Recombinant Clostridium kluyveri Peptide chain release factor 1 (prfA)
Description
Introduction to Clostridium kluyveri and Peptide Chain Release Factors
Clostridium kluyveri is a strict anaerobic bacterium first isolated by H.A. Barker in 1937 from canal mud in Delft, The Netherlands. It possesses unique metabolic characteristics among clostridia, notably its ability to grow anaerobically on ethanol and acetate as sole energy sources, producing butyrate, caproate, and hydrogen as fermentation products . The complete genome of C. kluyveri was sequenced in 2008, revealing a circular chromosome of 3.96 Mbp and one circular 59-kb plasmid, providing valuable insights into its metabolic capabilities and evolutionary relationships .
Peptide chain release factors (RFs) are essential proteins that direct the termination of translation in response to specific peptide chain termination codons. In bacteria, these proteins recognize stop codons in mRNA and trigger the hydrolysis of the ester bond between the completed polypeptide chain and the tRNA, resulting in the release of the newly synthesized protein . Bacterial release factors are classified into two types: RF1 (prfA) and RF2 (prfB), which recognize different stop codons with some overlap in function.
Structure and Characteristics of C. kluyveri prfA
The peptide chain release factor 1 (prfA) from Clostridium kluyveri is a full-length protein comprising 357 amino acid residues. The primary sequence of C. kluyveri prfA has been thoroughly characterized and is available in protein databases under UniProt accession number A5N3J7 .
Protein Characteristics
The key physical and biochemical properties of recombinant C. kluyveri prfA are summarized in Table 1.
Table 1: Physical and Biochemical Properties of Recombinant C. kluyveri prfA
| Property | Characteristic |
|---|---|
| Number of amino acids | 357 |
| Molecular weight | Approximately 40 kDa |
| Purity (commercial preparations) | >85% (SDS-PAGE) |
| UniProt accession number | A5N3J7 |
| Source organism | Clostridium kluyveri (strain ATCC 8527 / DSM 555 / NCIMB 10680) |
| Expression region | 1-357 (full length) |
| Subcellular localization | Cytoplasmic |
Production and Purification of Recombinant C. kluyveri prfA
Recombinant C. kluyveri prfA can be expressed and purified from various host systems, with yeast and E. coli being the most common expression platforms due to their ability to produce high yields with relatively short turnaround times .
Expression Systems
Multiple expression systems have been utilized for producing recombinant C. kluyveri prfA, each with distinct advantages for specific applications:
Table 2: Comparison of Expression Systems for Recombinant C. kluyveri prfA
| Expression System | Advantages | Disadvantages | Typical Yield |
|---|---|---|---|
| E. coli | High yield, rapid growth, cost-effective | Limited post-translational modifications | High |
| Yeast | Proper folding, some post-translational modifications | Longer production time than E. coli | Medium-High |
| Insect cells | Enhanced post-translational modifications | More complex, higher cost | Medium |
| Mammalian cells | Most native-like post-translational modifications | Highest complexity and cost, lowest yield | Low |
The choice of expression system depends on the specific requirements for downstream applications. For structural studies and basic biochemical characterization, E. coli or yeast-expressed prfA is often sufficient . For applications requiring specific post-translational modifications, insect cells with baculovirus or mammalian expression systems may be preferred, despite their higher cost and complexity.
Function and Mechanism of Action
Peptide chain release factor 1 (prfA) plays a critical role in the termination phase of protein translation. In bacteria, prfA specifically recognizes the stop codons UAA and UAG (while RF2/prfB recognizes UAA and UGA), then catalyzes the hydrolysis of the peptidyl-tRNA bond to release the completed polypeptide chain from the ribosome .
Molecular Mechanism
The molecular mechanism of prfA action involves several key steps:
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Recognition of the stop codon in the mRNA at the ribosomal A site
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Conformational changes in the ribosome and prfA protein
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Positioning of conserved motifs near the peptidyl transferase center
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Catalysis of the hydrolytic reaction that cleaves the ester bond between the nascent peptide and the tRNA
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Release of the completed polypeptide chain
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Dissociation of the release factor from the ribosome
This process requires precise molecular interactions and conformational changes that are highly conserved across bacterial species, explaining the significant sequence homology observed between prfA proteins from different bacteria .
