Recombinant Kocuria rhizophila Peptide chain release factor 1 (prfA)

What is the general genomic structure of Kocuria rhizophila?

The genome of K. rhizophila (specifically strain BT304) consists of a single circular chromosome of 2,763,150 bp with a relatively high GC content of 71.2% . The genome contains 2,359 coding sequences, 51 tRNA genes, and 9 rRNA genes . Comparative genomic analysis with other K. rhizophila strains (such as DC2201, FDAARGOS 302, and G2) shows high similarity with OrthoANI values exceeding 97.8% . The genomic content is particularly rich in genes related to amino acid, carbohydrate, and protein metabolism, with specific emphasis on pathways involving branched chain amino acid biosynthesis and degradation .

What are the recommended methods for isolating and purifying recombinant K. rhizophila prfA?

For isolating and purifying recombinant K. rhizophila prfA, researchers should consider a multi-step approach:

• Gene Cloning: First, amplify the prfA gene from K. rhizophila genomic DNA using PCR with primers designed based on the genome sequence data available (GenBank Accession Number CP030039) . Design primers to include appropriate restriction sites for subsequent cloning.

• Expression Vector Construction: Clone the amplified prfA gene into a suitable expression vector (such as pET series for E. coli expression systems) with an appropriate affinity tag (His-tag is commonly used).

• Transformation and Expression: Transform the recombinant vector into a compatible expression host (E. coli BL21(DE3) is often used). Induce protein expression with IPTG or another appropriate inducer based on the vector system.

• Cell Lysis: Harvest cells and lyse using either sonication or chemical methods in a buffer optimized for protein stability (typically phosphate or Tris-based buffers with appropriate salt concentration).

• Purification: Utilize affinity chromatography (Ni-NTA for His-tagged proteins) followed by size exclusion chromatography to achieve high purity. Consider ion exchange chromatography as an additional purification step if necessary.

• Quality Control: Verify protein identity and purity using SDS-PAGE, Western blotting, and mass spectrometry.

This protocol should be optimized based on specific laboratory conditions and the particular characteristics of K. rhizophila prfA .

How can researchers effectively design experiments to study the functional activity of recombinant prfA from K. rhizophila?

To effectively study the functional activity of recombinant prfA from K. rhizophila, researchers should implement the following experimental design strategies:

• In vitro Translation Termination Assays: Establish an in vitro translation system using either purified ribosomes or commercially available translation kits. Design synthetic mRNAs containing UAA or UAG stop codons followed by reporter sequences. Measure termination efficiency by quantifying released peptides versus read-through products.

• Peptidyl-tRNA Hydrolysis Assays: Use fluorescently labeled peptidyl-tRNA substrates to directly measure the hydrolytic activity of purified prfA. Monitor the release of the fluorescent peptide over time to determine kinetic parameters.

• Binding Affinity Studies: Employ surface plasmon resonance (SPR) or microscale thermophoresis (MST) to determine the binding affinity of prfA to ribosomes and to different stop codon contexts.

• Mutagenesis Analysis: Create site-directed mutants of key residues predicted to be involved in stop codon recognition or peptidyl-tRNA hydrolysis based on structural models or sequence alignments with well-characterized release factors. Compare their activities to the wild-type protein.

• Complementation Studies: Test the ability of K. rhizophila prfA to complement prfA-deficient bacterial strains, which would demonstrate functional conservation across species.

These approaches should be accompanied by appropriate controls and statistical analyses to ensure robust and reproducible results .

What computational tools are most effective for analyzing K. rhizophila prfA structure and predicting functional domains?

For analyzing K. rhizophila prfA structure and predicting functional domains, researchers should employ a comprehensive suite of computational tools:

• Sequence Analysis:

  • BLAST and HMMER for identifying homologous sequences

  • Multiple Sequence Alignment tools (MUSCLE, Clustal Omega) for comparing with prfA from other bacterial species

  • ConSurf for identifying evolutionarily conserved residues

• Structural Prediction:

  • AlphaFold2 or RoseTTAFold for generating accurate protein structure predictions

  • SWISS-MODEL for homology modeling based on known bacterial release factor structures

  • PyMOL or UCSF Chimera for structural visualization and analysis

• Functional Domain Prediction:

  • InterProScan to identify conserved domains

  • MOTIF Search for identifying functional motifs

  • PredictProtein for secondary structure and functional site prediction

• Molecular Dynamics Simulations:

  • GROMACS or AMBER for studying conformational changes and dynamics

  • FoldX for assessing the stability of the predicted structures and mutant variants

• Codon Recognition Analysis:

  • specialized tools for predicting stop codon recognition domains based on known bacterial release factor structures

These computational approaches should be integrated with experimental validation to ensure accurate structural and functional characterization of K. rhizophila prfA .

