Recombinant Shewanella pealeana Peptide chain release factor 1 (prfA)

What is Shewanella pealeana and what ecological niches does it occupy?

Shewanella pealeana is a mesophilic, facultatively anaerobic, psychrotolerant bacterium originally isolated from the accessory nidamental gland of the squid Loligo pealei . This Gram-negative rod-shaped bacterium (2.0-3.0 microns long, 0.4-0.6 micron wide) belongs to the gamma-3 subclass of Proteobacteria . S. pealeana is notable for its ability to reduce elemental sulfur, which was the basis for its selection from the microbial community of the squid .

The bacterium grows optimally at temperatures between 25-30°C and pH 6.5-7.5 in media containing 0.5 M NaCl . While initially isolated from a marine organism, members of the Shewanella genus have since been found in various aquatic environments including both marine and freshwater habitats . This ecological versatility makes S. pealeana an interesting subject for studying bacterial adaptation to different environmental conditions.

What is the function of peptide chain release factor 1 (prfA) in bacterial translation?

Peptide chain release factor 1 (prfA) plays a crucial role in the termination phase of protein synthesis in bacteria. The protein recognizes the stop codons UAA and UAG in messenger RNA, facilitating the release of the completed polypeptide chain from the ribosome. This process is essential for proper protein synthesis and bacterial survival.

In Shewanella species, prfA functions similarly to other bacterial systems but may have adapted specific features related to the organism's environmental adaptations. For instance, given S. pealeana's psychrotolerant nature (ability to grow at low temperatures), its prfA might exhibit structural and functional adaptations that maintain efficient translation termination across a broader temperature range compared to mesophilic bacteria.

How does the genomic context of prfA in S. pealeana compare to other bacterial species?

While specific genomic information about prfA in S. pealeana is limited in these search results, comparative genomics approaches can be informative. In many bacteria, prfA is often part of operons containing genes involved in translation. Genomic analyses of related Shewanella species, such as S. oneidensis MR-1 and S. putrefaciens W3-18-1, have revealed extensive horizontal gene transfer events that contribute to their metabolic versatility and environmental adaptation .

The genomic context of prfA in S. pealeana likely reflects both its evolutionary history within the Shewanella genus and functional constraints related to its role in translation termination. Analysis of the genomic neighborhood of prfA can provide insights into potential co-regulated genes and functional relationships specific to S. pealeana's niche adaptation.

What expression systems and conditions are optimal for recombinant S. pealeana prfA production?

For recombinant expression of S. pealeana prfA, E. coli-based expression systems are commonly employed with the following considerations:

• Vector selection: pET-based expression vectors under the control of T7 promoter systems typically provide high yields for bacterial proteins.

• Host strain optimization: BL21(DE3) or Rosetta strains are preferred, with the latter being advantageous if the prfA gene contains rare codons that could limit expression in standard E. coli strains.

• Temperature optimization: Given S. pealeana's psychrotolerant nature, expression at lower temperatures (16-20°C) after induction may improve protein folding and solubility .

• Media composition: Supplementation with NaCl (0.3-0.5 M) may help replicate S. pealeana's natural environment and potentially improve protein stability .

• Induction conditions: Lower IPTG concentrations (0.1-0.5 mM) with extended expression times (16-20 hours) at reduced temperatures often yield better results for recombinant proteins from psychrotolerant organisms.

What purification strategies are most effective for recombinant S. pealeana prfA?

Effective purification of recombinant S. pealeana prfA typically involves a multi-step approach:

Including reducing agents such as DTT or β-mercaptoethanol (1-5 mM) in all buffers helps maintain protein stability. For psychrotolerant proteins like those from S. pealeana, maintaining lower temperatures (4°C) throughout purification is critical to preserve native conformation and activity.

How can the activity of recombinant S. pealeana prfA be assayed in vitro?

In vitro activity assessment of recombinant S. pealeana prfA can be performed using several complementary approaches:

• Ribosome-dependent peptide release assay: This assay measures the ability of prfA to catalyze the release of a peptidyl-tRNA from ribosomes at stop codons.

  • Pre-termination complexes are assembled using purified ribosomes, mRNA containing stop codons, and radiolabeled peptidyl-tRNA.

  • Release activity is quantified by measuring the release of radiolabeled peptide upon addition of purified prfA.

• GTPase activation assay: Since prfA functions together with the GTPase RF3, measuring GTP hydrolysis rates in reconstituted systems can provide insights into prfA function.