Functional Domains
Sequence analysis of C. kluyveri prfA reveals several functionally important domains and motifs that are conserved across bacterial species:
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N-terminal domain: Involved in stop codon recognition
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Central domain: Contains catalytic motifs for peptidyl-tRNA hydrolysis
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C-terminal domain: Mediates interactions with the ribosome and other translation factors
These domains work in concert to ensure accurate translation termination at appropriate stop codons, preventing readthrough errors that could lead to extended, potentially non-functional proteins.
Comparative Analysis with prfA from Other Species
Recombinant prfA proteins have been produced from numerous bacterial species, allowing for comparative analyses that illuminate both conserved features and species-specific adaptations in translation termination mechanisms.
Sequence Homology
Sequence alignments reveal significant homology between C. kluyveri prfA and release factors from other bacterial species. Particularly high levels of conservation are observed in the functional domains responsible for stop codon recognition and peptidyl-tRNA hydrolysis.
Table 3: Comparison of prfA Amino Acid Lengths Across Selected Bacterial Species
The variations in sequence length reflect species-specific adaptations while maintaining the core functional domains required for translation termination.
Applications in Research and Biotechnology
Recombinant C. kluyveri prfA has numerous applications in both basic research and biotechnology fields.
Research Applications
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Translation Termination Studies: Recombinant prfA serves as a model protein for investigating the mechanisms of translation termination in prokaryotes.
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Structural Biology: Purified recombinant prfA enables crystallographic and cryo-EM studies to elucidate the three-dimensional structure and dynamics of release factors in complex with ribosomes.
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Evolutionary Studies: Comparative analysis of prfA sequences across bacterial species provides insights into the evolution of translation termination mechanisms.
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Protein-Protein Interaction Studies: Recombinant prfA facilitates the investigation of interactions with other components of the translation machinery.
Biotechnological Applications
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In vitro Translation Systems: Recombinant prfA is a key component in cell-free protein synthesis systems used for the production of proteins difficult to express in vivo.
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Antibiotic Development: Given the essential role of release factors in bacterial protein synthesis, recombinant prfA serves as a potential target for novel antibiotic development.
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Synthetic Biology: Engineered variants of prfA can be employed in synthetic biology applications to control translation termination or to repurpose stop codons for the incorporation of non-canonical amino acids.
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Immunological Studies: Purified recombinant prfA can be used as an antigen for the development of antibodies against C. kluyveri.
Future Research Directions
Despite the significant advances in our understanding of bacterial release factors, several aspects of C. kluyveri prfA warrant further investigation:
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Structural Characterization: High-resolution structural studies of C. kluyveri prfA alone and in complex with ribosomes would provide deeper insights into species-specific aspects of translation termination.
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Regulation of Expression: Studies on the regulation of prfA expression in C. kluyveri would enhance our understanding of translation control mechanisms in this organism.
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Post-translational Modifications: Investigation of potential post-translational modifications of prfA in C. kluyveri and their functional implications remains an interesting area for future research.
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Engineered Variants: Development of engineered variants with altered codon specificity or catalytic efficiency could open new avenues for biotechnological applications.
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Interaction with Antibiotics: Understanding how existing or novel antibiotics interact with C. kluyveri prfA could contribute to the development of new antimicrobial strategies targeting anaerobic pathogens.
Product Specs
Target Background
KEGG: ckl:CKL_3708
STRING: 431943.CKL_3708
Q&A
What is Peptide Chain Release Factor 1 (PrfA) in Clostridium kluyveri?
Peptide Chain Release Factor 1 (PrfA) in Clostridium kluyveri is a critical protein involved in translation termination during protein biosynthesis. The recombinant form consists of 357 amino acids and functions by recognizing stop codons (UAA and UAG) in messenger RNA, catalyzing the release of newly synthesized peptide chains from the ribosome . This protein plays an essential role in ensuring accurate translation termination, which is crucial for proper protein synthesis and cellular function in this anaerobic bacterium. The complete amino acid sequence begins with MLERLNFIEN and continues through a series of conserved domains necessary for stop codon recognition and peptidyl-tRNA hydrolysis .