How does K. rhizophila prfA compare with peptide chain release factors from other bacterial species in terms of structure and function?

Comparing K. rhizophila prfA with peptide chain release factors from other bacterial species reveals important evolutionary and functional insights:

This comparative analysis provides valuable context for understanding the evolution and specialized functions of translation termination factors across bacterial lineages .

What experimental approaches are recommended for investigating potential interactions between K. rhizophila prfA and other components of the translation machinery?

To investigate interactions between K. rhizophila prfA and other translation components, researchers should employ multiple complementary approaches:

• Cryo-Electron Microscopy (Cryo-EM):

  • Prepare ribosome complexes with bound prfA at termination codons

  • Determine the structure at near-atomic resolution

  • Analyze conformational changes in both prfA and the ribosome during termination

• Co-Immunoprecipitation (Co-IP) and Pull-down Assays:

  • Use tagged prfA to identify interacting partners from K. rhizophila cellular extracts

  • Confirm specific interactions with purified components (ribosomes, ribosomal proteins, other factors)

  • Employ mass spectrometry for comprehensive identification of interaction partners

• Surface Plasmon Resonance (SPR) and Microscale Thermophoresis (MST):

  • Quantitatively measure binding kinetics between prfA and purified ribosomal components

  • Determine the effects of different nucleotides, antibiotics, or mutations on these interactions

  • Compare binding profiles with release factors from other bacterial species

• Crosslinking Mass Spectrometry (XL-MS):

  • Identify specific contact residues between prfA and ribosomal components

  • Map the interaction network within the termination complex

  • Validate functional importance through mutagenesis of identified contact points

• Fluorescence Resonance Energy Transfer (FRET):

  • Monitor real-time conformational changes during termination

  • Analyze the dynamics of prfA binding and release

  • Study the effects of different stop codon contexts on interaction kinetics

These approaches would provide comprehensive insights into how K. rhizophila prfA integrates into the translation machinery, potentially revealing unique features compared to well-studied model organisms .

How can researchers address data contradictions when analyzing the function of recombinant K. rhizophila prfA in different experimental systems?

When confronting contradictory data regarding recombinant K. rhizophila prfA function across different experimental systems, researchers should implement a systematic approach to resolution:

• Standardization of Experimental Protocols:

  • Establish unified protein expression and purification protocols

  • Implement consistent buffer conditions and assay parameters

  • Develop reference standards for activity measurements

  • Share standardized reagents between laboratories

• Multi-System Validation:

  • Test prfA function in multiple experimental platforms (in vitro, E. coli-based, cell-free systems)

  • Compare homologous versus heterologous expression systems

  • Evaluate the impact of different affinity tags and their positions

  • Assess native versus recombinant protein activities when possible

• Statistical and Computational Analysis:

  • Apply meta-analysis techniques to integrate data from multiple studies

  • Develop mathematical models to explain system-dependent variations

  • Use Bayesian approaches to update confidence in specific hypotheses

  • Implement machine learning to identify patterns in contradictory results

• Targeted Investigation of Confounding Variables:

  • Examine post-translational modifications across different expression systems

  • Investigate the influence of buffer components and pH on activity

  • Assess the impact of contaminating proteins or nucleic acids

  • Evaluate temperature-dependent effects on structure and function

• Collaborative Cross-Laboratory Validation:

  • Organize multi-laboratory testing of identical samples

  • Implement blind testing protocols to minimize bias

  • Share raw data and analytical methods between research groups

  • Document all experimental variables in standardized formats

By systematically addressing these aspects, researchers can resolve apparent contradictions and develop a more coherent understanding of K. rhizophila prfA function across different experimental conditions .

What can comparative genomic analysis reveal about the evolution of prfA in K. rhizophila and related Actinobacteria?

