Recombinant Pseudomonas phage Pf1 Uncharacterized protein ORF424

How does ORF424 protein fit within the evolutionary context of Pseudomonas filamentous phages?

ORF424 is a component of Pseudomonas phage Pf1, which belongs to evolutionary lineage I of Pseudomonas filamentous (Pf) bacteriophages. Phylogenetic analysis has revealed that Pf phages exist in two distinct evolutionary lineages (I and II) with different structural and morphogenesis properties despite sharing integration sites in host chromosomes . The most studied Pf phages (Pf1, Pf4, and Pf5) all belong to lineage I.

Comparative genomic analysis between Pf1, Pf4, and Pf5 shows conserved synteny and varying degrees of sequence identity, with Pf5 being intermediate in size (10,675 bp) between Pf1 (7,349 bp) and Pf4 (12,437 bp) . The ORF424 gene has homologs in other Pf phages, corresponding to PA0726 in PAO1 strain (Pf4 phage) and PA14_48910 in PA14 strain (Pf5 phage) .

What storage and handling protocols optimize ORF424 protein stability for experimental use?

For optimal stability and activity of recombinant ORF424 protein, follow these evidence-based storage and handling protocols:

• Upon receipt, briefly centrifuge the vial to bring contents to the bottom

• Reconstitute the lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended)

• Aliquot for long-term storage at -20°C/-80°C to avoid repeated freeze-thaw cycles

• For short-term use, working aliquots can be stored at 4°C for up to one week

• Storage buffer should be Tris/PBS-based with 6% Trehalose at pH 8.0

Repeated freeze-thaw cycles significantly reduce protein activity and should be strictly avoided. When handling the protein for experiments, maintain cold chain protocols whenever possible.

What are the recommended methods for detecting ORF424 in clinical Pseudomonas aeruginosa isolates?

For detecting ORF424 and related Pf phage sequences in clinical isolates, a PCR-based approach has been validated. Universal primers targeting a conserved region of the ORF424 gene and its homologs can identify Pf-like phages regardless of the specific variant. For more specific detection, phage-specific primers can distinguish between Pf1, Pf4, and Pf5.

Universal Pf Phage PCR Protocol:
PCR amplification using primers targeting the conserved Zot-toxin-like gene (ORF424 in Pf1, PA0726 in PAO1, and PA14_48910 in Pf5) can detect the presence of any of these filamentous phages .

For comprehensive detection and differentiation of Pf phages in clinical isolates, researchers should implement a multiplex PCR approach using the following primer sets:

A combined protocol using universal Pf primers along with Pf4-specific primers at 53°C annealing temperature has shown reliable results in differentiating Pf phage types .

What expression systems yield highest activity and purity of recombinant ORF424 protein?

E. coli expression systems have proven effective for producing recombinant ORF424 protein with high yield and purity. The optimal expression protocol includes:

• Cloning the full-length ORF424 sequence (1-424 amino acids) fused to an N-terminal His-tag

• Expression in E. coli using appropriate induction parameters

• Purification using affinity chromatography targeting the His-tag

• Quality assessment using SDS-PAGE (should achieve >90% purity)

• Formulation in Tris/PBS-based buffer with 6% Trehalose at pH 8.0

This approach consistently yields functional protein with purity greater than 90% as determined by SDS-PAGE analysis.

How can researchers effectively analyze ORF424's role in biofilm formation?

To investigate ORF424's role in biofilm formation, researchers should implement a multifaceted experimental approach:

• Genetic manipulation studies:

  • Generate ORF424 deletion mutants in Pseudomonas strains

  • Create overexpression constructs with inducible promoters

  • Develop complementation systems to verify phenotypes

• Biofilm assessment protocols:

  • Crystal violet staining for quantitative biofilm mass measurement

  • Confocal laser scanning microscopy for structural analysis

  • Flow cell systems for dynamic biofilm development assessment

• Comparative analysis:

  • Test biofilm formation in wild-type vs. mutant strains

  • Evaluate biofilm characteristics under various environmental conditions

  • Assess impact of exogenously added purified ORF424 protein

Studies have shown that Pf phages significantly increase biofilm mass formation not only in P. aeruginosa but also in various bacteria and fungi including Candida albicans . The experimental design should include controls to differentiate direct protein effects from those mediated by the intact phage structure.

