Recombinant Sorangium cellulosum Ribosome-recycling factor (frr)

What is the Ribosome-recycling Factor (frr) in Sorangium cellulosum?

The ribosome-recycling factor (RRF), also known as ribosome-releasing factor, is a protein that plays a crucial role in protein synthesis by dissociating ribosomes from mRNA after the termination of translation. This process effectively "recycles" ribosomes for new rounds of translation. In Escherichia coli, the frr gene product has been demonstrated to be essential for cell growth . The S. cellulosum RRF consists of 185 amino acids and likely performs similar essential functions in translation termination and ribosome recycling within this organism.

Why is the frr gene essential for bacterial survival?

Studies in E. coli have definitively established that the frr gene is essential for cell growth . When researchers constructed an E. coli strain (MC1061-2) carrying a frame-shifted frr in the chromosome and wild-type frr on a temperature-sensitive plasmid, the strain exhibited temperature-sensitive growth. Importantly, all spontaneously formed thermoresistant colonies derived from this strain carried wild-type frr, either in the bacterial chromosome (by re-exchange) or in plasmids that had become temperature-resistant. These observations confirmed that frr is an essential gene for cell growth . The critical function of RRF in recycling ribosomes after translation termination makes it indispensable for continuous protein synthesis and therefore cell viability.

What expression systems are optimal for producing recombinant S. cellulosum RRF?

Based on available product information, recombinant S. cellulosum RRF has been successfully expressed in yeast expression systems . While the specific yeast strain is not detailed, common choices include Saccharomyces cerevisiae and Pichia pastoris for heterologous protein expression. When planning expression experiments, researchers should consider:

• Codon optimization for the host organism

• Selection of appropriate promoters and signal sequences

• Growth conditions optimization (temperature, pH, media composition)

• Induction parameters if using inducible promoters

• Purification strategy design based on the chosen expression system

The resulting recombinant protein can be purified to >85% purity as verified by SDS-PAGE .

How should researchers properly store and handle recombinant S. cellulosum RRF?

For optimal stability and activity retention of recombinant S. cellulosum RRF, follow these evidence-based storage and handling guidelines:

• Storage temperature: Store at -20°C, or -80°C for extended storage

• Reconstitution: Briefly centrifuge vials before opening to bring contents to the bottom

• Concentration: Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Cryoprotectant: Add glycerol to 5-50% final concentration (50% is recommended)

• Aliquoting: Prepare small working aliquots to avoid repeated freeze-thaw cycles

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

• Shelf life: Approximately 6 months for liquid form at -20°C/-80°C; 12 months for lyophilized form

Repeated freezing and thawing should be strictly avoided as it can lead to protein denaturation and loss of activity .

What techniques are available for genetic manipulation of S. cellulosum?

Genetic manipulation of S. cellulosum has historically been challenging, but several established methods now exist:

• Conjugation-based gene transfer: Recombinant vectors derived from broad-host-range mobilizable plasmids (e.g., pSUP2021) can be transferred from E. coli to S. cellulosum via IncP-mediated conjugation. These vectors can integrate into the chromosome by homologous recombination and remain stably maintained .

• Dual antibiotic selection: Research indicates that using dual selection antibiotics can improve conjugation efficacy in S. cellulosum .

• Autonomous replication: Some plasmids capable of autonomous replication in S. cellulosum have been developed, including those that can induce expression of reporter proteins like GFP .

These genetic tools provide researchers with methods to introduce, delete, or modify genes in S. cellulosum, including the frr gene, for functional studies.

How can RNA isolation and real-time PCR be optimized for studying frr gene expression in S. cellulosum?

For accurate quantification of frr gene expression in S. cellulosum, researchers should implement the following optimized RNA isolation and real-time PCR protocol:

• Cell collection: Harvest cells in mid-logarithmic growth phase to ensure active gene expression

• RNA extraction: Use Trizol LS reagent (Invitrogen) or equivalent for total RNA extraction

• DNA removal: Treat RNA samples with DNase I (e.g., Fermentas) to eliminate genomic DNA contamination

• cDNA synthesis: Use Mu-MLV reverse transcriptase (Fermentas) or similar high-fidelity enzyme

• Real-time PCR: Employ SYBR Green PCR Master Mix (ABI) with an appropriate instrument such as ABI Prism SDS 7000

• Primer design: Design amplicons to cover the middle of the frr gene using software like PerlPrimer

• Controls: Include multiple technical replicates, no-template controls, and normalization with stable reference genes

This protocol has been successfully applied in studies of S. cellulosum gene expression, detecting transcripts from over 90% of the coding sequences in the genome .

What comparative genomic approaches can provide insights into S. cellulosum RRF function?

S. cellulosum possesses an extraordinarily large genome (14.7 million base pairs) , making comparative genomic approaches particularly valuable for understanding RRF function in this organism:

• Sequence homology analysis: Compare the S. cellulosum RRF sequence with well-characterized RRFs from model organisms like E. coli to identify conserved functional domains

• Genomic context analysis: Examine gene clusters surrounding the frr gene to identify potential co-regulated genes involved in translation or related processes

• Transcriptomic comparison: Analyze expression patterns of frr under different environmental conditions (e.g., pH variations) using RNA-Seq, which has successfully detected 90.7% of coding sequences in S. cellulosum

• Phylogenetic analysis: Construct evolutionary trees of RRF sequences across diverse bacterial species to understand the evolutionary relationships and potentially unique adaptations in S. cellulosum

• Structural prediction: Employ bioinformatic tools to predict structural features of S. cellulosum RRF and compare with crystallized structures from other bacteria

These approaches can elucidate both conserved and unique aspects of RRF function in S. cellulosum, contributing to broader understanding of translation termination across bacterial species.