• Temperature-dependent activity profiling: Given S. pealeana's psychrotolerant nature , assaying prfA activity across a temperature range (4-37°C) can reveal adaptations specific to this species' environmental niche.

• Competition assays with RF2: Assessing how S. pealeana prfA competes with RF2 (which recognizes UGA stop codons) at UAA stop codons (recognized by both factors) can provide insights into the evolution of translation termination mechanisms in this species.

How might S. pealeana's respiratory versatility influence prfA function and expression?

S. pealeana demonstrates remarkable respiratory versatility, growing anaerobically through the reduction of iron, manganese, nitrate, fumarate, trimethylamine-N-oxide, thiosulfate, and elemental sulfur . This metabolic flexibility suggests sophisticated regulatory networks that likely influence translation, including termination processes mediated by prfA.

Research approaches to investigate this relationship include:

• Comparing prfA expression levels under different respiratory conditions using RT-qPCR and western blotting.

• Investigating post-translational modifications of prfA that might occur under specific respiratory conditions.

• Examining ribosome profiles and translation termination efficiency across different respiratory states to determine if prfA activity is modulated in response to electron acceptor availability.

• Creating reporter systems with prfA-dependent readthrough to assess in vivo activity under various respiratory conditions.

The unique ability of Shewanella species to utilize diverse electron acceptors may have driven evolutionary adaptations in their translation machinery, including specialized features of prfA that warrant investigation.

What structural adaptations might S. pealeana prfA exhibit related to psychrotolerance?

Given S. pealeana's ability to grow at lower temperatures, its prfA likely possesses structural adaptations that maintain functionality in cold environments. Research approaches to investigate these adaptations include:

• Comparative structural analysis: Using homology modeling and, ideally, solving the crystal structure of S. pealeana prfA to compare with mesophilic homologs.

• Thermostability studies: Measuring the thermal denaturation profiles of recombinant S. pealeana prfA compared to homologs from mesophilic bacteria.

• Temperature-dependent activity assays: Systematically comparing peptide release activity at different temperatures (4-37°C) between S. pealeana prfA and homologs from mesophilic relatives.

• Molecular dynamics simulations: Investigating the conformational flexibility and solvent interactions of S. pealeana prfA at different temperatures to identify key adaptations for cold activity.

Typical adaptations in psychrotolerant proteins include increased flexibility of catalytic regions, decreased surface charge, modified hydrophobic cores, and altered amino acid composition that together maintain function at lower temperatures.

How does the codon usage in S. pealeana affect the optimization of recombinant prfA expression?

Shewanella species often exhibit codon usage patterns that differ from common expression hosts like E. coli. This can significantly impact recombinant expression efficiency. Based on comparative genomic analyses of Shewanella species , researchers should consider:

• Codon optimization strategies: Analyzing the native S. pealeana prfA sequence for rare codons and potentially redesigning the sequence for expression in E. coli while maintaining the amino acid sequence.

• Expression host selection: Using specialized E. coli strains (e.g., Rosetta) that supply rare tRNAs if codon optimization is not desired.

• Impact on protein folding: Investigating whether alterations in translation rate due to codon usage affect the folding and activity of recombinant prfA.

• Native context considerations: Examining whether S. pealeana has evolved specialized mechanisms for efficient translation of its native prfA sequence that might be lost in heterologous expression systems.

How can researchers address contradictory results in prfA activity assays?

When facing contradictory results in prfA activity assays, researchers should implement a systematic troubleshooting approach:

• Standardize experimental conditions: Ensure consistent buffer composition, pH, salt concentration, and temperature across experiments, as S. pealeana prfA activity may be particularly sensitive to these parameters given its psychrotolerant nature .

• Assess protein quality: Implement multiple quality control measures including SDS-PAGE, size exclusion chromatography, and thermal shift assays to verify protein integrity before activity testing.

• Control for contextual factors: Test activity in the presence of different ribosome sources, as S. pealeana prfA may have evolved specificity for its native ribosomal components.

• Benchmark against controls: Always include positive controls (e.g., E. coli prfA) in parallel assays to validate experimental systems.

• Implement orthogonal assays: Use multiple independent assay formats to cross-validate activity measurements, as single assay types may be subject to specific interferences.

Careful documentation of all experimental variables and iterative refinement of protocols are essential for resolving contradictory results when working with specialized proteins like S. pealeana prfA.

What bioinformatic approaches are most valuable for analyzing S. pealeana prfA sequence and function?