What is the proposed functional role of ORF424 in Pseudomonas phage biology?

• Structural role: As a component of the filamentous phage, ORF424 may contribute to phage assembly or stability

• Host interaction: The Zot-toxin similarity suggests potential involvement in modifying host cell properties

• Phage replication: May participate in the replication cycle of the phage within the host

Further functional characterization through domain analysis, structural studies, and interaction mapping is needed to fully elucidate ORF424's specific role in phage biology.

How does ORF424 and other Pf phage proteins contribute to Pseudomonas pathogenicity?

Filamentous phages like Pf1 containing ORF424 contribute significantly to Pseudomonas aeruginosa pathogenicity through several mechanisms:

• Biofilm enhancement: Pf phages promote biofilm formation through interaction with host and bacterial biopolymers (mucin, actin, DNA, and glycosaminoglycans) to assemble highly structured liquid crystals that enhance biofilm adhesion .

• Antibiotic tolerance: The high negative charge density of Pf phages allows them to sequester cationic antibacterial agents, including aminoglycoside antibiotics and host antimicrobial peptides, potentially contributing to antibiotic resistance .

• Immune evasion: P. aeruginosa producing Pf phages at levels comparable to those in biofilms is less invasive, less inflammatory, and more resistant to phagocytosis by macrophages, suggesting a role in immune evasion that facilitates chronic infections .

• Small colony variant (SCV) formation: Filamentous phages like Pf4 (related to Pf1) mediate the formation of small-colony variants in biofilms, which are associated with antibiotic resistance and persistence .

The specific contribution of ORF424 to these pathogenicity mechanisms requires further investigation, but as a component of the Pf1 phage, it is likely involved in at least some of these processes.

What is the relationship between ORF424 and small colony variant (SCV) formation in Pseudomonas biofilms?

While direct evidence linking ORF424 specifically to small colony variant (SCV) formation is limited, research has established that filamentous phages containing this protein play crucial roles in SCV development:

• Filamentous bacteriophage Pf4 (related to Pf1 containing ORF424) mediates the formation of SCVs in P. aeruginosa PAO1 biofilms .

• SCVs represent a morphology type associated with antibiotic resistance and persistence in biofilm infections.

• Studies have detected the production of replicative form (RF) of Pf5 phage in both wild-type and SCV bacteria , suggesting active phage replication occurring in both phenotypes.

• The mechanism may involve phage-induced alterations in gene expression, cell surface properties, or signaling pathways.

For researchers investigating this relationship, experimental approaches should include:

• Comparison of ORF424 expression levels between normal and SCV phenotypes

• Assessment of SCV formation rates in strains with modified ORF424 expression

• Evaluation of the impact of purified ORF424 protein on SCV induction

• Analysis of genetic and proteomic differences between wild-type and SCV phenotypes

How can researchers investigate interactions between ORF424 and host cellular components?

To elucidate interactions between ORF424 and host cellular components, implement these advanced methodological approaches:

• Protein-protein interaction studies:

  • Co-immunoprecipitation with tagged ORF424

  • Yeast two-hybrid screening against P. aeruginosa proteome

  • Bacterial two-hybrid systems for in vivo interaction validation

  • Pull-down assays with purified ORF424 as bait

• Localization studies:

  • Immunofluorescence microscopy with anti-ORF424 antibodies

  • Fluorescent protein fusions for real-time tracking

  • Subcellular fractionation followed by Western blotting

  • Electron microscopy with immunogold labeling

• Functional validation:

  • CRISPR interference to modulate expression of potential interactors

  • Site-directed mutagenesis of key ORF424 domains

  • Competition assays with peptide fragments

  • Heterologous expression systems to isolate specific interactions

The high negative charge density of Pf phages suggests potential interactions with cationic host molecules . Research should prioritize investigating interactions with antimicrobial peptides, DNA-binding proteins, and membrane components to understand the protein's role in pathogenicity and biofilm formation.

What approaches can resolve the three-dimensional structure of ORF424 protein?