How does C. kluyveri PrfA differ from the PrfA protein in Listeria monocytogenes?
Despite sharing the same abbreviation, these are functionally distinct proteins in different bacterial species. C. kluyveri PrfA functions as a peptide chain release factor involved in translation termination, whereas Listeria monocytogenes PrfA serves as a master virulence regulator . The L. monocytogenes PrfA controls virulence gene expression through interactions with DNA and is regulated by glutathione and inhibitory oligopeptides . In contrast, C. kluyveri PrfA interacts with the ribosome to facilitate the termination of protein synthesis when stop codons are encountered. This fundamental difference highlights the importance of specifying the organism when discussing proteins with similar nomenclature.
What is the significance of C. kluyveri as a research organism?
Clostridium kluyveri is a strict anaerobe with unique metabolic properties, making it valuable for various biotechnological applications. This organism has been used as a source of enzymes for analytical purposes and stereospecific hydrogenation reactions . Its ability to convert ethanol and acetate to butyrate, caproate, and hydrogen represents a distinctive fermentation pathway with potential industrial applications . The genome of C. kluyveri (3.96 Mbp chromosome and a 59-kb plasmid) provides insights into its metabolic versatility, including ethanol-acetate fermentation mechanisms and nitrogen fixation capabilities . Understanding the function of essential proteins like PrfA in this organism contributes to our knowledge of protein synthesis in anaerobic bacteria with unique metabolic capabilities.
What expression systems are optimal for producing recombinant C. kluyveri PrfA?
For research applications requiring recombinant C. kluyveri PrfA, multiple expression systems can be employed, each with specific advantages depending on experimental needs. Commonly used expression hosts include:
| Expression System | Advantages | Considerations |
|---|---|---|
| E. coli | Rapid growth, high yields, established protocols | May lack post-translational modifications |
| Yeast | Eukaryotic post-translational modifications, proper folding | Longer production time, more complex media requirements |
| Baculovirus | Insect cell expression, complex folding capabilities | Technical complexity, higher cost |
| Mammalian cells | Most sophisticated folding and modification machinery | Slowest production, highest cost |
For most basic structural and functional studies, E. coli expression systems typically provide sufficient yield and quality . The addition of affinity tags, such as the His-tag described in the available recombinant product, facilitates purification while minimally impacting function in most applications . When selecting an expression system, researchers should consider the specific downstream applications and whether authentic folding or post-translational modifications are critical to the protein's activity being studied.
What purification strategies are most effective for recombinant PrfA?
Purification of His-tagged recombinant C. kluyveri PrfA (aa 1-357) typically employs immobilized metal affinity chromatography (IMAC) as the primary capture step, followed by additional purification steps to achieve research-grade purity. An optimized purification protocol involves:
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Cell lysis under native conditions (buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and protease inhibitors)
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IMAC using Ni-NTA resin with gradient elution (10-250 mM imidazole)
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Size exclusion chromatography to remove aggregates and isolate monomeric protein
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Optional ion exchange chromatography for removal of nucleic acid contamination
For functional studies, maintaining reducing conditions with 1-5 mM DTT or 2-mercaptoethanol is recommended to preserve the native conformation of the protein. Final purity should be assessed by SDS-PAGE and Western blotting, with expected yield of 2-5 mg of purified protein per liter of culture when using optimized E. coli expression conditions .
How can researchers verify the functional activity of purified recombinant PrfA?
Verifying the functional activity of purified recombinant C. kluyveri PrfA requires assays that assess its peptide release activity. A comprehensive functional assessment includes:
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In vitro translation termination assay: Using pre-formed ribosome-mRNA complexes with a stop codon in the A site and measuring the release of a fluorescently labeled peptide from the peptidyl-tRNA.
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Stop codon specificity assessment: Testing the protein's activity across different stop codons (UAA, UAG) to confirm its expected specificity profile.
