Recombinant Delftia acidovorans Phasin (phaP)

What is Delftia acidovorans phasin (phaP) and what is its primary role in bacterial cells?

Delftia acidovorans phasin belongs to the broader family of phasin proteins (PhaPs), which are PHA granule-associated proteins that localize on the surfaces of polyhydroxyalkanoate granules in bacterial cells. Like other characterized phasins, the primary function of D. acidovorans phaP is likely control of surface properties of PHA granules. These proteins bind strongly to the hydrophobic surfaces of growing PHA granules to block the binding of other proteins, thereby influencing granule development through hydrophobic aggregation of small granules .

Typical phasin functions include:

• Preventing coalescence of PHA granules

• Controlling granule size and number

• Stabilizing the PHA granule interface

• Potential modulation of PHA synthase activity

• Preventing non-specific protein binding to granules

How does D. acidovorans phasin compare structurally to other well-characterized phasins?

While the search results don't provide specific structural characteristics of D. acidovorans phasin, comparisons with other phasins provide context. Most phasins, including those from Ralstonia eutropha (PhaP1 Re), have a molecular mass of approximately 20 kDa. In contrast, Aeromonas caviae phasin (PhaP Ac) is a smaller protein with a molecular mass of 13 kDa and shows low sequence identity (13.1%) to PhaP1 Re .

Given that D. acidovorans PHA synthase (PhaC Da) is a class I synthase with a unique insertion sequence of 40 amino acids located at the C-terminus of the active center cysteine, and has relatively low amino acid sequence identity to PhaC Re (50.7%) and higher similarity to PhaC Ac (31.1%), its corresponding phasin may have distinctive structural features that reflect its species-specific functions .

What genetic and genomic context surrounds the phaP gene in D. acidovorans?

D. acidovorans has a single circular chromosome of 6,685,842 bp with 66.7% G+C content, containing 6,028 predicted genes (5,931 protein-encoding with 4,425 assigned to putative functions) . While the specific genetic organization of the PHA metabolism genes in D. acidovorans isn't detailed in the search results, in many bacteria, PHA-related genes are often clustered in operons or functional gene clusters.

The D. acidovorans genome contains various biodegradation pathways, including those for aromatic compounds like phenanthrene and benzoate . These metabolic capabilities may interact with PHA metabolism, as breakdown products can potentially serve as substrates for PHA synthesis.

What expression systems are most effective for recombinant D. acidovorans phasin production?

Based on successful approaches with related phasins, the following expression systems would be appropriate for D. acidovorans phasin:

When designing expression constructs, researchers should consider:

• Affinity tags (His, GST, MBP) for purification

• Tag position (N or C-terminal) to minimize functional interference

• Codon optimization for the expression host

• Inducible promoter systems for controlled expression

What purification strategies yield the highest purity and activity for recombinant D. acidovorans phasin?

While the search results don't provide specific purification protocols for D. acidovorans phasin, successful purification strategies for similar proteins typically follow this workflow:

• Initial clarification:

  • Cell lysis under conditions that maintain phasin solubility

  • Centrifugation to remove cellular debris

• Primary capture:

  • Affinity chromatography (Ni-NTA for His-tagged constructs)

  • Ion exchange chromatography based on phasin's isoelectric point

• Polishing steps:

  • Size exclusion chromatography to remove aggregates

  • Additional ion exchange step if necessary

Critical considerations include:

• Buffer composition to maintain solubility (may require detergents or salt)

• Phasin's potential to associate with hydrophobic cellular components

• Activity assays to confirm functional integrity after purification

For in vitro activity studies, similar approaches to those used for A. caviae phasin could be employed, including polymerization activity assays with purified PHA synthase .

What analytical methods are most effective for characterizing recombinant D. acidovorans phasin?

Multiple complementary techniques should be employed:

Structural Characterization:

Functional Characterization:

• PHA binding assays to quantify granule interaction

• Surface plasmon resonance for binding kinetics

• In vitro polymerization activity assays to assess effects on PhaC activity

• Fluorescence microscopy with labeled phasin to visualize PHA interactions

Advanced Imaging Techniques:

• Confocal laser scanning microscopy (CLSM) and scanning transmission X-ray microscopy (STXM), which have been successfully used to study D. acidovorans biofilms, could be adapted for phasin-PHA interaction studies .

How does D. acidovorans phasin affect PHA synthase activity and substrate specificity?

Studies with A. caviae phasin (PhaP Ac) demonstrated two key findings:

• PhaP Ac significantly activated its cognate PHA synthase (PhaC Ac), increasing activity 3.0-fold when added at the beginning of polymerization

• PhaP Ac decreased the activities of non-cognate PHA synthases, including PhaC Re and PhaC Da, by approximately 10-fold

This suggests species-specific interactions between phasins and PHA synthases. Additionally, PhaP-activated PhaC Ac showed a slight shift in substrate preference toward 3-hydroxyhexanoyl-CoA (C6) .