Determining the three-dimensional structure of ORF424 requires a multi-technique approach:

• X-ray crystallography:

  • Express and purify large quantities of ORF424 (>5mg)

  • Screen multiple crystallization conditions (pH, salt, precipitants)

  • Optimize crystal growth for high-resolution diffraction

  • Consider heavy atom derivatives for phasing

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Produce isotopically labeled protein (13C, 15N)

  • Perform sequential assignment of backbone and side-chain resonances

  • Collect distance restraints through NOE experiments

  • Generate solution structure through computational modeling

• Cryo-electron microscopy:

  • Particularly useful if ORF424 forms oligomeric structures

  • Prepare vitrified samples on appropriate grids

  • Collect high-resolution image data

  • Perform 3D reconstruction and model building

• Computational approaches:

  • Homology modeling based on related Zot-toxin structures

  • Ab initio protein folding algorithms

  • Molecular dynamics simulations for flexibility analysis

  • Integrated modeling combining experimental constraints

Since ORF424 has been identified as a Zot-toxin-like protein , structural comparison with known Zot toxins may provide insights into functional domains and mechanisms of action.

How can researchers differentiate between the effects of ORF424 and other Pf phage proteins in experimental systems?

Differentiating the specific effects of ORF424 from other Pf phage proteins requires carefully designed experimental approaches:

• Genetic dissection:

  • Create targeted gene knockouts of ORF424 while maintaining other phage genes

  • Generate complementation constructs expressing only ORF424

  • Develop inducible expression systems for controlled ORF424 production

  • Design domain-swap chimeras to isolate functional regions

• Biochemical separation:

  • Purify recombinant ORF424 for use in isolation

  • Compare effects of whole phage particles versus purified ORF424

  • Use antibody-mediated neutralization to selectively block ORF424 function

  • Employ size exclusion chromatography to separate phage components

• Comparative analysis across phage variants:

  • Utilize natural variation in ORF424 sequences across Pf1, Pf4, and Pf5

  • Analyze effects in heterologous hosts expressing only ORF424

  • Compare phenotypes between strains carrying different Pf phage lineages

  • Examine cross-complementation between phage variants

• Temporal analysis:

  • Study the kinetics of ORF424 expression during phage infection

  • Monitor phenotypic changes in relation to ORF424 production timeline

  • Use pulse-chase experiments to track ORF424 throughout infection cycle

Research has shown that Pf phages exist as two evolutionary lineages with substantially different structural and morphogenesis properties , making comparative studies between lineages particularly valuable for isolating ORF424-specific effects.

How does ORF424 compare structurally and functionally to homologous proteins in other phages?

ORF424 belongs to a family of proteins found across filamentous Pseudomonas phages with varying degrees of conservation:

Comparative analysis between these homologs reveals:

• Conserved synteny across the phage genomes

• Differential expression patterns depending on growth phase and stress conditions

• Potential adaptation to different host strain backgrounds

The evolutionary conservation of this protein across different Pseudomonas phages suggests an important role in phage biology and host-phage interactions.

What is the prevalence of ORF424-containing phages across clinical Pseudomonas aeruginosa isolates?

The prevalence of filamentous phages containing ORF424 and related proteins in clinical isolates is significant and epidemiologically relevant:

Studies have employed PCR detection methods using universal primers targeting the conserved gene corresponding to ORF424 in Pf1, PA0726 in PAO1, and PA14_48910 in Pf5 . This universal approach allows researchers to detect the presence of any Pf-like phage regardless of the specific variant.

While comprehensive epidemiological data is still emerging, research suggests:

• Pf phages are widely distributed among clinical isolates of P. aeruginosa

• Biofilm-related infections (such as those from cystic fibrosis patients and catheter-associated infections) show particularly high prevalence of these phages

• The distribution varies between different clinical settings and geographical regions

For researchers studying the epidemiology of these phages, screening methodologies should include:

• PCR detection using both universal and phage-specific primers

• Analysis of small colony variants (SCVs) in primary isolation plates

• Correlation of phage presence with antibiotic resistance profiles

• Genomic sequencing to identify integrated prophage regions

How do environmental factors influence ORF424 expression and function in Pseudomonas biofilms?