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GTP hydrolysis assay: Monitoring PrfA-mediated GTP hydrolysis in the presence of ribosomes and appropriate stop codons.
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Ribosome binding studies: Using surface plasmon resonance or other binding assays to verify interaction with bacterial ribosomes.
A functional recombinant PrfA should demonstrate concentration-dependent peptide release activity with kinetic parameters comparable to those of the native protein. Thermal stability assays (differential scanning fluorimetry) can provide additional confirmation of proper folding and stability under various buffer conditions.
How does the structure of C. kluyveri PrfA relate to its function in translation termination?
The structure-function relationship in C. kluyveri PrfA centers on several conserved domains critical for its peptide release activity. Based on homology modeling and structural studies of related bacterial release factors, the protein contains:
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Domain 1: Contains the N-terminal region involved in stop codon recognition
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Domain 2: Houses the GGQ motif essential for catalyzing peptide release through hydrolysis of the peptidyl-tRNA ester bond
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Domain 3: Contributes to ribosomal binding and positioning
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Domain 4: Connects domains 2 and 3, facilitating the conformational changes needed during termination
The sequence provided (MLERLNFIEN KYEELSIKIS DPTIIADQKK...) reveals conserved regions that align with functional domains in homologous proteins . X-ray crystallography studies of related bacterial release factors have shown that these proteins undergo significant conformational changes upon binding to the ribosome, positioning the catalytic GGQ motif precisely at the peptidyl transferase center to facilitate peptide release. This conformational flexibility is likely conserved in C. kluyveri PrfA and essential for its function.
What role might C. kluyveri PrfA play in the organism's unique metabolic processes?
While PrfA's primary function involves translation termination, its role may have special significance in C. kluyveri's distinctive metabolism. This anaerobic bacterium employs unusual fermentation pathways, including the conversion of ethanol and acetate to butyrate and caproate . These metabolic processes require precise regulation of numerous enzymes, including:
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Alcohol dehydrogenases
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Aldehyde dehydrogenases
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Thiolases
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Energy-converting NADH:ferredoxin oxidoreductase complexes
The efficient translation of these metabolic enzymes, including accurate termination facilitated by PrfA, is crucial for maintaining C. kluyveri's energy metabolism . Research examining differential expression of PrfA under various growth conditions could provide insights into how translation regulation adapts to support the organism's metabolic flexibility. Additionally, the accurate termination of protein synthesis prevents ribosome stalling and the production of aberrant proteins, which could be particularly important in an organism with such specialized metabolic pathways.
How can structural biology approaches advance our understanding of C. kluyveri PrfA?
Advanced structural biology approaches can significantly enhance our understanding of C. kluyveri PrfA's molecular mechanisms:
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Cryo-electron microscopy (cryo-EM): This technique can capture PrfA in complex with the ribosome at near-atomic resolution, revealing the precise interactions during translation termination. Specifically, it can visualize how PrfA positions its catalytic center relative to the peptidyl-tRNA.
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X-ray crystallography: Obtaining high-resolution crystal structures of PrfA in different conformational states could reveal the structural transitions that occur during its functional cycle.
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Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This method can map the dynamics and conformational changes in PrfA upon binding to different ligands or under various conditions.
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Nuclear magnetic resonance (NMR) spectroscopy: For studying specific domains or smaller fragments of PrfA, NMR can provide insights into local structural dynamics and interactions.
These approaches would be particularly valuable for comparing C. kluyveri PrfA with release factors from other organisms, potentially revealing adaptations specific to C. kluyveri's unusual metabolism or environmental niche.
How does C. kluyveri PrfA compare to release factors in other Clostridium species?
Peptide chain release factors show varying degrees of conservation across Clostridium species, reflecting evolutionary adaptations to different ecological niches and metabolic strategies. Comparative analysis reveals:
These variations may reflect adaptations to different translational requirements or regulatory mechanisms. C. kluyveri's distinctive metabolic capabilities, including its ethanol and acetate fermentation pathway, may have influenced subtle evolutionary adaptations in its translation termination machinery . Further comparative genomic and structural analyses could reveal how these differences correlate with the unique physiological properties of each species.