Similar experiments could determine whether D. acidovorans phasin:

• Activates its cognate PhaC Da

• Influences substrate specificity

• Shows different effects depending on when it's added during polymerization

What molecular mechanisms explain how phasins modulate PHA granule formation?

Based on the general understanding of phasin function and the specific findings for A. caviae phasin, several mechanisms likely apply to D. acidovorans phasin:

• Direct enzyme activation: Phasins may interact directly with PHA synthase to enhance catalytic efficiency, as demonstrated by the 3.0-fold activation of PhaC Ac by PhaP Ac .

• Surface modification: By binding to nascent PHA granules, phasins modify surface properties, preventing non-specific protein interactions and granule coalescence.

• Substrate channeling: Phasins might facilitate the delivery of hydrophobic substrates to the PHA synthase active site, potentially explaining the shift in substrate preference observed with PhaP Ac .

• Conformational effects: Phasin binding may induce favorable conformational changes in PHA synthase, enhancing its catalytic properties.

Further studies using techniques like protein-protein interaction assays, site-directed mutagenesis, and structural biology approaches would help elucidate the specific mechanisms for D. acidovorans phasin.

How does recombinant expression of D. acidovorans phasin affect PHA production in heterologous hosts?

While specific data for D. acidovorans phasin isn't available in the search results, findings with A. caviae phasin provide valuable insights:

• Expression of PhaP Ac in E. coli TOP10 increased PHA production by up to 2.3-fold in strains expressing the cognate PhaC Ac

• PHA production was only slightly increased in strains expressing the non-cognate PhaC Re

This suggests that the most significant enhancement occurs with cognate phasin-synthase pairs. Similar experiments co-expressing D. acidovorans phasin with its cognate PHA synthase in heterologous hosts would likely reveal comparable effects.

Key experimental design considerations include:

• Optimization of expression levels for both phasin and synthase

• Appropriate carbon sources for PHA accumulation

• Analysis of PHA quantity and composition

How do the functional properties of D. acidovorans phasin compare to phasins from other bacterial species?

The search results provide comparative data that allows us to position D. acidovorans phasin within the context of other phasins:

The low sequence identity between A. caviae and R. eutropha phasins (13.1%) suggests considerable diversity among phasins from different bacterial species. Given the evolutionary relationships between these bacteria, D. acidovorans phasin likely has unique properties that reflect its specific role in PHA metabolism.

What are the key differences in experimental approaches when working with D. acidovorans phasin compared to other phasins?

Based on the properties of D. acidovorans and related species from the search results:

• Cultivation conditions: D. acidovorans has been studied under various conditions, including phenanthrene degradation , which may influence PHA metabolism and phasin expression. Researchers should consider these metabolic capabilities when designing experiments.

• PHA composition: D. acidovorans PHA synthase (PhaC Da) is a class I synthase with unique properties , potentially producing PHAs with different compositions than other bacteria. Analytical methods should be optimized accordingly.

• Biofilm formation: D. acidovorans forms biofilms that have been studied using advanced microscopy techniques . The potential role of phasin in biofilm formation could be a unique research direction.

• Environmental adaptation: Given D. acidovorans' ability to degrade various pollutants , its PHA metabolism may be adapted to specific environmental niches, potentially influencing phasin function.

What contradictory findings exist in the literature regarding phasin function that might apply to D. acidovorans phasin?

The search results highlight a potentially contradictory finding:

• PhaP Ac activates its cognate PhaC Ac but inhibits non-cognate PHA synthases including PhaC Da

This species-specific effect raises questions about the evolutionary divergence of phasin function and the molecular basis for these opposing effects. For D. acidovorans phasin research, this suggests:

• The need to test both activation and inhibition effects on various PHA synthases

• Potential complex interactions when multiple phasins are present

• Evolutionary adaptation of phasin-synthase interactions within specific metabolic contexts

What experimental design considerations are critical when investigating D. acidovorans phasin interactions with PHA synthase?

Based on successful approaches used with A. caviae phasin , researchers should consider:

• Timing of interaction studies:

  • Effect of adding phasin at different stages (prepolymerization vs. during elongation)

  • Time-resolved studies to capture dynamic interactions

• Substrate diversity:

  • Testing multiple hydroxyalkanoyl-CoA substrates (C4-C6)

  • Examining potential shifts in substrate preference

• Protein variants:

  • Wild-type vs. tagged constructs

  • Truncated versions to identify functional domains

  • Site-directed mutants to probe specific interactions

• Assay conditions:

  • Buffer composition optimization

  • Enzyme:phasin ratio variation

  • Temperature and pH optimization

The finding that PhaP Ac activated polymer-elongating PhaC Ac but not PhaC Re highlights the importance of testing different experimental conditions to fully characterize phasin effects.

