Recombinant Enterobacteria phage PRD1 Protein P34 (XXXIV)

What is the genomic context of Protein P34 in the PRD1 genome?

Protein P34 (XXXIV) would be encoded by gene XXXIV in the PRD1 genome. The PRD1 genome is a linear double-stranded DNA molecule approximately 15 kbp in length with covalently attached priming proteins at both 5′ termini. Like other phage genes, gene XXXIV would be part of the genomic sequence that has been fully mapped. For context, other PRD1 proteins mentioned in research include at least 25 gene products identified through genetic and biochemical analysis of nonsense mutants . The sequencing of the genome led to the firm assignment of 33 genes and open reading frames (ORFs) . This suggests that P34 would be among the later-characterized proteins in the PRD1 genome.

What methodologies are recommended for expressing and purifying recombinant PRD1 proteins?

Expression and purification of recombinant PRD1 proteins typically follow these methodological steps:

• Cloning Strategy Design: The gene encoding P34 should be amplified by PCR from PRD1 genomic DNA using specific primers containing appropriate restriction sites.

• Expression System Selection: For PRD1 proteins, bacterial expression systems using pSU18/pSU19 vectors have been demonstrated effective, as seen with other PRD1 proteins .

• Protein Induction and Expression: After transformation into appropriate E. coli strains, protein expression can be induced using IPTG if under a lac promoter system.

• Purification Protocol:

  • Initial clarification by centrifugation

  • Affinity chromatography (if tagged)

  • Size exclusion chromatography for final purification

• Verification: SDS-PAGE and Western blotting using antibodies against the protein or tag.

Similar protocols have been employed for other PRD1 proteins, where targeted mutagenesis systems combining in vitro manipulation and in vivo recombination were developed to overcome challenges associated with the hydrophobic terminal proteins that complicate handling of the genome in vitro .

How do researchers assess protein-protein interactions between PRD1 structural proteins?

Protein-protein interactions between PRD1 structural proteins can be assessed through multiple complementary approaches:

• Co-immunoprecipitation (Co-IP): Using antibodies against known PRD1 proteins to precipitate potential binding partners, followed by mass spectrometry or Western blot analysis.

• Yeast Two-Hybrid Screening: This method has been applied to identify interactions between various phage proteins.

• Cryo-EM Structural Analysis: High-resolution cryo-electron microscopy has been particularly valuable for determining protein interactions in phage PRD1 and related phages. For example, the structure of PR772 (related to PRD1) was determined at 2.75 Å resolution, revealing interactions between various capsid proteins .

• Cross-linking Mass Spectrometry: Chemical cross-linking combined with mass spectrometry can identify proteins in close proximity.

• Surface Plasmon Resonance (SPR): To quantify binding affinities between purified proteins.

In practice, researchers have identified interactions such as those between protein P30 and the penton proteins (P5/P31) through hydrophobic interactions observed in structural studies .

What structural and functional domains are predicted for Protein P34 based on comparative analysis with other PRD1 proteins?

While specific structural information about P34 is not directly available, comparative analysis with other PRD1 proteins can provide insights:

Predicted Structural Features:

• N-terminal Domain: Many PRD1 proteins like P5 and P31 have specialized N-terminal domains with DNA-binding capacity or protein-protein interaction functions. For instance, terminal proteins (TPs) of related phages possess N-terminal domains with sequence-independent DNA-binding capacity .

• C-terminal Domain: Some PRD1 proteins show distinct C-terminal domains with specialized functions, as seen in P5's host recognition domain .

• Secondary Structure Prediction: Based on patterns observed in other membrane-associated PRD1 proteins, P34 might contain:

  • Alpha-helical transmembrane segments if membrane-associated

  • Beta-sheet structures if involved in capsid formation

  • Coiled-coil motifs if participating in oligomerization

Functional Domain Prediction:
Based on the roles of other PRD1 proteins, P34 might contain domains involved in:

• DNA binding (similar to terminal proteins)

• Membrane association (like P32)

• Structural stabilization (similar to P30)

• Specialized vertex functions (like P2, P5, P31)

A comprehensive bioinformatic analysis combining sequence alignment, secondary structure prediction, and 3D modeling would be essential for characterizing P34's domains in the absence of experimental structures.

How does mutagenesis of PRD1 structural proteins affect viral assembly and infectivity?

Mutagenesis studies of PRD1 structural proteins have revealed critical insights into viral assembly and infectivity mechanisms:

Methodological Approach:

• Targeted Gene Disruption: Using targeted mutagenesis systems based on combined in vitro manipulation and in vivo recombination, as developed for the PRD1 genome .