What methodologies are most appropriate for studying evolutionary relationships of translation factors across bacterial species?
For investigating the evolutionary relationships of translation factors like PrfA across bacterial species, several complementary approaches yield the most comprehensive insights:
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Phylogenetic analysis: Construction of maximum likelihood or Bayesian phylogenetic trees based on multiple sequence alignments of release factors from diverse bacterial lineages.
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Synteny analysis: Examination of genomic context conservation around the prfA gene across related species to identify patterns of gene arrangement preservation or rearrangement.
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Selection pressure analysis: Calculation of dN/dS ratios to identify regions under purifying or positive selection, revealing functionally critical domains.
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Ancestral sequence reconstruction: Computational inference of ancestral release factor sequences to trace the evolutionary trajectory of functional changes.
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Domain architecture comparison: Analysis of domain conservation, fusion events, or domain shuffling across bacterial lineages.
When applied to C. kluyveri PrfA, these methods could reveal how this translation termination factor has co-evolved with the organism's specialized metabolic systems. For instance, the integration of phylogenetic analysis with structural information can identify if specific residue changes correlate with C. kluyveri's unique fermentation capabilities described in the literature .
What are the major technical challenges in studying C. kluyveri proteins like PrfA?
Researchers face several significant technical challenges when working with proteins from C. kluyveri:
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Anaerobic culturing requirements: As a strict anaerobe, C. kluyveri requires specialized anaerobic cultivation techniques, making native protein isolation laborious .
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Recombinant expression optimization: Achieving proper folding and activity in heterologous expression systems can be challenging, particularly for proteins involved in specialized cellular processes like translation termination .
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Functional reconstitution: Creating in vitro systems that accurately recapitulate the translation termination environment requires purified ribosomes, translation factors, and appropriate mRNA substrates.
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Structural analysis complexities: Capturing the conformational changes that occur during PrfA's functional cycle requires sophisticated structural biology approaches and often yields incomplete pictures of the dynamic process.
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Limited genetic tools: Compared to model organisms, genetic manipulation of C. kluyveri to study PrfA function in vivo is challenging, though advances in CRISPR-based technologies offer new opportunities.
These challenges have contributed to knowledge gaps regarding the specific adaptations of C. kluyveri's translation machinery to its unique metabolism and environmental niche.
How might systems biology approaches enhance our understanding of PrfA's role in C. kluyveri?
Systems biology approaches offer powerful frameworks for contextualizing PrfA's function within C. kluyveri's broader cellular processes:
These approaches could help bridge the gap between molecular understanding of PrfA function and its broader implications for C. kluyveri's ecological and biotechnological significance.
What emerging technologies might advance research on translation termination factors in anaerobic bacteria?
Several cutting-edge technologies show particular promise for advancing research on translation termination factors in anaerobic bacteria like C. kluyveri:
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Time-resolved cryo-EM: Capturing structural snapshots of PrfA during the termination process at millisecond timescales could reveal the dynamic conformational changes essential for function.
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Single-molecule fluorescence resonance energy transfer (smFRET): This technique can monitor PrfA conformational dynamics and interactions with the ribosome in real-time at the single-molecule level.
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Microfluidics-based translation systems: Miniaturized platforms for studying translation in controlled environments could enable high-throughput analysis of PrfA variants or different environmental conditions.
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In-cell NMR spectroscopy: This emerging approach could potentially monitor structural changes in isotopically labeled PrfA within living C. kluyveri cells under various metabolic states.
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AI-driven structural prediction: Tools like AlphaFold2 are increasingly capable of predicting protein structures and complexes, potentially offering insights into PrfA-ribosome interactions when experimental data is challenging to obtain.
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CRISPR interference (CRISPRi) systems for anaerobes: Development of tunable gene repression tools for anaerobic bacteria would enable precise control over prfA expression to study its role in cellular physiology.
These technologies could collectively address current knowledge gaps regarding how translation termination in C. kluyveri is integrated with its unique metabolic capabilities and environmental adaptations .
