Recombinant Pseudomonas phage Pf1 Uncharacterized protein ORF83

What is the predicted structure and function of Pseudomonas phage Pf1 ORF83?

ORF83 is an uncharacterized protein in Pseudomonas phage Pf1 with no clearly defined function based on sequence homology alone. Structural prediction algorithms suggest it may contain domains similar to those found in other filamentous phage proteins involved in replication or assembly processes. The protein likely plays a role in one of several critical phage functions: DNA replication, phage assembly, or host interaction. Based on the genomic organization of filamentous phages like Pf, small ORFs often encode proteins with regulatory functions or proteins that interact with host machinery during infection and replication cycles .

To begin characterizing this protein, researchers should conduct computational analyses including protein sequence alignment with known phage proteins, secondary structure prediction, and identification of conserved motifs or domains. Unlike many characterized phage proteins, ORF83 lacks homology to proteins with known functions in databases, making experimental characterization essential for understanding its role.

How does ORF83 compare to other uncharacterized proteins in filamentous phages?

Filamentous phages across different bacterial species contain numerous uncharacterized ORFs with potentially similar functions. Comparative genomic analysis of Pf1 ORF83 with proteins from related filamentous phages such as Pf4, CTXφ, and VGJφ reveals limited sequence homology but potentially conserved structural features. Unlike the well-characterized initiator proteins of CTXφ and VGJφ that contain the catalytic module SIYNK, ORF83 lacks this sequence motif but may contain other functional domains .

When analyzing ORF83 in relation to other phage proteins, researchers should consider not only sequence similarity but also genomic context, as genes with related functions are often clustered together in phage genomes. The positioning of ORF83 in relation to genes involved in replication, structural components, or host interaction may provide insight into its function.

What are the predicted physicochemical properties of ORF83?

ORF83 is predicted to be a small protein (approximately 83 amino acids, as suggested by its designation) with specific physicochemical properties that influence its experimental handling. Key properties include:

These properties should be considered when designing expression and purification protocols. The absence of predicted transmembrane domains suggests ORF83 is likely soluble, which is advantageous for recombinant expression in E. coli or other host systems.

How might ORF83 interact with the UvrD helicase during phage replication?

Recent findings demonstrate that Pseudomonas filamentous phage replication depends on the host UvrD helicase, which is primarily known for its role in DNA repair . ORF83 may potentially interact with UvrD during rolling circle replication (RCR). To investigate this interaction, researchers should consider the following experimental approaches:

• Co-immunoprecipitation assays using tagged versions of ORF83 and UvrD

• Bacterial two-hybrid screening to detect direct protein-protein interactions

• In vitro helicase activity assays to determine if ORF83 enhances or inhibits UvrD activity

• DNA binding assays to assess if ORF83 recognizes specific sequences at the origin of replication

If ORF83 interacts with UvrD, it might function as an accessory protein that regulates helicase activity or helps localize the helicase to the phage replication origin. Understanding this interaction could reveal novel mechanisms of phage DNA replication and potentially identify new targets for controlling Pseudomonas biofilm formation.

What role might ORF83 play in Pf phage-mediated biofilm formation and virulence?

Pf filamentous phage plays a critical role in P. aeruginosa biofilm development, which contributes to bacterial persistence and antibiotic resistance . ORF83 could potentially influence biofilm formation through several mechanisms:

• Modification of extracellular DNA arrangement in the biofilm matrix

• Interaction with bacterial surface proteins involved in attachment

• Regulation of phage production or superinfection during biofilm maturation

• Modulation of host gene expression related to virulence factor production

To investigate these possibilities, researchers should consider comparing biofilm formation between wild-type phage and phage with mutations in ORF83. Confocal microscopy combined with fluorescently tagged ORF83 could reveal localization patterns within biofilms. Additionally, transcriptomic analysis of host cells infected with wild-type versus ORF83-mutant phage could identify changes in expression patterns of virulence-related genes.

How does ORF83 contribute to the phage life cycle in different environmental conditions?

Environmental factors significantly influence phage-host interactions and may alter the function or importance of ORF83. Researchers investigating this question should examine how different conditions affect ORF83 expression and function:

Understanding how ORF83 functions under different environmental conditions could provide insights into the ecological role of Pf phage in natural and clinical settings. This knowledge might lead to strategies for disrupting phage-mediated bacterial adaptations to hostile environments.

What are the optimal conditions for recombinant expression of Pf1 ORF83?