• Phenotype Analysis:

  • Plaque formation assays

  • Electron microscopy to examine particle morphology

  • DNA packaging tests

  • Host range determination

Key Findings from Similar Proteins:

• P32 Disruption: Disruption of gene XXXII resulted in a mutant phenotype defective in phage reproduction. P32-deficient particles exhibited normal assembly but showed defects in phage DNA injection capability. Specifically, the phage membrane was unable to undergo structural transformation from spherical to tubular form, suggesting P32's role in DNA ejection rather than host cell penetration .

• P31 Function: Studies with PRD1 mutants lacking P31 (sus525) showed particles without vertex complexes, demonstrating its essential role in vertex formation .

• P5 Substitution: When P31 replaced P5 to form the penton, intact but non-infectious viral particles were produced due to the lack of the viral receptor-binding protein P2 that normally binds to P5 .

This methodological framework could be applied to investigate P34's role through similar disruption experiments, examining effects on assembly, structural integrity, and infection capability.

What is the role of PRD1 proteins in membrane transformation during infection?

PRD1 proteins play crucial roles in membrane transformation during infection, a process that might involve P34:

Mechanism of Membrane Transformation:

• Initial Recognition: The infection process begins with specific recognition of the receptor by adsorption protein P2 .

• Conformational Change Cascade: This interaction triggers conformational changes, resulting in P2 detachment from the particle and signaling other vertex proteins .

• Vertex Destabilization: The vertex becomes metastable, leading to release of peripentonal capsid protein trimers .

• Membrane Transformation: The phage membrane undergoes structural transformation from a spherical vesicle to a tubular form .

• DNA Injection: The vertex opening enables the membranous tube to protrude from the particle, facilitating DNA entry into the host .

Protein-Specific Contributions:

• P32: Essential for membrane transformation from spherical to tubular form during DNA ejection .

• Vertex Complex Proteins (P5/P31): Form the heteropentameric base that interacts with the viral membrane, indicating a possible mechanism for initiating structural changes during infection .

• P2: Host-recognition protein that triggers the initial conformational changes .

If P34 is membrane-associated, it might participate in this transformation process, potentially stabilizing intermediate structures or facilitating the required conformational changes.

What techniques are most effective for visualizing PRD1 protein localization during viral assembly and infection?

For visualizing PRD1 protein localization during viral assembly and infection, researchers employ multiple complementary imaging techniques:

Advanced Microscopy Approaches:

• Cryo-Electron Microscopy (Cryo-EM): Provides near-atomic resolution of viral structures. Applied to PRD1-related phage PR772, achieving 2.75 Å resolution of the virion and 2.3 Å resolution of the protein capsid .

• Cryo-Electron Tomography: Allows visualization of structural transitions during infection.

• Super-Resolution Fluorescence Microscopy:

  • PALM (Photoactivated Localization Microscopy)

  • STORM (Stochastic Optical Reconstruction Microscopy)

  • These techniques overcome the diffraction limit, enabling tracking of individual proteins.

• Correlative Light-Electron Microscopy (CLEM): Combines fluorescence and electron microscopy to track labeled proteins and correlate their locations with ultrastructural features.

Protein Labeling Strategies:

• Fluorescent Fusion Proteins: C- or N-terminal tagging with fluorescent proteins (ensuring functionality is maintained).

• Immunofluorescence with Specific Antibodies: For tracking native proteins without modifications.

• Click Chemistry Approaches: Using bioorthogonal reactions to label proteins with minimal structural disruption.

Research Findings:
Studies have shown that terminal proteins (TPs) of phages like φ29 and PRD1 associate with the host bacterial nucleoid independently of other viral-encoded proteins . Further analysis revealed that the TP recruits phage DNA polymerase to the bacterial nucleoid, and both proteins are later redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent manner . Similar approaches could be applied to study P34 localization.

How should researchers design experiments to determine if Protein P34 is essential for viral replication?

To determine if Protein P34 is essential for viral replication, researchers should implement a systematic experimental design:

Experimental Approach:

• Gene Disruption Strategy:

  • Insert a marker gene (like lacZα) into gene XXXIV, following methodologies used for other PRD1 genes

  • Create a complementation system with P34 expressed in trans

• Construction of Recombinant Phage:

  • Use recombination between wild-type genome and a plasmid carrying the disrupted gene XXXIV

  • Select recombinant phages using appropriate markers (e.g., blue/white screening if using lacZα)

• Phenotypic Analysis Pipeline:

• Complementation Testing:

  • Express P34 from an inducible plasmid in host cells

  • Assess whether viral replication is restored when P34 is provided in trans

This experimental design mirrors successful approaches used to characterize gene XXXII, where disruption revealed P32's essential role in phage reproduction despite not affecting particle assembly .

