Recombinant Renibacterium salmoninarum Ribosome-recycling factor (frr)

What is Ribosome-recycling factor (frr) and what is its function in Renibacterium salmoninarum?

Ribosome-recycling factor (frr), originally called ribosome releasing factor, is an essential bacterial protein responsible for disassembling post-termination complexes during protein synthesis. In R. salmoninarum, as in other bacteria, frr functions to release ribosomes from mRNA after translation termination, allowing them to participate in new rounds of protein synthesis. The frr protein from R. salmoninarum is 185 amino acids long with a molecular mass of approximately 20.6 kDa . Structurally, RRF is a near-perfect mimic of tRNA, which explains its ability to interact with the ribosome . The disassembly of post-termination complexes by RRF depends on elongation factor G (EF-G), GTP, and the RRF protein itself, ultimately releasing ribosomes from mRNA either as 70S ribosomes or as 50S subunits, depending on various factors including mRNA configuration and Mg²⁺ concentration .

Why is studying recombinant frr from R. salmoninarum important for fish pathogen research?

R. salmoninarum is the causative agent of bacterial kidney disease (BKD), a major pathogen affecting salmonid fish species worldwide . Studying recombinant frr is important because:

• R. salmoninarum is slow-growing and difficult to culture in vitro, which has restricted our knowledge of its pathogenicity mechanisms .

• Understanding essential bacterial proteins like frr provides insights into potential antimicrobial targets.

• Recombinant expression allows researchers to study proteins from pathogens that are otherwise challenging to work with due to slow growth or specialized culture requirements.

• Research on frr contributes to our understanding of protein synthesis in this economically important fish pathogen, potentially informing disease control strategies for aquaculture .

What are the recommended expression systems for producing recombinant R. salmoninarum frr?

For expressing recombinant R. salmoninarum frr, E. coli-based expression systems are commonly used due to their efficiency and ease of manipulation. Based on research with other R. salmoninarum proteins:

Recommended protocol:

• Vector selection: pET expression systems are suitable for frr expression, as they provide high-level expression under tight control.

• Host strain: E. coli K12 host vector systems have been successfully used for R. salmoninarum gene libraries . BL21(DE3) strains are recommended for protein expression due to their deficiency in lon and ompT proteases.

• Codon optimization: Consider codon optimization as R. salmoninarum has a high G+C content, which may affect expression efficiency in E. coli.

• Expression conditions:

  • Induce with 0.5-1.0 mM IPTG

  • Express at lower temperatures (16-25°C) to enhance solubility

  • Include 5-10% glycerol in lysis buffers to improve stability

• Purification strategy: Use affinity tags such as His6 for one-step purification via IMAC. For functional studies, consider tag removal using a protease cleavage site.

Research with other R. salmoninarum proteins has shown that recombinant proteins can be successfully expressed and purified from E. coli systems while maintaining structural and functional properties .

How can I verify the functional activity of recombinant R. salmoninarum frr protein?

Verifying functional activity of recombinant frr requires assessing its ability to disassemble post-termination complexes. Based on methodologies used for RRF from other organisms:

Functional assay procedures:

• Ribosome recycling assay:

  • Prepare post-termination complexes using purified ribosomes, mRNA with a stop codon, and appropriate release factors

  • Add recombinant frr, EF-G, and GTP

  • Monitor dissociation by sucrose density gradient centrifugation

  • In functional assays, RRF should convert bound 70S ribosomes into free ribosomes or subunits

• Complementation assay:

  • Use temperature-sensitive E. coli mutants with defective frr

  • Transform with a plasmid expressing R. salmoninarum frr

  • Test growth at non-permissive temperatures

  • Functional frr will rescue growth at restrictive temperatures

• In vitro translation enhancement:

  • Measure protein synthesis rates in a reconstituted translation system with and without recombinant frr

  • Functional frr should stimulate protein synthesis 4- to 7-fold when ribosomes are limiting

• Ribosome binding assays:

  • Assess binding of labeled frr to ribosomes using filter binding or surface plasmon resonance

  • Compare binding affinity to known RRF proteins from model organisms

How does RRF depletion affect translation in bacterial systems and what implications does this have for R. salmoninarum research?