Expressing uncharacterized phage proteins like ORF83 presents several challenges that require optimization of expression systems and conditions. For successful recombinant expression of ORF83, consider the following methodological approach:

• Expression System Selection: E. coli BL21(DE3) is generally suitable for initial expression trials. For proteins difficult to express in prokaryotic systems, consider P. aeruginosa-based expression systems which may provide native folding machinery.

• Fusion Tag Strategy: Employ a dual tagging approach with an N-terminal 6xHis tag and C-terminal FLAG tag to ensure purification of full-length protein and protection against proteolysis .

• Codon Optimization: Analyze the ORF83 sequence for rare codons that might impede expression in E. coli. Custom synthesis of a codon-optimized gene may improve expression yields.

• Expression Conditions: Test multiple induction temperatures (18°C, 25°C, 30°C, 37°C) and IPTG concentrations (0.1-1.0 mM) to optimize soluble protein yield.

• Solubility Enhancement: If inclusion body formation occurs, addition of solubility-enhancing fusion partners such as SUMO, MBP, or TrxA may improve soluble expression.

The purification protocol should include immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography to ensure high purity. For proteins with challenging expression profiles, consider cell-free protein synthesis systems which can overcome toxicity issues associated with membrane-interactive proteins.

How can researchers verify the functional activity of recombinant ORF83?

Determining functional activity of an uncharacterized protein presents significant challenges. For ORF83, a systematic approach to functional verification should include:

• DNA Binding Assays: Use electrophoretic mobility shift assays (EMSA) with Pf1 phage DNA fragments to test if ORF83 binds DNA, particularly at the origin of replication.

• Protein-Protein Interaction Studies: Perform pull-down assays to identify host or phage proteins that interact with ORF83. Focus particularly on UvrD helicase and known replication proteins .

• In vitro Replication Assays: Develop a cell-free system using purified components to test if ORF83 influences rolling circle replication efficiency.

• Structural Analysis: Employ circular dichroism and thermal shift assays to assess protein folding and stability. X-ray crystallography or NMR spectroscopy can provide detailed structural information if sufficient quantities of purified protein are available.

• Complementation Studies: Express ORF83 in Pseudomonas strains with the corresponding gene deleted from the prophage to test for restoration of phenotypes such as biofilm formation.

Each functional assay should include appropriate positive and negative controls. For example, when testing DNA binding, include a known DNA-binding protein as a positive control and an unrelated protein as a negative control.

What genetic manipulation approaches are most effective for studying ORF83 function in vivo?

Understanding ORF83 function requires sophisticated genetic approaches for in vivo studies. The following methodological strategies are recommended:

• Gene Deletion: Create a clean deletion of ORF83 in the Pf1 prophage using CRISPR-Cas9 or homologous recombination techniques. Analyze the resulting phenotype for defects in phage production, biofilm formation, or host cell physiology.

• Complementation Analysis: Reintroduce wild-type or mutated versions of ORF83 to the deletion strain to confirm phenotypes and identify essential residues or domains.

• Fluorescent Protein Fusion: Generate C-terminal or N-terminal fusions with fluorescent proteins (ensuring function is maintained) to track localization of ORF83 during infection and replication cycles.

• Inducible Expression Systems: Use tetracycline-responsive or arabinose-inducible promoters to control ORF83 expression levels, allowing analysis of dose-dependent effects.

• Site-Directed Mutagenesis: Create targeted mutations in predicted functional domains to identify critical residues for ORF83 activity.

For P. aeruginosa, which can be challenging for genetic manipulation, electroporation of suicide vectors followed by sucrose counter-selection is often effective for creating chromosomal modifications. When working with phage genes, consider the potential impact on phage induction and superinfection, which might complicate phenotypic analysis.

What bioinformatic approaches are most useful for predicting ORF83 structure and function?

In the absence of experimental data, computational methods can provide valuable insights into ORF83's potential structure and function:

• Homology Modeling: While ORF83 lacks close homologs with solved structures, remote homology detection methods like HHpred can identify distant structural relatives. Focus on comparison with proteins from other filamentous phages, particularly those with similar genome organization to Pf1.

• Ab Initio Structure Prediction: Use AlphaFold2 or RoseTTAFold to generate predicted structures, which have shown remarkable accuracy even for proteins with no detectable sequence homology to known structures.

• Functional Site Prediction: Employ tools like ConSurf, COACH, and 3DLigandSite to identify conserved surface residues and potential ligand-binding pockets.

