Recombinant Salmonella arizonae Ribosome-recycling factor (frr)

What is Ribosome-recycling factor and what is its primary function in bacterial cells?

Ribosome-recycling factor (RRF), the product of the frr gene, is responsible for the dissociation of ribosomes from mRNA after the termination of translation. It functions to "recycle" ribosomes, enabling them to participate in subsequent rounds of protein synthesis. This process is essential for efficient translation in bacterial cells. Studies in Escherichia coli have established that frr is an essential gene for cell growth, as demonstrated through experiments with temperature-sensitive strains carrying frame-shifted frr in the chromosome . Without functional RRF, ribosomes remain bound to mRNA after termination, preventing their participation in new rounds of translation and ultimately leading to growth arrest.

What are the recommended storage and handling protocols for recombinant Salmonella arizonae RRF?

For optimal maintenance of recombinant Salmonella arizonae RRF activity, the following storage and handling protocols are recommended:

Specific handling recommendations include:

• Brief centrifugation of the vial prior to opening to bring contents to the bottom

• Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of glycerol to a final concentration of 5-50% (optimal: 50%) for long-term storage

• Aliquoting to minimize freeze-thaw cycles, as repeated freezing and thawing is not recommended

The shelf life depends on multiple factors including storage state, buffer ingredients, storage temperature, and the intrinsic stability of the protein. Generally, liquid preparations maintain integrity for approximately 6 months at -20°C/-80°C, while lyophilized forms remain stable for up to 12 months at the same temperatures .

What expression systems are most effective for producing recombinant Salmonella arizonae Ribosome-recycling factor?

While commercial recombinant Salmonella arizonae Ribosome-recycling factor is typically produced in yeast expression systems , researchers investigating the protein's structure-function relationships may consider alternative expression approaches. Based on studies of RRF from related bacterial species, the following expression systems can be considered:

• E. coli-based expression systems: These offer high yield and simplified purification for bacterial proteins. Commonly used strains include BL21(DE3) with pET vector systems containing a T7 promoter.

• Yeast expression systems: These provide eukaryotic post-translational processing capability, which may be beneficial for certain applications. Saccharomyces cerevisiae or Pichia pastoris systems are frequently employed.

• Cell-free protein synthesis: This approach can be advantageous for proteins that might affect cellular viability when overexpressed in living cells.

When designing expression constructs, researchers should consider incorporating affinity tags (His-tag, GST-tag) to facilitate purification while ensuring these modifications don't interfere with protein function through appropriate control experiments.

How does Salmonella arizonae RRF compare phylogenetically to RRF proteins from other Salmonella subspecies?

Salmonella arizonae (also known as Salmonella enterica subspecies arizonae or Salmonella subgroup IIIa) occupies a phylogenetically distinct position within the Salmonella genus. Comparative genomic analyses place S. arizonae between Salmonella subgroup I (which includes human pathogens) and S. bongori (usually non-pathogenic to humans) . This intermediate phylogenetic position makes S. arizonae RRF particularly interesting for evolutionary studies.

Core genome phylogenetic analyses reveal that S. arizonae possesses unique genetic characteristics that distinguish it from other Salmonella subspecies. Of the 4,203 protein-coding genes identified in the S. arizonae genome, 926 genes appear to be specific to this subspecies, while 2,823 genes are common across S. arizonae, S. bongori, and Salmonella subgroup I representatives . This genomic architecture reflects the evolutionary history of S. arizonae and potentially influences the structure and function of proteins like RRF.

While the RRF protein is highly conserved across bacterial species due to its essential function, subtle sequence variations may reflect adaptations to specific ecological niches or host ranges. Detailed comparative analyses of RRF sequences across Salmonella subspecies would provide valuable insights into the evolutionary pressures acting on this critical protein.

What insights does the study of Salmonella arizonae RRF provide into bacterial evolution and speciation?