What are the challenges in discriminating between structural and functional roles of PRD1 proteins in the viral life cycle?

Discriminating between structural and functional roles of PRD1 proteins presents several methodological challenges:

Key Challenges:

• Dual Roles: Many phage proteins serve both structural and functional purposes. For example, P5 forms part of the vertex structure but also plays a functional role in host recognition .

• Interdependence: Disruption of one protein often affects the incorporation or function of others, making it difficult to isolate specific effects. As seen with P31, which is necessary for vertex complex formation .

• Assembly Prerequisites: Some proteins may be essential for assembly but not directly involved in structure, like scaffold proteins that are not present in mature virions.

• Technical Limitations:

  • Resolution limits in structural studies

  • Challenges in creating viable mutants for essential proteins

  • Difficulty in purifying membrane-associated proteins

Methodological Solutions:

• Temperature-Sensitive Mutants: Allow protein function at permissive temperatures but not at restrictive temperatures.

• Inducible Expression Systems: Control protein levels during different stages of infection.

• Domain-Specific Mutations: Target specific functional domains while preserving structural roles.

• In vitro Reconstitution: Assemble viral components in controlled conditions to assess structural contributions.

• Real-Time Imaging: Track protein dynamics during infection to distinguish between assembly and functional roles.

Researchers studying PRD1 have employed strategies like targeted mutagenesis systems based on combined in vitro manipulation and in vivo recombination to overcome challenges associated with the hydrophobic terminal proteins that complicate handling of the genome .

How can researchers compare evolutionary conservation of PRD1 Protein P34 across the Tectiviridae family?

To analyze evolutionary conservation of PRD1 Protein P34 across the Tectiviridae family, researchers should employ the following bioinformatic and experimental approaches:

Computational Analysis Protocol:

• Sequence Retrieval:

  • Obtain sequences of P34 homologs from all available Tectiviridae genomes

  • Include related phages like PR772, which shows high structural similarity to PRD1

• Multiple Sequence Alignment (MSA):

  • Use algorithms specifically optimized for viral proteins (MUSCLE, MAFFT)

  • Manually curate alignments to account for insertions/deletions

• Conservation Analysis:

  • Calculate sequence identity and similarity percentages

  • Identify highly conserved residues and motifs

  • Generate conservation scores for each position

• Phylogenetic Analysis:

  • Construct phylogenetic trees using maximum likelihood or Bayesian methods

  • Compare P34 evolution to whole-genome phylogeny to identify potential horizontal gene transfer events

Structural Comparison Approach:

• Homology Modeling:

  • Create structural models of P34 homologs based on related phage proteins

  • Compare predicted secondary and tertiary structures

• Structural Alignment:

  • Align predicted structures to identify conserved structural elements

  • Calculate RMSD values between structural models

Experimental Validation:

• Complementation Experiments:

  • Test if P34 homologs from different Tectiviridae can complement P34-deficient PRD1

  • Assess functional conservation through phenotypic rescue

• Domain Swapping:

  • Create chimeric proteins with domains from different P34 homologs

  • Determine which domains are functionally interchangeable

This systematic approach would reveal the evolutionary pressures acting on P34 and identify which regions are essential for function across the Tectiviridae family.

What are common pitfalls when attempting to express recombinant PRD1 proteins in E. coli, and how can they be addressed?

Expressing recombinant PRD1 proteins in E. coli presents several challenges, particularly with membrane-associated or structural proteins:

Common Expression Challenges and Solutions:

PRD1-Specific Considerations:
For PRD1 proteins, researchers have successfully employed specialized approaches:

• The conventional mutagenesis systems based on in vitro manipulation of isolated DNA are challenging due to the covalently attached priming proteins at both 5′ termini of the PRD1 genome, which complicate handling .

• Targeted mutagenesis systems combining in vitro manipulation and in vivo recombination have been developed specifically for the PRD1 genome to overcome these challenges .

• For membrane-associated proteins (which P34 might be), successful expression might require:

  • Detergent screening to identify optimal solubilization conditions

  • Nanodisc or liposome reconstitution for functional studies

  • Co-expression with other viral proteins that might form functional complexes

How should researchers interpret conflicting results between in vitro and in vivo studies of PRD1 protein function?