Comparative genomic analysis of prfA in K. rhizophila and related Actinobacteria provides valuable evolutionary insights:

• Conservation Patterns: Analysis of K. rhizophila strains with high genomic similarity (>97.8% OrthoANI) allows identification of highly conserved regions within prfA that likely represent functionally critical domains. The table below shows genome comparison metrics among K. rhizophila strains:

• Selective Pressure Analysis: Calculation of dN/dS ratios (non-synonymous to synonymous substitution rates) across prfA sequences from different Actinobacteria species can identify regions under purifying, neutral, or positive selection, indicating functional constraints or adaptive evolution.

• Horizontal Gene Transfer Assessment: Analysis of GC content, codon usage bias, and phylogenetic incongruence can reveal potential horizontal gene transfer events affecting prfA evolution in Actinobacteria. K. rhizophila's high GC content (71.2%) creates a distinctive genomic context for prfA evolution.

• Coevolution with Translation Machinery: Investigation of correlated evolutionary changes between prfA and other translation components (ribosomes, tRNAs, other factors) across Actinobacteria can reveal coevolutionary networks maintaining translational fidelity.

• Environmental Adaptation Signatures: Comparison of prfA sequences from K. rhizophila strains isolated from diverse environments (bovine gut, soil, slaughterhouse walls, etc.) might reveal environment-specific adaptations in translation termination mechanisms.

This evolutionary analysis provides context for understanding prfA function and can guide experimental approaches for characterizing its specific attributes in K. rhizophila .

How does the high GC content (71.2%) of the K. rhizophila genome influence the expression and function of recombinant prfA?

The high GC content (71.2%) of the K. rhizophila genome significantly impacts recombinant prfA expression and function through multiple mechanisms:

• Codon Usage Challenges:

  • The GC-rich coding sequences in K. rhizophila utilize different codon frequencies compared to common expression hosts like E. coli (50.8% GC)

  • This disparity can lead to translational pausing, premature termination, or protein misfolding when expressing K. rhizophila proteins in heterologous systems

  • For optimal expression, codon optimization aligning with the host's preferences may be necessary

• mRNA Secondary Structure Effects:

  • High GC content promotes stable secondary structures in mRNA

  • These structures can impede ribosome binding and translation initiation

  • They may also cause ribosomal stalling during elongation

  • Strategic introduction of silent mutations may be required to reduce problematic mRNA structures

• DNA Amplification and Cloning Difficulties:

  • GC-rich templates are challenging for PCR amplification due to stable secondary structures and higher denaturation temperatures

  • Special PCR protocols using DMSO, betaine, or specialized polymerases may be necessary

  • Sequencing GC-rich regions often requires specialized techniques

• Protein Structural Considerations:

  • The GC bias influences amino acid composition, potentially leading to higher proportions of amino acids encoded by GC-rich codons (Ala, Gly, Pro, Arg)

  • This compositional bias may affect protein folding, stability, and activity when expressed in different cellular environments

• Expression System Selection:

  • Expression hosts with higher GC content (like Streptomyces with ~70% GC) may be more suitable than E. coli for functional expression

  • Cell-free expression systems can be optimized with specific tRNAs and factors to accommodate GC-rich transcripts

Addressing these challenges requires an integrated approach combining codon optimization, expression host selection, and buffer formulation tailored to the characteristics of K. rhizophila prfA .

What are the potential applications of recombinant K. rhizophila prfA in studying antibiotic mechanisms targeting bacterial translation?

Recombinant K. rhizophila prfA offers valuable applications for studying translation-targeting antibiotics:

• Novel Antibiotic Screening Platform:

  • Develop high-throughput assays using purified K. rhizophila prfA to identify compounds that specifically inhibit translation termination

  • Compare effects on prfA from different bacterial sources to identify species-specific inhibitors

  • Screen for compounds that modulate stop codon readthrough, potentially useful for treating genetic diseases

• Mechanism of Action Studies:

  • Investigate how established antibiotics (aminoglycosides, macrolides, tetracyclines) affect prfA binding and activity

  • Elucidate structural interactions between antibiotics and the termination complex using cryo-EM and other structural techniques

  • Understand the molecular basis of antibiotic selectivity between bacterial and eukaryotic translation systems

• Resistance Mechanism Investigation:

  • Compare prfA function between antibiotic-sensitive and resistant K. rhizophila strains