The study of Salmonella arizonae RRF contributes to our understanding of bacterial evolution and speciation in several ways:

• Evolutionary conservation of essential functions: The frr gene encoding RRF is essential for bacterial growth, as demonstrated in E. coli studies . The conservation of this gene across diverse bacterial species, including S. arizonae, highlights the fundamental importance of ribosome recycling in bacterial physiology.

• Adaptive evolution: Comparative analysis of RRF sequences can reveal signatures of positive or purifying selection, providing insights into how this protein has evolved under different ecological pressures. S. arizonae's intermediate phylogenetic position makes it particularly valuable for such evolutionary studies.

• Genomic context: The genomic context of the frr gene in S. arizonae compared to other Salmonella subspecies can illuminate processes of genome reorganization during speciation. Whole-genome phylogenetic analyses have revealed that S. arizonae occupies a distinct position in Salmonella evolution, positioned between Salmonella subgroup I and S. bongori .

• Pathogenicity evolution: S. arizonae contains both shared and unique pathogenicity factors compared to other Salmonella subspecies. While Salmonella pathogenicity islands (SPIs) 1 and 2 are present across all Salmonella including S. arizonae, some effectors appear to be lost in this lineage. Additionally, SPI-20, encoding a type VI secretion system, is exclusive to S. arizonae and maintained across all sampled genomes . Understanding how essential proteins like RRF function within this unique genomic context can provide insights into the evolution of bacterial pathogenicity.

How can recombinant Salmonella arizonae RRF be used to study translation termination mechanisms?

Recombinant Salmonella arizonae RRF provides a valuable tool for investigating translation termination mechanisms through several experimental approaches:

• In vitro translation systems: Purified recombinant Salmonella arizonae RRF can be incorporated into cell-free translation systems to study its role in ribosome recycling after termination. By manipulating RRF concentrations or using mutant variants, researchers can assess how alterations in RRF activity affect the efficiency of ribosome dissociation from mRNA.

• Structural studies: High-resolution structural determination through X-ray crystallography or cryo-electron microscopy can elucidate the specific interactions between Salmonella arizonae RRF and ribosomal components. These structural insights can be compared with RRF from other bacterial species to identify conserved and divergent features.

• Site-directed mutagenesis: By creating targeted mutations in recombinant Salmonella arizonae RRF, researchers can identify critical residues for function and characterize their specific roles in ribosome binding and dissociation. The full protein sequence provided for the recombinant protein (MISDIRKDAE VRMEKCVEAF KTQISKVRTG RASPSLLDGI VVEYYGTPTP LRQLASVTVE DSRTLKINVF DRSMGPAVEK AIMASDLGLN PSSAGTDIRV PLPPLTEERR KDLTKIVRGE AEQARVAVRN VRRDANDKVK ALLKDKAISE DDDRRSQEEV QKMTDAAIKK VDAALADKEA ELMQF) serves as an excellent reference for designing such mutations.

• Interaction studies: Techniques such as surface plasmon resonance or isothermal titration calorimetry can quantify the binding dynamics between Salmonella arizonae RRF and ribosomal components, providing insights into the kinetics and thermodynamics of these interactions.

What are the implications of studying Salmonella arizonae RRF for antimicrobial drug development?

Ribosome-recycling factor represents a potential target for antimicrobial development due to its essential role in bacterial translation and its absence in eukaryotic cells. Research on Salmonella arizonae RRF has several implications for drug discovery:

• Target validation: Studies establishing the essentiality of frr in E. coli suggest that RRF would be an effective antibiotic target. Confirmation of this essentiality in Salmonella arizonae would further validate RRF as a drug target for Salmonella infections.

• Structure-based drug design: The availability of recombinant Salmonella arizonae RRF enables structural studies that can guide the design of small-molecule inhibitors. By identifying binding pockets unique to bacterial RRF, researchers can develop compounds that specifically interfere with ribosome recycling.