When faced with conflicting results between in vitro and in vivo studies of PRD1 protein function, researchers should apply the following interpretative framework:

Sources of Discrepancies:

• Contextual Differences:

  • In vivo systems include the complete viral and cellular machinery

  • In vitro systems may lack essential cofactors or interacting partners

• Concentration Effects:

  • Non-physiological protein concentrations in vitro may drive non-native interactions

  • In vivo concentration gradients and compartmentalization affect function

• Post-translational Modifications:

  • In vivo modifications may be absent in in vitro systems

  • Bacterial expression might lack modifications present in natural host

• Structural Constraints:

  • Proteins may adopt different conformations in crowded cellular environments

  • Membrane proteins particularly sensitive to lipid environment differences

Methodological Approach to Resolution:

• Validation Through Multiple Techniques:

  • Confirm in vitro findings with multiple biochemical approaches

  • Validate in vivo observations through genetic complementation

• Bridging Experiments:

  • Develop semi-in vitro systems (cell extracts, permeabilized cells)

  • Reconstitute minimal systems with purified components

• Structural Verification:

  • Use structural techniques (cryo-EM, X-ray crystallography) to verify protein conformations

  • Compare structures in different environments

Case Study Application:
For PRD1 proteins, in vitro studies of DNA replication mechanisms have been extensive, while in vivo organization of the proteins involved has been less well-characterized . Research bridging this gap showed that terminal proteins (TPs) associate with the host bacterial nucleoid independently of other viral-encoded proteins in vivo, providing context for interpreting previous in vitro findings .

What strategies can overcome challenges in detecting low-abundance viral proteins like P34 in infected cells?

Detecting low-abundance viral proteins in infected cells requires specialized techniques to enhance sensitivity and specificity:

Advanced Detection Strategies:

• Enrichment Techniques:

  • Subcellular Fractionation: Isolate relevant cellular compartments where P34 is expected to localize

  • Immunoprecipitation: Use antibodies against predicted interacting partners

  • Density Gradient Centrifugation: Separate viral components based on density

• High-Sensitivity Protein Detection:

  • Selected Reaction Monitoring (SRM): Targeted mass spectrometry for specific peptides

  • Parallel Reaction Monitoring (PRM): Higher specificity variation of SRM

  • Digital ELISA: Single-molecule detection of proteins

• Signal Amplification Methods:

  • Tyramide Signal Amplification (TSA): Enhances immunodetection sensitivity

  • Proximity Ligation Assay (PLA): Detects protein-protein interactions with single-molecule sensitivity

  • Click Chemistry: Metabolic labeling of newly synthesized proteins

• Genetic Engineering Approaches:

  • Epitope Tagging: Add detectable tags that don't disrupt function

  • Inducible Overexpression: Controlled expression to facilitate detection

  • Fluorescent Protein Fusion: For live-cell imaging with signal accumulation

Optimization Parameters:

Temporal Considerations:
Studies of phage PRD1 have shown that viral proteins redistribute during infection. For example, terminal proteins recruit phage DNA polymerase to the bacterial nucleoid, and both are later redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent manner . Therefore, time-course experiments with multiple sampling points are crucial for detecting transient or redistributed proteins.

How might structural studies of PRD1 P34 inform the development of phage-based biotechnological applications?

Structural characterization of PRD1 P34 could significantly advance phage-based biotechnological applications through multiple pathways:

Potential Biotechnological Applications:

• Phage Display Platform Enhancement:

  • If P34 is involved in capsid formation, understanding its structure could allow for strategic modifications to display foreign peptides

  • Rational design of insertion sites based on structural data could improve display efficiency

• Targeted Delivery Systems:

  • If P34 participates in host recognition or membrane transformation, its structure could inform the development of phage-based delivery vehicles

  • Structural knowledge could guide modifications to alter host range or delivery efficiency

• Antimicrobial Development:

  • Structural insights into P34's role in infection might reveal targets for inhibiting phage-resistant bacterial strains

  • Understanding structural interactions could lead to novel antimicrobial peptides derived from functional domains

• Nanotechnology Applications:

  • PRD1's unique structure with internal membrane has applications in nanoparticle design

  • P34 structural data could inform the engineering of stable virus-like particles with customized properties

Research Methodology for Structural Characterization:

• Integrated Structural Biology Approach:

  • X-ray crystallography of purified P34

  • Cryo-EM of P34 in context of the viral particle

  • NMR for dynamic regions and interactions

  • Molecular dynamics simulations to predict functional movements

• Structure-Function Correlation:

  • Site-directed mutagenesis guided by structural data

  • Functional assays to correlate structural features with specific activities

  • Interaction mapping with other viral and host proteins

The structural characterization methodology would build upon successful approaches used for related phages. For example, electron cryo-microscopy of bacteriophage PR772 achieved 2.75 Å resolution of the virion and 2.3 Å resolution of the protein capsid, revealing the composition and structure of the vertex complex along with protein conformations crucial for maintaining capsid architecture .