Environmental factors significantly impact the expression and function of ORF424 and related phage components in Pseudomonas biofilms:

• Growth phase regulation:

  • Expression levels are highly growth-phase dependent

  • Stationary phase planktonic cells show elevated expression

  • Dynamic regulation throughout biofilm development stages

• Stress response activation:

  • Cell envelope stress induces transcriptional upregulation

  • Phage exposure triggers expression changes

  • Antibiotic exposure, particularly aminoglycosides, influences expression patterns

• Biofilm microenvironment effects:

  • Oxygen gradients within biofilms affect expression patterns

  • Nutrient limitation alters phage activity and protein production

  • Matrix component interactions modify protein function

• Host strain background influence:

  • Clinical isolates show different expression patterns compared to laboratory strains

  • Genetic background of the host affects phenotypic outcomes

  • Strain-specific regulatory networks modulate expression

These findings suggest that ORF424 functions within a complex regulatory network responsive to environmental cues and stress conditions. Researchers should consider these factors when designing experiments to study ORF424 function in different contexts.

What potential therapeutic applications could exploit ORF424 in combating Pseudomonas infections?

Understanding ORF424 and Pf phage biology opens several potential therapeutic avenues:

• Anti-biofilm strategies:

  • Targeting ORF424 or its interactions may disrupt biofilm formation

  • Modulating Pf phage activity could reduce biofilm stability

  • Combination approaches with conventional antibiotics may enhance penetration

• Immunomodulatory approaches:

  • Neutralizing antibodies against ORF424 might reduce immune evasion

  • Vaccine strategies targeting conserved epitopes across Pf phages

  • Restoring phagocytic clearance of Pseudomonas in chronic infections

• Diagnostic applications:

  • ORF424 detection as a biomarker for antibiotic resistance risk

  • Monitoring Pf phage activity to predict infection chronicity

  • Stratifying patients based on phage presence for personalized treatment

The high negative charge density of Pf phages and their ability to sequester cationic antibacterial agents suggests that neutralizing these phages might enhance the efficacy of aminoglycoside antibiotics and host antimicrobial peptides in treating resistant infections.

How can researchers optimize experimental systems to study ORF424's role in antibiotic resistance?

To effectively investigate ORF424's contribution to antibiotic resistance, implement these methodological approaches:

• Genetic manipulation systems:

  • Construct inducible expression systems for controlled ORF424 production

  • Generate knockout mutants in both laboratory and clinical isolates

  • Develop complementation systems with wild-type and mutant variants

• Resistance phenotype characterization:

  • Determine Minimum Inhibitory Concentrations (MICs) using standard protocols

  • Assess tolerance to different antibiotic classes, particularly aminoglycosides

  • Measure antibiotic penetration into biofilms with and without ORF424 expression

• Sequestration activity quantification:

  • Develop in vitro binding assays between purified ORF424 and antibiotics

  • Measure changes in antibiotic availability in the presence of the protein

  • Correlate binding capacity with resistance phenotypes

• Clinical correlation studies:

  • Compare ORF424 expression levels with antibiotic treatment outcomes

  • Analyze isolates from treatment failures for phage-related changes

  • Longitudinal sampling during antibiotic therapy to track evolutionary changes

Research has demonstrated that deletion of phage-related operons can alter tobramycin resistance in clinical isolates of P. aeruginosa , highlighting the importance of phage components in antibiotic resistance mechanisms.

What novel biotechnological applications might exploit ORF424's structural and functional properties?

The unique properties of ORF424 and Pf phages suggest several potential biotechnological applications:

• Biofilm engineering:

  • Controlled expression for modulating biofilm architecture in beneficial biofilms

  • Development of anti-biofilm coatings for medical devices

  • Creation of structured biofilms for bioremediation applications

• Drug delivery systems:

  • Engineering phage-based nanoparticles incorporating ORF424

  • Exploiting liquid crystal formation properties for controlled release

  • Targeting specific bacterial populations in polymicrobial contexts

• Protein scaffolding technology:

  • Using filamentous phage structure as nanoscale building blocks

  • Incorporation of ORF424 as functional domains in chimeric proteins

  • Development of self-assembling biomaterials with ordered structures

• Biosensing applications:

  • Detection of interaction partners in environmental samples

  • Monitoring phage activity in industrial or clinical settings

  • Development of reporter systems based on phage biology

The ability of Pf phages to form liquid crystal structures with highly organized architecture makes them particularly interesting for materials science applications requiring ordered nanoscale assemblies.