• Species-specific targeting: Comparative analyses of RRF across bacterial species can reveal unique structural features in Salmonella arizonae RRF that might be exploited for developing narrow-spectrum antibiotics targeting Salmonella species specifically.

• Resistance mechanism studies: RRF mutations conferring resistance to inhibitors can be characterized using recombinant protein expression systems, providing insights into potential resistance mechanisms and guiding the development of more robust antimicrobial compounds.

• Combination therapy approaches: Understanding how RRF inhibition affects bacterial physiology may reveal synergistic targets for combination therapy, potentially enhancing the efficacy of existing antibiotics against Salmonella infections.

What are common challenges in working with recombinant Salmonella arizonae RRF and how can they be addressed?

Researchers working with recombinant Salmonella arizonae RRF may encounter several challenges that can affect experimental outcomes:

• Protein stability issues:

  • Challenge: Recombinant RRF may lose activity during storage or experimental manipulation.

  • Solution: Strict adherence to storage recommendations is essential. Store at -20°C for standard use or -80°C for extended storage. Working aliquots should be kept at 4°C for no more than one week. Adding glycerol to a final concentration of 5-50% can enhance stability during storage .

• Functional assay limitations:

  • Challenge: Assessing RRF activity can be complex, requiring reconstituted translation systems.

  • Solution: Develop simplified functional assays, such as ribosome binding assays or fluorescence-based interaction studies, that can serve as proxies for full functional assessment.

• Protein purity considerations:

  • Challenge: Commercial recombinant preparations typically guarantee >85% purity by SDS-PAGE , which may be insufficient for certain applications.

  • Solution: Additional purification steps, such as size exclusion chromatography or ion exchange chromatography, may be necessary to achieve higher purity for structural or interaction studies.

• Tag interference:

  • Challenge: Affinity tags used in recombinant protein production may interfere with RRF function or interactions.

  • Solution: Compare tagged and tag-cleaved versions of the protein in functional assays to assess potential interference. Consider using cleavable tags and removing them before critical experiments.

How can researchers optimize experimental protocols for studying Salmonella arizonae RRF interactions with ribosomes?

Optimizing experimental protocols for studying Salmonella arizonae RRF interactions with ribosomes requires careful consideration of several factors:

How does Salmonella arizonae genomic context influence RRF function compared to other bacterial species?

The genomic context of Salmonella arizonae provides a unique environment that may influence RRF function in several ways:

• Evolutionary positioning: S. arizonae occupies an intermediate phylogenetic position between Salmonella subgroup I (human pathogens) and S. bongori (generally non-pathogenic to humans) . This evolutionary position may be reflected in specific adaptations of essential proteins like RRF.

• Genome structure and organization: The S. arizonae genome has been fully sequenced, revealing 4,574,836 bp with 4,203 protein-coding genes, 82 tRNA genes, and 7 rRNA operons . This genomic architecture may influence the expression and regulation of essential genes like frr.

• Subspecies-specific genes: Analysis of the S. arizonae genome has identified 926 genes specific to this subspecies . While frr itself is highly conserved due to its essential function, subspecies-specific genes may interact with or regulate RRF activity in ways unique to S. arizonae.

• Pathogenicity islands: S. arizonae contains both shared and unique pathogenicity factors. While SPI-1 and SPI-2 are present across all Salmonella including S. arizonae, some effectors appear to be lost in this lineage. Additionally, SPI-20, encoding a type VI secretion system, is exclusive to S. arizonae . These pathogenicity factors may indirectly influence translation efficiency and thus RRF function under infection conditions.

• Comparative genomics approach: A study comparing core gene data of S. arizonae RKS2983, S. bongori NCTC 12419, and S. typhimurium LT2 identified 2,823 genes common to all three genomes . This comparative approach can reveal how conserved genes like frr function within different genomic contexts.

What can comparative analyses of RRF across Salmonella subspecies reveal about functional adaptation?