Comprehensive bioinformatic analysis of S. pealeana prfA should include:

• Phylogenetic analysis: Constructing phylogenetic trees of prfA sequences across the Shewanella genus and related bacteria to understand evolutionary relationships and potential functional divergence.

• Structural prediction: Using tools like AlphaFold2 or RoseTTAFold to predict the three-dimensional structure of S. pealeana prfA, particularly focusing on domains involved in stop codon recognition.

• Comparative sequence analysis: Identifying conserved and divergent residues through multiple sequence alignments with prfA proteins from bacteria inhabiting different environments.

• Codon usage analysis: Examining the codon usage patterns in the prfA gene compared to highly expressed genes in S. pealeana to understand translational optimization.

• Genomic context exploration: Analyzing genes upstream and downstream of prfA to identify potential operonic structures and co-regulated genes that might provide insights into specialized functions.

These approaches can reveal unique adaptations of S. pealeana prfA related to its ecological niche in marine environments and its psychrotolerant characteristics .

How might CRISPR-Cas9 technology be leveraged to study prfA function in S. pealeana?

CRISPR-Cas9 technology offers powerful approaches for investigating prfA function in S. pealeana:

• Gene knockdown studies: Creating conditional knockdown strains of prfA to study its essentiality and the cellular consequences of reduced prfA activity.

• Domain-specific mutagenesis: Introducing precise mutations in functional domains of prfA to assess their impact on translation termination efficiency and stop codon specificity.

• Tagged variant generation: Creating genomically encoded tagged versions of prfA for in vivo localization and interaction studies without overexpression artifacts.

• Promoter replacement: Substituting the native prfA promoter with controllable promoters to study the effects of altered expression levels on cellular physiology.

• Comparative editing: Performing parallel edits of prfA in S. pealeana and related Shewanella species to compare phenotypic effects across environmental adaptations.

Implementation requires optimization of transformation protocols for S. pealeana, which may be challenging given its marine origin and potential restriction barriers. Electroporation methods adapted for marine bacteria, potentially including protocols that account for high salinity preferences (0.5 M NaCl) , would likely be necessary.

What insights might comparative proteomics provide about prfA function in S. pealeana?

Comparative proteomics approaches can yield valuable insights into prfA function and regulation in S. pealeana:

• Translation termination fidelity assessment: Quantitative proteomics to identify proteins showing evidence of stop codon readthrough or premature termination when prfA function is perturbed.

• Interactome mapping: Proximity-labeling approaches (BioID or APEX) coupled with mass spectrometry to identify proteins interacting with prfA under different environmental conditions.

• Condition-dependent expression analysis: Quantitative proteomics comparing prfA expression levels across different growth conditions, particularly varying temperature and electron acceptor availability based on S. pealeana's known physiological characteristics .

• Post-translational modification mapping: Phosphoproteomics and other PTM analyses to identify regulatory modifications on prfA that might control its activity in response to environmental factors.

• Ribosome profiling: Analyzing ribosome occupancy patterns at stop codons to assess translation termination efficiency in vivo under various conditions.

These approaches can reveal how S. pealeana has adapted its translation termination mechanisms to function optimally in its ecological niche, particularly in relation to its psychrotolerance and respiratory versatility .

How might S. pealeana prfA research contribute to understanding bacterial adaptation to extreme environments?

Research on S. pealeana prfA has broader implications for understanding bacterial adaptation to challenging environments:

• Cold adaptation mechanisms: As a psychrotolerant organism , S. pealeana likely possesses specialized adaptations in its translation machinery, including prfA, that maintain functionality at lower temperatures. These adaptations could provide insights applicable to other cold-adapted species.

• Responsive translation systems: S. pealeana's ability to thrive with diverse electron acceptors suggests sophisticated regulatory networks that may extend to translation termination. Understanding how prfA function is maintained across metabolic states could reveal principles of bacterial metabolic flexibility.

• Symbiotic relationship adaptations: Originally isolated from squid nidamental glands , S. pealeana may have evolved specialized translation termination features related to host interaction, potentially revealing principles of bacterial-host co-evolution.

• Evolutionary plasticity: Comparative studies of prfA across Shewanella species from marine and freshwater environments could illuminate how translation termination systems evolve during adaptation to new ecological niches.

• Biotechnological applications: Insights from S. pealeana prfA could inform the design of engineered translation systems functioning under non-standard conditions, including low temperatures or varying redox states.