RRF depletion has profound effects on translation in bacterial systems, as demonstrated by ribosome profiling studies in E. coli:

Effects of RRF depletion:

• Accumulation of post-termination complexes: Ribosomes accumulate at stop codons and in 3'-UTRs, as shown by increased ribosome density in these regions .

• Translation blockage: Elongating ribosomes are unable to complete translation because they are blocked by non-recycled ribosomes at stop codons .

• Ribosome queueing: RRF depletion causes ribosomes to stack upstream of stop codons, indicating that earlier ribosomes translating the same mRNA cannot finish translation when recycling is impaired .

• Effects on gene expression: Loss of recycling leads to significant changes in gene expression, including dramatic upregulation of ribosome rescue factors .

Implications for R. salmoninarum research:

• As an essential protein in bacteria, frr could be a potential antimicrobial target for controlling R. salmoninarum infections.

• The slow growth rate of R. salmoninarum might be partially explained by inefficient translation processes, including ribosome recycling.

• Understanding frr function in R. salmoninarum could provide insights into its unique physiological adaptations, such as intracellular survival and chronic infection establishment.

• The impact of frr on translational coupling could affect expression of virulence factors in operons, potentially influencing pathogenicity.

What structural and functional differences might exist between R. salmoninarum frr and homologs from other bacterial species?

Analyzing the sequence and predicted structure of R. salmoninarum frr reveals both conservation and potential differences compared to other bacterial RRFs:

Comparative analysis:

Functional implications:

• Subtle structural differences may affect the efficiency of ribosome recycling, potentially contributing to the slow growth rate of R. salmoninarum.

• Species-specific interactions with EF-G could influence the energy requirements for ribosome recycling.

• Differences in thermal stability might reflect adaptation to the temperature range of salmonid hosts.

• Understanding these differences could guide the development of selective inhibitors that target R. salmoninarum frr without affecting commensal bacteria.

What challenges might researchers face when working with recombinant R. salmoninarum frr and how can they be addressed?

Common challenges and solutions:

• Protein solubility issues:

  • Challenge: R. salmoninarum proteins may form inclusion bodies when overexpressed.

  • Solution: Express at lower temperatures (16-20°C), reduce inducer concentration, use solubility-enhancing fusion tags (SUMO, MBP), or optimize buffer conditions (include glycerol, mild detergents, or arginine) .

• Protein stability:

  • Challenge: Functional integrity may be compromised during purification.

  • Solution: Include protease inhibitors, maintain samples at 4°C, and consider adding stabilizing agents like glycerol or specific divalent cations.

• Functional assay sensitivity:

  • Challenge: Detecting ribosome recycling activity may require sensitive methods.

  • Solution: Implement fluorescence-based assays or use ribosome profiling techniques to detect changes in ribosome distribution on mRNAs .

• Contamination with host RRF:

  • Challenge: E. coli-derived RRF may co-purify with recombinant R. salmoninarum frr.

  • Solution: Use stringent purification conditions, implement multiple chromatography steps, or design expression constructs with unique tags that allow selective purification.

• Accurate concentration determination:

  • Challenge: Quantifying active protein versus total protein.

  • Solution: Use activity-based assays alongside standard protein quantification methods to determine the fraction of functionally active protein.

How can researchers interpret contradictory data when studying the function of recombinant R. salmoninarum frr?

