, this would suggest species-specific coupling mechanisms

  • Particular attention should be paid to operons involved in:

    • Biocontrol functions (e.g., antifungal compound production)

    • Host interaction factors

    • Stress response pathways

• Operon structure analysis:

  • Genome-wide analysis of intergenic regions in B. ambifaria operons

  • Correlation between operon structure and gene function

  • Comparison with other Burkholderia species and more distant bacteria

This research would contribute to understanding how translational coupling mechanisms might vary across bacterial species and potentially reveal adaptations specific to B. ambifaria's ecological niches.

How does RRF depletion affect global gene expression in B. ambifaria?

Investigating the genome-wide effects of RRF depletion in B. ambifaria would provide insights into both general translation mechanisms and species-specific responses:

• Expected primary effects based on E. coli studies:

  • Accumulation of ribosomes in 3′-UTRs as post-termination complexes fail to be recycled

  • Ribosome queuing upstream of stop codons as elongating ribosomes are blocked by non-recycled ribosomes

  • Potential ribosome stalling at specific sequence contexts

• Secondary effects on gene expression:

  • In E. coli, RRF depletion led to significant upregulation of ribosome rescue factors, including a 39-fold increase in ArfA levels

  • B. ambifaria-specific responses might include changes in:

    • Expression of plant-interaction factors

    • Virulence-associated genes

    • Stress response pathways

• Methodology for global analysis:

  • Construction of conditional RRF depletion strains using:

    • Inducible antisense RNA

    • CRISPR interference (CRISPRi)

    • Controllable degradation systems

  • Multi-omics approaches:

    • RNA-seq for transcriptome analysis

    • Ribosome profiling for translation analysis

    • Proteomics for protein abundance changes

    • Metabolomics for downstream metabolic effects

• Data analysis framework:

  • Time-course sampling to distinguish primary from secondary effects

  • Pathway enrichment analysis to identify affected cellular processes

  • Comparison with similar datasets from other bacterial species

This comprehensive analysis would provide insights into how B. ambifaria's translation machinery responds to recycling defects and potentially reveal unique adaptations related to its ecological versatility.

How does B. ambifaria RRF contribute to the organism's environmental adaptability?

The dual lifestyle of B. ambifaria as both a plant-beneficial microbe and human pathogen raises questions about how its translational machinery, including RRF, adapts to different environments:

• Environmental responsiveness of RRF:

  • Analysis of frr gene expression under conditions mimicking:

    • Plant rhizosphere (temperature, pH, nutrient availability)

    • Human host environments (temperature, immune factors)

    • Stress conditions (oxidative stress, antimicrobial exposure)

  • Potential temperature-dependent structural adaptations of RRF protein

  • Interaction with environment-specific translation factors

• Contribution to stress response and adaptation:

  • Role in recovery of translation following:

    • Temperature shifts when transitioning between environments

    • pH changes in different niches

    • Nutrient limitation scenarios

  • Potential specialized functions in recycling stalled ribosomes under stress

• Experimental approaches:

  • GFP-tagged B. ambifaria strains (similar to those described in ) with modified RRF levels to monitor colonization efficiency in plant tissues

  • Competition assays between wild-type and RRF-altered strains in different environments

  • In vitro translation assays under varying conditions to assess RRF activity

  • Structural studies of RRF under different environmental conditions

• Potential adaptations to investigate:

  • Thermostability differences compared to RRF from strictly human or environmental pathogens

  • Protein-protein interactions unique to B. ambifaria RRF

  • Regulatory mechanisms controlling RRF levels in different environments

Understanding how RRF contributes to B. ambifaria's remarkable environmental adaptability could provide insights into bacterial adaptation mechanisms and potentially reveal new approaches for controlling pathogenicity while preserving beneficial functions.

What advanced structural biology approaches can reveal new insights about B. ambifaria RRF function?

Cutting-edge structural biology techniques can provide unprecedented insights into the molecular mechanisms of B. ambifaria RRF:

• Cryo-electron microscopy (cryo-EM) approaches:

  • Single-particle analysis of RRF-ribosome complexes at different functional states

  • Time-resolved cryo-EM to capture transient intermediates in the recycling process

  • Visualization of RRF interaction with other factors (EF-G, ribosome rescue factors)

  • Sample preparation considerations:

    • Crosslinking to stabilize transient complexes

    • GTP analogs to trap specific states

    • Nanodisc embedding for membrane-associated translation studies

• Single-molecule techniques:

  • Fluorescence resonance energy transfer (FRET) to monitor:

    • RRF binding and dissociation kinetics

    • Conformational changes during recycling

    • Effects of mutations on function

  • Optical tweezers or magnetic tweezers to measure forces involved in ribosome splitting

  • Total internal reflection fluorescence (TIRF) microscopy to visualize individual recycling events

• Advanced computational approaches:

  • Molecular dynamics simulations to model:

    • RRF interaction with the ribosome

    • Conformational changes during function

    • Effects of environmental conditions (temperature, pH) on structure

  • Machine learning approaches to predict functional hotspots

  • Computational design of modified RRF with enhanced or altered functions

• In-cell structural approaches:

  • Cryo-electron tomography of B. ambifaria cells to visualize RRF-ribosome interactions in situ

  • Proximity labeling (BioID, APEX) to map the spatial organization of the recycling machinery

  • In-cell NMR to monitor RRF dynamics in the cellular environment

These advanced structural approaches would provide mechanistic insights into RRF function and potentially reveal B. ambifaria-specific adaptations that contribute to its environmental versatility.

Table 1: Comparison of RRF Properties Across Bacterial Species

*Estimated based on typical bacterial RRF properties; requires experimental confirmation.

Table 2: Methods for Studying RRF Function in B. ambifaria

Table 3: Potential B. ambifaria RRF Research Applications