What advanced analytical techniques provide the most insight into D. acidovorans phasin structure-function relationships?

To thoroughly characterize D. acidovorans phasin:

• Structural analysis:

  • X-ray crystallography or cryo-electron microscopy for high-resolution structure

  • Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

  • SAXS for solution behavior and conformational changes upon binding

• Interaction studies:

  • Cross-linking mass spectrometry to identify interaction interfaces

  • Microscale thermophoresis for binding affinity measurements

  • Förster resonance energy transfer (FRET) for real-time interaction dynamics

• Advanced microscopy:

  • Super-resolution microscopy to visualize phasin distribution on PHA granules

  • Scanning transmission X-ray microscopy (STXM), which has been successfully applied to D. acidovorans biofilms

  • Atomic force microscopy to examine phasin effects on PHA surface properties

• Computational approaches:

  • Molecular dynamics simulations of phasin-PHA interactions

  • Protein-protein docking to predict phasin-synthase binding modes

  • Evolutionary analysis to identify conserved functional motifs

How can researchers effectively isolate the specific effects of D. acidovorans phasin from other cellular factors?

Designing controlled experiments requires:

• In vitro reconstitution systems:

  • Purified components (phasin, PHA synthase, substrates)

  • Synthetic PHA granules or model hydrophobic surfaces

  • Defined buffer conditions to eliminate cellular variables

• Genetic approaches in vivo:

  • Clean deletion mutants of phaP in D. acidovorans

  • Complementation with wild-type and mutant phasin variants

  • Heterologous expression in minimal genetic backgrounds

• Control experiments:

  • Other phasins (PhaP Ac, PhaP1 Re) as comparative controls

  • Non-phasin hydrophobic proteins as specificity controls

  • Careful titration of phasin concentrations

• Time-resolved studies:

  • Inducible expression systems for temporal control

  • Sampling at multiple time points during PHA accumulation

  • Real-time monitoring of granule formation

What are common pitfalls in purifying active recombinant D. acidovorans phasin, and how can they be overcome?

Although specific challenges for D. acidovorans phasin aren't detailed in the search results, common issues with similar proteins include:

• Solubility challenges:

  • Use solubility-enhancing fusion partners (MBP, SUMO)

  • Optimize expression temperature (typically lower temperatures improve solubility)

  • Include mild detergents or higher salt concentrations in purification buffers

• Non-specific binding:

  • Phasins inherently bind hydrophobic surfaces, which can complicate purification

  • Include washing steps with low concentrations of non-ionic detergents

  • Use high-salt washes to reduce non-specific ionic interactions

• Activity loss during purification:

  • Minimize freeze-thaw cycles

  • Include stabilizing agents in storage buffers

  • Validate activity after each purification step

• Aggregation during concentration:

  • Use gradual concentration methods

  • Include glycerol or other stabilizing agents

  • Monitor aggregation state by dynamic light scattering

How can researchers troubleshoot inconsistent results in D. acidovorans phasin functional assays?

Based on the variability observed in phasin studies :

• Protein quality control:

  • Verify protein homogeneity by size exclusion chromatography

  • Check for degradation products by SDS-PAGE

  • Validate correct folding by circular dichroism

• Assay standardization:

  • Standardize enzyme:phasin ratios

  • Control the timing of phasin addition precisely

  • Maintain consistent substrate quality and concentration

• Technical variability:

  • Include internal controls in each experiment

  • Use technical replicates to assess method reproducibility

  • Perform biological replicates with independently prepared proteins

• Data analysis:

  • Apply appropriate statistical methods

  • Account for lag phases in polymerization kinetics

  • Consider biphasic behavior in activation/inhibition studies

Studies with A. caviae phasin showed that timing was critical—adding phasin at the beginning of polymerization versus during elongation produced different effects .

What strategies can overcome technical challenges in studying D. acidovorans phasin in vivo?

Building on approaches used for studying D. acidovorans in biofilms and other contexts :

• Genetic manipulation challenges:

  • Optimize transformation methods specific to D. acidovorans

  • Develop or adapt genetic tools (plasmids, transposons)

  • Use inducible promoters for controlled expression

• Detection and visualization:

  • Develop specific antibodies against D. acidovorans phasin

  • Create fluorescent protein fusions that maintain function

  • Adapt STXM and CLSM protocols used for D. acidovorans biofilms

• Metabolic complexity:

  • Control carbon source availability to direct metabolism

  • Consider the impact of D. acidovorans' degradative capabilities on PHA accumulation

  • Monitor metabolic state using appropriate markers

• Biofilm considerations:

  • Account for biofilm formation in experimental design

  • Develop protocols for PHA analysis in biofilm context

  • Consider spatial heterogeneity in biofilm samples