When faced with contradictory data regarding recombinant R. salmoninarum frr function, researchers should consider:

Systematic approach to resolving contradictions:

• Validate protein integrity:

  • Confirm protein purity by SDS-PAGE and mass spectrometry

  • Verify proper folding using circular dichroism or fluorescence spectroscopy

  • Assess oligomeric state using size exclusion chromatography or analytical ultracentrifugation

• Check experimental conditions:

  • RRF function is dependent on factors like Mg²⁺ concentration, which can critically affect results

  • Temperature, pH, and buffer composition can influence activity measurements

  • Presence/absence of GTP and EF-G will significantly impact functional assays

• Consider methodological limitations:

  • Different assays measure different aspects of RRF function

  • In vitro systems may not fully recapitulate the in vivo environment

  • High-salt conditions in ribosome profiling experiments can dissociate post-termination complexes, affecting interpretations

• Address biological context:

  • RRF interacts with multiple components of the translation machinery

  • Species-specific differences in ribosomal proteins may affect heterologous assays

  • Contradictory results might reflect genuine biological complexity rather than experimental error

• Reconcile findings with literature:

  • Recent studies suggest that some traditional views of RRF function may need revision

  • For instance, research showing that RRF depletion did not significantly affect translational coupling efficiency challenges previous models

What is the potential role of frr in R. salmoninarum pathogenesis and host-pathogen interactions?

While direct evidence for frr's role in R. salmoninarum pathogenesis is limited, its essential function in protein synthesis suggests several potential contributions:

Potential roles in pathogenesis:

• Adaptation to intracellular environment:

  • R. salmoninarum is a facultative intracellular pathogen

  • Efficient ribosome recycling may be crucial for protein synthesis under the resource-limited conditions inside host cells

  • Specialized adaptations in frr could contribute to the bacterium's ability to persist within macrophages

• Regulation of virulence factor expression:

  • The expression of the major soluble antigen (MSA), a key virulence factor in R. salmoninarum, could be influenced by translational efficiency

  • frr might affect translational coupling in operons containing virulence genes

  • Both msa1 and msa2 genes are needed for full virulence, suggesting complex translational regulation

• Response to host immune defenses:

  • During infection, pathogens must rapidly adjust protein synthesis in response to host defenses

  • Efficient ribosome recycling is crucial during stress responses when rapid reprogramming of the proteome is required

  • Studies have shown that nutritional immunomodulation affects Atlantic salmon's response to R. salmoninarum, which could involve changes in bacterial translation efficiency

• Potential as a therapeutic target:

  • As an essential gene with structural differences from host factors, frr could be targeted for antimicrobial development

  • Inhibition of ribosome recycling would affect multiple aspects of bacterial physiology simultaneously

  • The slow growth rate of R. salmoninarum might make it particularly vulnerable to translation-targeting antimicrobials

How might studying recombinant R. salmoninarum frr contribute to understanding the evolution and adaptation of this fish pathogen?

Studying recombinant R. salmoninarum frr can provide insights into evolutionary aspects of this pathogen:

Evolutionary insights:

Can recombinant R. salmoninarum frr be used to develop improved diagnostic tools for bacterial kidney disease?

While frr is not currently used in diagnostic applications for R. salmoninarum, recombinant frr could potentially contribute to diagnostic development:

Potential diagnostic applications:

• Development of anti-frr antibodies:

  • Recombinant frr could be used to generate specific antibodies

  • These antibodies might complement existing diagnostic tools that target other R. salmoninarum antigens

  • Immunoassays targeting multiple bacterial proteins simultaneously could increase diagnostic sensitivity

• PCR-based detection:

  • Current PCR methods for R. salmoninarum detection target the major surface antigen gene

  • The essential nature of frr makes it a stable target that is unlikely to be lost

  • Designing primers specific to unique regions of the R. salmoninarum frr gene could provide an alternative molecular diagnostic approach

• Comparison with existing methods:

  • Current detection methods for R. salmoninarum include culture, ELISA, fluorescent antibody techniques (FAT), and PCR

  • Each method has limitations: culture is slow (requiring 5-15 days even with optimized media), ELISA may detect non-viable bacteria, and PCR may detect dead bacterial cells

  • frr-based detection could potentially complement these approaches for a more comprehensive assessment

• Quantitative applications:

  • qPCR targeting frr could potentially provide information about bacterial load

  • The copy number of frr (single-copy gene) makes it suitable for quantitative applications

  • Correlation between frr expression levels and bacterial metabolic state could potentially distinguish active from dormant infections

What methodological approaches are most effective for studying the structure-function relationship of recombinant R. salmoninarum frr?