How might researchers investigate interactions between S. cellulosum RRF and the translation machinery?

To characterize interactions between S. cellulosum RRF and other components of the translation apparatus, researchers can employ these methodological approaches:

• In vitro binding assays: Use purified recombinant S. cellulosum RRF and isolated ribosomes to measure direct binding interactions through techniques such as:

  • Surface plasmon resonance

  • Isothermal titration calorimetry

  • Microscale thermophoresis

• Structural studies: Analyze RRF-ribosome complexes using:

  • Cryo-electron microscopy

  • X-ray crystallography (if suitable crystals can be obtained)

  • Hydrogen-deuterium exchange mass spectrometry

• Functional assays: Develop in vitro translation termination and ribosome recycling assays using:

  • Purified translation components

  • Reporter systems to measure recycling efficiency

  • Competition experiments with other translation factors

• Crosslinking approaches: Employ chemical or photo-crosslinking to capture transient interactions between RRF and ribosomal components or other factors

These methods can provide comprehensive insights into the molecular mechanisms of RRF function in the complex process of translation termination and ribosome recycling.

How does S. cellulosum RRF compare to RRF proteins from other bacterial species?

While the search results don't provide direct comparative data between S. cellulosum RRF and other bacterial RRFs, several analytical approaches can address this question:

• Sequence alignment: The 185-amino acid sequence of S. cellulosum RRF can be aligned with RRFs from other species to identify:

  • Conserved residues essential for function

  • Variable regions that may confer species-specific properties

  • Potential structural differences

• Functional complementation: Experimental testing of whether S. cellulosum RRF can rescue growth in E. coli strains with temperature-sensitive frr mutations

• Evolutionary rate analysis: Comparison of evolutionary conservation patterns between RRFs from different bacterial phyla

• Structure-function correlation: Analysis of how any sequence differences might affect the known functional domains of RRF

This comparative analysis could reveal adaptations in S. cellulosum RRF related to the organism's unique environmental adaptability, such as its ability to grow across a wide pH range .

What insights might S. cellulosum RRF provide into the evolution of translation machinery in complex bacteria?

S. cellulosum represents an interesting model for studying translation machinery evolution due to several unique characteristics:

• Extraordinary genome size: At 14,782,125 base pairs, the S. cellulosum genome is 1.75 megabases larger than previously reported bacterial genomes, containing 11,599 coding sequences

• Environmental adaptability: S. cellulosum exhibits alkaline-adaptive properties and can grow across a wide pH range

• Genome expansion mechanisms: The genome contains massive duplications and horizontally transferred genes

• Complex regulation: The organism possesses numerous protein kinases, sigma factors, and transcriptional regulators

Studying RRF in this context could reveal how essential translation components are maintained despite extensive genomic expansion and adaptation. This may provide insights into the evolution of core cellular machinery in bacteria with complex lifestyles and environmental adaptations.

What are the critical quality control parameters for recombinant S. cellulosum RRF preparations?

To ensure experimental reproducibility and reliability when working with recombinant S. cellulosum RRF, researchers should implement these quality control measures:

Regular monitoring of these parameters is essential for maintaining consistency across experiments and ensuring that observed effects can be attributed to the biological activity of RRF rather than preparation artifacts.

What common challenges might researchers encounter when working with S. cellulosum RRF?

Based on the available information and general considerations for working with recombinant proteins from non-model organisms, researchers might encounter these challenges:

• Expression optimization: S. cellulosum proteins may require codon optimization for efficient expression in common host systems

• Solubility issues: Maintaining protein solubility during expression, purification, and storage

• Stability concerns: The recommended storage conditions (-20°C/-80°C with glycerol) suggest potential stability challenges

• Activity assessment: Developing reliable assays to confirm that recombinant RRF retains its native function

• Species-specific interactions: S. cellulosum RRF may have evolved specific interactions with its native translation machinery that aren't recapitulated in heterologous systems

• Post-translational modifications: If present in the native protein, these might be absent in recombinant versions depending on the expression system

Addressing these challenges requires careful optimization of experimental conditions and thorough quality control at each stage of the research process.

How might studying S. cellulosum RRF contribute to antibiotic development research?

The essential nature of the ribosome recycling factor for bacterial survival makes it a potential target for novel antimicrobial development. Research in this direction could explore:

• Structural analysis of S. cellulosum RRF to identify unique features that might be exploited for selective targeting

• High-throughput screening for compounds that specifically inhibit RRF-ribosome interactions

• Comparative studies between RRFs from different bacterial species to design broad-spectrum or species-specific inhibitors

• Investigation of natural compounds from S. cellulosum (known for producing bioactive secondary metabolites) that might target RRF in competing microorganisms

• Testing whether existing translation-targeting antibiotics interact with RRF function

This research could be particularly valuable given the emergence of antibiotic resistance and the need for new antimicrobial targets and compounds.

What unexplored aspects of S. cellulosum RRF function warrant further investigation?

Several promising research avenues remain unexplored based on the available information:

• Regulatory mechanisms: How expression of the frr gene is regulated under different environmental conditions, particularly given S. cellulosum's ability to adapt to varying pH levels

• Protein-protein interactions: Identification of S. cellulosum-specific binding partners that might confer unique properties to its translation termination process

• Role in stress responses: Whether RRF function is modulated during various cellular stresses, potentially contributing to the organism's environmental adaptability

• Post-translational modifications: Whether S. cellulosum RRF undergoes modifications that affect its activity or regulation

• Structural dynamics: How the protein's conformation changes during the ribosome recycling process, potentially using techniques like single-molecule FRET

These investigations could provide valuable insights not only into S. cellulosum biology but also into fundamental aspects of bacterial translation termination and adaptability.