• Molecular Dynamics Simulations: Once a structural model is available, MD simulations can provide insights into protein flexibility, potential conformational changes, and binding site accessibility.

• Co-evolution Analysis: Methods like EVcouplings can identify co-evolving residues that might indicate functional contacts within the protein or with interaction partners.

These computational predictions should guide experimental design, particularly for site-directed mutagenesis studies targeting predicted functional sites. The integration of computational and experimental approaches is essential for uncharacterized proteins like ORF83.

How does ORF83 compare to the initiator proteins identified in other filamentous phages?

The initiator proteins of filamentous phages are critical for initiating rolling circle replication. Based on the limited information available, ORF83 may have functional similarities to known initiator proteins despite sequence divergence:

• Domain Architecture: Unlike the initiator proteins of CTXφ and VGJφ that contain a Pfam02486 domain and the catalytic module SIYNK, ORF83 lacks these specific signatures but may contain alternative functional motifs .

• Genomic Context: The positioning of ORF83 in the Pf1 genome relative to other functional genes may provide clues about its role. In many filamentous phages, genes with related functions cluster together.

• Structural Comparison: Though primary sequence similarity is limited, structural prediction may reveal similar folding patterns between ORF83 and known initiator proteins.

To experimentally investigate whether ORF83 functions similarly to known initiator proteins, researchers should test its ability to bind to the Pf1 origin of replication and its catalytic activity in nicking DNA to initiate replication. Complementation experiments with known initiator proteins could also reveal functional equivalence despite sequence divergence.

What is known about post-translational modifications of ORF83 and their significance?

Post-translational modifications (PTMs) can significantly impact protein function, but little is known about PTMs in phage proteins, including ORF83. Researchers should consider:

• Prediction of PTM Sites: Use computational tools to predict potential sites for phosphorylation, acetylation, methylation, and other common modifications based on sequence motifs.

• Mass Spectrometry Analysis: Perform LC-MS/MS analysis of purified ORF83 expressed in P. aeruginosa to identify actual PTMs present in the native host environment.

• Site-Directed Mutagenesis: Create alanine substitutions at predicted PTM sites to assess their functional importance.

• Temporal Analysis: Investigate whether PTMs change during different stages of infection or under different environmental conditions.

A comparative PTM analysis between ORF83 expressed in E. coli versus P. aeruginosa might reveal host-specific modifications that affect protein function. This information could be crucial for understanding regulatory mechanisms controlling ORF83 activity during phage infection and replication.

How does ORF83 contribute to antibiotic resistance in P. aeruginosa infections?

Pf filamentous phages are known to influence P. aeruginosa biofilm formation, which contributes to antibiotic resistance . The potential role of ORF83 in this process has significant clinical implications:

• Biofilm Structure Modification: ORF83 may affect the arrangement of extracellular DNA or proteins in biofilms, altering antibiotic penetration.

• Stress Response Modulation: The protein might influence bacterial stress responses that contribute to tolerance or persistence during antibiotic treatment.

• Horizontal Gene Transfer: If ORF83 influences phage production or DNA transfer, it could indirectly affect the spread of resistance genes in bacterial populations.

Experimental approaches to investigate these possibilities include comparing antibiotic susceptibility profiles between wild-type and ORF83 mutant strains, analyzing biofilm architecture using confocal microscopy with fluorescent antibiotic probes, and measuring expression of resistance genes in the presence and absence of functional ORF83.

What are the implications of ORF83 for phage therapy development?

Understanding ORF83 function has potential implications for phage therapy approaches targeting P. aeruginosa infections:

• Phage Engineering: If ORF83 influences host range or replication efficiency, modifications to this gene could potentially enhance therapeutic efficacy.

• Resistance Mechanisms: Knowledge of how ORF83 interacts with host factors might reveal potential resistance mechanisms against phage therapy.

• Biofilm Disruption: If ORF83 is involved in biofilm formation or maintenance, targeting this protein could enhance the effectiveness of combined phage-antibiotic treatments.

• Safety Considerations: Understanding the full range of ORF83 functions is essential for assessing the safety of Pf phages in therapeutic applications, particularly if the protein influences horizontal gene transfer or virulence.

Researchers developing phage therapy approaches should consider creating ORF83 variants with enhanced activity or altered specificity, potentially expanding the therapeutic potential of engineered Pf phages against resistant P. aeruginosa infections.