What experimental approaches would best determine if P34 participates in phage DNA replication or packaging?

To determine if P34 participates in phage DNA replication or packaging, researchers should implement a multi-faceted experimental approach:

DNA Replication Investigation:

• In Vitro Replication Assays:

  • Reconstitute minimal replication system with purified components

  • Assess if adding purified P34 affects replication efficiency

  • Use template DNA containing PRD1 terminal sequences

• DNA Binding Studies:

  • Electrophoretic mobility shift assays (EMSA) with labeled DNA

  • Surface plasmon resonance to measure binding kinetics

  • Identify sequence preferences through SELEX or similar methods

• Protein Interaction Mapping:

  • Co-immunoprecipitation with known replication proteins

  • Yeast two-hybrid screening against replication machinery components

  • Crosslinking mass spectrometry to identify interaction interfaces

DNA Packaging Investigation:

• In Vitro Packaging Systems:

  • Establish cell-free packaging assays with procapsids

  • Test if P34 antibodies inhibit packaging

  • Assess packaging efficiency with and without P34

• Real-Time Imaging:

  • Fluorescently label DNA and track packaging in the presence or absence of P34

  • Use high-speed atomic force microscopy to visualize packaging dynamics

• Structural Localization:

  • Immuno-electron microscopy to localize P34 in particles

  • Cryo-EM reconstruction of particles at different stages of packaging

Functional Genetics Approach:

Research on related phage proteins provides methodological precedents. For instance, studies of terminal proteins (TPs) in phages φ29 and PRD1 showed they associate with the host bacterial nucleoid independently of other viral proteins, with the TP N-terminal domain possessing sequence-independent DNA-binding capacity essential for efficient DNA replication .

How might comparative studies between PRD1 P34 and related proteins in other phages advance our understanding of Tectiviridae evolution?

Comparative studies between PRD1 P34 and related proteins in other phages could provide significant insights into Tectiviridae evolution through several research approaches:

Evolutionary Analysis Framework:

• Comprehensive Homology Search:

  • Beyond obvious homologs in closely related phages

  • Profile-based searches to identify distant relatives

  • Structure-based homology detection for proteins with low sequence similarity

• Functional Conservation Assessment:

  • Cross-complementation experiments between different phages

  • Domain-swapping to identify functionally equivalent regions

  • Biochemical activity comparison of purified homologs

• Structural Conservation Analysis:

  • Compare structures of homologous proteins across evolutionary distance

  • Identify conserved structural motifs despite sequence divergence

  • Map evolutionary changes onto structural models

Specific Research Questions to Address:

• Gene Acquisition Patterns:

  • Is P34 conserved across all Tectiviridae or restricted to specific lineages?

  • Does P34 show evidence of horizontal gene transfer from other viral families?

  • Are there structural adaptations in P34 correlating with host range?

• Structural-Functional Evolution:

  • Which domains of P34 show highest conservation (functional constraints)?

  • Which regions show highest variability (host adaptation, immune evasion)?

  • How does structural conservation compare to sequence conservation?

• Co-evolution Patterns:

  • Does P34 co-evolve with interacting partners in the viral particle?

  • Is there evidence of compensatory mutations between P34 and other proteins?

  • How do evolutionary rates of P34 compare to other structural proteins?

Methodological Approach:
Research has already established valuable approaches for comparative studies in PRD1 and related phages. For example, electron cryo-microscopy of bacteriophage PR772 (a PRD1 relative) revealed that the penton base is an asymmetric heteropentamer consisting of three copies of P5 and two copies of P31, different from previous predictions for PRD1 . This type of comparative structural analysis between related phages provides a model for studying P34 evolution.

Additionally, researchers have compared proteins between PRD1 and phage φ29, showing that despite infecting distantly related bacteria (Bacillus subtilis and Escherichia coli), their terminal proteins share functional properties in nucleoid association and DNA replication . This cross-phage functional comparison approach could be productively applied to P34 studies.