Comparative analyses of RRF across Salmonella subspecies can provide valuable insights into functional adaptation:

• Sequence conservation patterns: Analyzing the degree of sequence conservation in different regions of RRF across Salmonella subspecies can identify functionally critical domains versus regions that may have undergone adaptive evolution. The full protein sequence of Salmonella arizonae RRF (MISDIRKDAE VRMEKCVEAF KTQISKVRTG RASPSLLDGI VVEYYGTPTP LRQLASVTVE DSRTLKINVF DRSMGPAVEK AIMASDLGLN PSSAGTDIRV PLPPLTEERR KDLTKIVRGE AEQARVAVRN VRRDANDKVK ALLKDKAISE DDDRRSQEEV QKMTDAAIKK VDAALADKEA ELMQF) provides a reference point for such analyses.

• Host range correlations: S. arizonae is frequently associated with reptile reservoirs but can cause illness in some mammals, including humans . By comparing RRF sequences across Salmonella subspecies with different host preferences, researchers may identify adaptations related to host-specific translation regulation.

• Expression pattern differences: Although RRF is essential across bacterial species, its expression level and regulation may vary. Comparative transcriptomic analyses could reveal subspecies-specific patterns in frr expression that correlate with ecological niches or pathogenicity.

• Interaction network variations: RRF functions within a complex network of translation factors. Comparative analyses can identify subspecies-specific variations in these interaction networks that may reflect functional adaptations.

• Structural variations: Even minor sequence variations in RRF across subspecies may lead to subtle structural differences that influence function. Comparative structural analyses using techniques like homology modeling can reveal how these variations might affect interaction with ribosomes.

What emerging technologies could advance the study of Salmonella arizonae RRF function and interactions?

Several emerging technologies hold promise for advancing our understanding of Salmonella arizonae RRF:

• Cryo-electron microscopy (Cryo-EM): Recent advances in cryo-EM resolution now allow visualization of ribosome-RRF complexes at near-atomic resolution. This technology could reveal the precise structural basis of Salmonella arizonae RRF interactions with ribosomes in different functional states.

• Single-molecule fluorescence resonance energy transfer (smFRET): This technique enables real-time observation of RRF-ribosome interactions at the single-molecule level, providing insights into the dynamics and heterogeneity of these interactions that are not accessible through bulk measurements.

• CRISPR-Cas9 gene editing: Precise genome editing of Salmonella arizonae could enable the creation of conditional frr mutants or tagged versions of RRF at the genomic level, allowing for more physiologically relevant studies of RRF function.

• Ribosome profiling: This technique provides genome-wide information on ribosome positioning and can reveal how RRF deficiency or mutation affects translation termination and ribosome recycling across the transcriptome.

• Computational approaches: Advanced molecular dynamics simulations can model the interactions between Salmonella arizonae RRF and ribosomes, predicting functional impacts of mutations or potential binding sites for inhibitors.

What are the potential applications of Salmonella arizonae RRF research in synthetic biology and biotechnology?

Research on Salmonella arizonae RRF has potential applications in several areas of synthetic biology and biotechnology:

• Engineered translation systems: Understanding the specific properties of Salmonella arizonae RRF could inform the design of optimized translation systems for synthetic biology applications, particularly those requiring efficient ribosome recycling.

• Protein expression optimization: Modulating RRF activity or expression could potentially enhance protein production in bacterial expression systems by improving ribosome recycling efficiency.

• Development of biosensors: RRF-ribosome interactions could be engineered to create biosensors for detecting specific molecular targets, based on changes in these interactions upon target binding.

• Minimal cell design: Essential genes like frr are critical components in minimal genome projects. Insights from Salmonella arizonae RRF could inform the design of minimal cells with optimized translation machinery.

• Directed evolution platforms: RRF could serve as a target for directed evolution approaches aimed at creating bacterial strains with enhanced translation properties for biotechnological applications.