Recombinant Bacillus thuringiensis subsp. konkukian Peptide chain release factor 1 (prfA)

What is the structure and function of prfA in Bacillus thuringiensis subsp. konkukian?

PrfA (peptide chain release factor 1) is a 355 amino acid protein that directs the termination of translation in response to stop codons. The AlphaFold DB computed structure model (AF-Q6HAV4-F1) shows a high confidence score (pLDDT) of 88.15, indicating reliable structural prediction . PrfA is essential for protein synthesis as it recognizes stop codons and facilitates the release of completed polypeptide chains from ribosomes.

Functionally, prfA has been implicated in cell division processes, with studies showing that mutations in prfA can inhibit cell division . The protein is categorized in the KEGG Orthology (KO) database under K02835 and is part of the genetic information processing pathway, specifically in translation .

How does prfA contribute to the biology of B. thuringiensis subsp. konkukian?

B. thuringiensis subsp. konkukian (strain 97-27) is notable for having been isolated from a case of severe human tissue necrosis, which is unusual as human infections by this organism are rare . This strain is closely related to B. anthracis based on phylogenetic analysis .

While prfA itself is not directly related to the pathogenicity of B. thuringiensis, its role in translation termination makes it essential for all protein synthesis, including virulence factors. Unlike the well-studied insecticidal crystal proteins (Cry proteins) that B. thuringiensis is known for, prfA functions in fundamental cellular processes necessary for bacterial survival and reproduction.

What expression systems are suitable for recombinant prfA production?

Based on established B. thuringiensis expression systems, researchers have several options:

• B. thuringiensis shuttle vectors: Plasmids like pHT3101 have been successfully used as shuttle vectors for protein expression in B. thuringiensis .

• Promoter selection: For constitutive expression, researchers can use the cryIIIA promoter, which is non-sporulation-dependent . For phase-specific expression, sporulation-specific promoters like Bt1 and Bt2 from strain HD73 can be utilized .

• E. coli expression systems: For high-yield production, E. coli systems with N-terminal tags (such as 6xHis-SUMO) can facilitate purification through IMAC (Immobilized Metal Affinity Chromatography), similar to methods used for other B. thuringiensis proteins .

How can site-directed mutagenesis of prfA be used to study translation termination mechanisms?

Researchers can employ strategic mutagenesis approaches to investigate prfA function:

For mutation construction, researchers should use overlap extension PCR with mutagenic primers targeting specific prfA regions. The mutated genes can be cloned into expression vectors like pCD4, which has been successfully used in B. thuringiensis . For phenotypic analysis, complementation studies in prfA-deficient strains can reveal functional impacts of mutations.

What is the comparative analysis of prfA between B. thuringiensis subsp. konkukian and other Bacillus species?

Phylogenetic analysis places B. thuringiensis subsp. konkukian closely related to B. anthracis , suggesting high conservation of essential proteins like prfA. Researchers should consider:

• Sequence homology analysis: Compare prfA sequences across the B. cereus group (including B. thuringiensis, B. cereus, and B. anthracis) to identify conserved domains and species-specific variations.

• Functional complementation: Test whether prfA from different Bacillus species can complement prfA mutations in B. thuringiensis subsp. konkukian.

• Structural comparison: Use the AlphaFold DB model (pLDDT score: 88.15) to compare with structures from other Bacillus species to identify potential functional differences.

How does prfA activity correlate with stress response and sporulation in B. thuringiensis?

B. thuringiensis undergoes significant physiological changes during sporulation, including the production of insecticidal crystal proteins . Researchers can investigate:

• Expression profiling: Measure prfA expression levels during different growth phases using RT-qPCR or RNA-seq approaches.

• Conditional knockdown: Use inducible antisense RNA systems to reduce prfA expression at specific growth stages.

• Proteomics analysis: Compare the proteome of wild-type and prfA-depleted strains during sporulation to identify proteins most affected by prfA activity.