  • Identify novel resistance mechanisms involving translation termination

  • Study how altered termination efficiency might compensate for antibiotic-induced translation defects

• Synthetic Biology Applications:

  • Engineer modified prfA variants with altered stop codon recognition profiles

  • Develop systems for expanded genetic code applications

  • Create translational switches responsive to small molecules for synthetic biology circuits

• Comparative Analysis with Clinically Relevant Bacteria:

  • Use K. rhizophila prfA as a model for understanding translation termination in pathogenic Actinobacteria

  • Identify unique features that might be exploited for selective targeting

These applications leverage the unique characteristics of K. rhizophila prfA and its industrial and potential probiotic relevance .

How might prfA function in K. rhizophila relate to its potential applications as a probiotic in livestock?

The function of prfA in K. rhizophila may significantly contribute to its potential as a probiotic in livestock through several mechanisms:

• Protein Synthesis Efficiency and Stress Adaptation:

  • Optimal translation termination via prfA contributes to efficient protein synthesis, enabling rapid adaptation to the changing gut environment

  • This may enhance K. rhizophila survival and colonization in the livestock intestinal tract

  • The BT304 strain isolated from bovine small intestine demonstrates adaptation to this specific niche

• Branched Chain Amino Acid (BCAA) Metabolism:

  • K. rhizophila possesses extensive genetic capacity for BCAA biosynthesis (20 genes) and degradation (52 genes)

  • Efficient translation termination through prfA is critical for expressing these metabolic enzymes

  • BCAA metabolism may contribute to beneficial interactions with the host by providing essential nutrients or regulating gut homeostasis

• Host-Microbe Interaction Factors:

  • The relatively small number of virulence-related genes (28 in strain BT304) suggests low pathogenic potential

  • Accurate translation of surface proteins and secreted factors regulated by proper termination may facilitate beneficial host interactions

  • Proteolytic activity observed in K. rhizophila isolates requires precisely translated enzymes for optimal function

• Competitive Advantage in the Gut Environment:

  • K. rhizophila demonstrates robust growth characteristics and adaptability

  • Efficient translation termination supports rapid protein synthesis required for competitive growth

  • The species shows good adaptation to various environments, including animal intestinal tracts

• Safety Profile Considerations:

  • Genomic analysis confirms the absence of antimicrobial resistance genes and amino acid decarboxylase activity in certain strains

  • Translation fidelity, influenced by prfA function, ensures accurate production of metabolites and prevents potentially harmful misfolded proteins

These aspects highlight how fundamental cellular processes like translation termination, mediated by prfA, underpin the probiotic potential of K. rhizophila in livestock applications .

What emerging technologies might enhance future research on K. rhizophila prfA and its role in bacterial translation?

Several cutting-edge technologies are poised to revolutionize research on K. rhizophila prfA and translation termination:

• Advanced Structural Biology Approaches:

  • Time-resolved cryo-EM to capture the dynamic process of translation termination

  • Microcrystal electron diffraction (MicroED) for determining structures of difficult-to-crystallize complexes

  • Integrative structural biology combining multiple data types (SAXS, NMR, XL-MS) for complete structural models

• Single-Molecule Techniques:

  • Single-molecule FRET to monitor real-time conformational changes during termination

  • Optical tweezers to measure forces involved in peptide release and ribosome recycling

  • Zero-mode waveguide technology for observing translation termination in real-time at single-molecule resolution

• Advanced Genetic and Genomic Tools:

  • CRISPR-Cas systems adapted for precise genome editing in K. rhizophila

  • Ribosome profiling to map translation termination events genome-wide

  • Translational recoding systems to study stop codon context effects

• Computational and AI-Based Methods:

  • Machine learning approaches to predict termination efficiency based on sequence context

  • Molecular dynamics simulations with enhanced sampling techniques

  • Quantum mechanical/molecular mechanical (QM/MM) calculations for studying the catalytic mechanism of peptide release

• High-Throughput Functional Genomics:

  • Deep mutational scanning of prfA to comprehensively map sequence-function relationships

  • Microfluidic-based assays for rapid phenotyping of thousands of prfA variants

  • Cell-free expression systems for high-throughput functional characterization

Integration of these technologies would provide unprecedented insights into the molecular mechanisms of translation termination in K. rhizophila and potential applications in biotechnology and probiotics .