To elucidate structure-function relationships in R. salmoninarum frr, researchers should consider:

Effective methodological approaches:

• Structural determination:

  • X-ray crystallography of purified recombinant frr, alone and in complex with ribosomal components

  • Cryo-electron microscopy of frr-ribosome complexes to visualize interactions in near-native states

  • NMR spectroscopy for dynamic aspects of frr function, particularly domain movements during ribosome binding

• Mutational analysis:

  • Site-directed mutagenesis of conserved residues based on sequence alignments with well-characterized RRFs

  • Creation of chimeric proteins combining domains from R. salmoninarum frr and other bacterial frr proteins

  • Alanine-scanning mutagenesis to identify functional hotspots

• Biochemical characterization:

  • Ribosome binding assays with wild-type and mutant frr proteins

  • GTPase stimulation assays to measure interaction with EF-G

  • Thermal shift assays to assess protein stability changes upon mutation

  • In vitro translation assays to quantify the functional impact of mutations

• Computational approaches:

  • Molecular dynamics simulations to predict conformational changes during ribosome recycling

  • Protein-protein docking to model interactions with EF-G and ribosomal components

  • Evolutionary coupling analysis to identify co-evolving residues important for function

  • Comparative structural modeling using known RRF structures as templates

What are the most promising future research directions for recombinant R. salmoninarum frr?

Several promising research directions could advance our understanding of R. salmoninarum frr:

• Development of specific inhibitors:

  • Design of small molecules that selectively target R. salmoninarum frr

  • Structure-based drug design approaches using high-resolution structures

  • Screening of natural product libraries for compounds that disrupt frr function

• Systems biology integration:

  • Ribosome profiling studies in R. salmoninarum under various conditions

  • Integration of frr function with global protein synthesis regulation

  • Modeling how translation efficiency affects R. salmoninarum growth and virulence

• Interaction networks:

  • Comprehensive mapping of protein-protein interactions involving frr

  • Investigation of potential regulatory factors that modulate frr activity

  • Characterization of the complete ribosome recycling machinery in R. salmoninarum

• Host-pathogen interface:

  • Examination of how host factors might interact with or target bacterial translation machinery

  • Study of frr function under conditions mimicking the intracellular environment

  • Investigation of potential cross-talk between bacterial and host translation systems

• Technological applications:

  • Exploration of frr as a potential vaccine component or drug target

  • Development of biosensors based on frr-ribosome interactions

  • Use of recombinant frr in in vitro translation systems for R. salmoninarum protein production

How can comparative studies of frr across different bacterial pathogens inform our understanding of R. salmoninarum pathogenesis?

Comparative studies of frr across bacterial pathogens could provide valuable insights:

• Evolutionary adaptations:

  • Comparison of frr sequences and structures from diverse pathogens could reveal convergent or divergent adaptations

  • Analysis of frr from other slow-growing pathogens (like Mycobacteria) might identify common features related to growth rate regulation

  • Understanding conserved versus variable regions could inform targeted drug design

• Functional conservation:

  • Determination of whether frr proteins from different pathogens are functionally interchangeable

  • Identification of species-specific interacting partners

  • Quantitative comparison of recycling efficiency across species

• Specialized adaptations:

  • Investigation of whether intracellular pathogens show common adaptations in frr function

  • Analysis of whether cold-adapted fish pathogens have similar modifications to frr

  • Examination of potential correlations between frr properties and bacterial persistence

• Translational control mechanisms:

  • Comparison of how different pathogens regulate translation through ribosome recycling

  • Investigation of whether translational coupling mechanisms differ between species

  • Analysis of the relationship between frr function and stress response across pathogens

• Therapeutic implications:

  • Identification of common vulnerable sites across pathogen frr proteins

  • Development of broad-spectrum versus selective targeting strategies

  • Understanding of resistance mechanisms that might emerge under selective pressure