What are the optimal conditions for expressing and purifying recombinant prfA?

Based on successful expression strategies for other B. thuringiensis proteins, researchers should consider:

For optimal activity, purified prfA should be stored in buffer containing 20mM Tris-HCl pH 7.5, 100mM KCl, 10mM MgCl₂, and 5mM β-mercaptoethanol. Flash-freeze aliquots in liquid nitrogen and store at -80°C to maintain activity.

What assays can be used to measure prfA activity in vitro?

Researchers can employ the following assays to characterize recombinant prfA activity:

• In vitro translation termination assay: Using purified ribosomes, mRNA with stop codons, and aminoacyl-tRNAs to measure release of completed peptides.

• Toe-printing assay: To determine prfA binding to ribosome-mRNA complexes at stop codons.

• GTP hydrolysis assay: Measuring GTP hydrolysis rates when prfA interacts with ribosomes and RF3.

• Thermal shift assay: To assess protein stability under different buffer conditions or in the presence of potential inhibitors.

• Surface plasmon resonance: For measuring binding kinetics between prfA and ribosomal components.

How can CRISPR-Cas9 be used to study prfA function in B. thuringiensis?

CRISPR-Cas9 offers powerful approaches for studying essential genes like prfA:

• Conditional knockdown: Design an inducible CRISPR interference (CRISPRi) system targeting prfA to reduce expression without complete knockout.

• Promoter replacement: Use CRISPR-Cas9 to replace the native prfA promoter with inducible promoters like the P₍hj3₎ promoter from B. thuringiensis, which has shown strong activity at different growth phases .

• Tagging strategy: Engineer a C-terminal fluorescent protein tag to monitor prfA localization during different growth phases and stress conditions.

• Point mutations: Create specific point mutations in the chromosomal prfA gene to study the effect of amino acid changes on function.

How might prfA function interact with the insecticidal properties of B. thuringiensis?

While prfA itself is not directly involved in insecticidal activity, as a translation termination factor, it plays a critical role in the expression of all proteins, including the Cry and Cyt toxins that give B. thuringiensis its insecticidal properties . Researchers could investigate:

• Impact on toxin expression: Whether prfA mutations affect the translation efficiency of cry genes, potentially altering toxin production levels.

• Stress response coordination: How prfA activity changes during the transition to sporulation phase when crystal proteins are produced .

• Regulatory networks: Potential interactions between translation termination efficiency and the expression of sporulation-specific genes involved in crystal protein formation.

What are the challenges and solutions for studying potentially essential genes like prfA?

Studying essential genes presents unique challenges:

How can structural biology approaches enhance our understanding of prfA function?

The available AlphaFold DB model (pLDDT: 88.15) provides a starting point, but researchers should consider:

• X-ray crystallography: Obtain high-resolution structures of prfA alone and in complex with ribosomal components to visualize functional interactions.

• Cryo-electron microscopy: Capture prfA in the act of terminating translation by resolving ribosome-prfA complexes.

• Hydrogen-deuterium exchange mass spectrometry: Map conformational changes in prfA upon binding to different substrates or under different conditions.

• Molecular dynamics simulations: Use the AlphaFold model to predict conformational changes during stop codon recognition and peptide release.

• NMR studies: Investigate the dynamic aspects of prfA function, particularly flexible regions that may undergo conformational changes during activity.

What emerging technologies could advance prfA research?

Researchers should consider these cutting-edge approaches:

• Single-molecule studies: Apply techniques like FRET to observe individual prfA molecules interacting with ribosomes in real-time.

• Ribosome profiling: Use Ribo-seq to map translation termination events genome-wide in different prfA mutant backgrounds.

• Synthetic biology approaches: Create minimal translation systems with purified components to study prfA function in controlled environments.

• Microfluidics: Develop high-throughput screening systems to identify compounds that modulate prfA activity, potentially as research tools.

• Integration with AI prediction tools: Combine experimental data with machine learning approaches to predict the impact of specific prfA mutations.