Recombinant Delftia acidovorans Acyl carrier protein (acpP)

What is Delftia acidovorans and what makes its Acyl carrier protein significant for research?

Delftia acidovorans is an aerobic, non-fermenting, Gram-negative bacillus belonging to the family Comamonadaceae within the Burkholderiales order of Betaproteobacteria . Although typically considered an environmental organism found in soil, water systems, and plant rhizospheres, it occasionally causes opportunistic infections in both immunocompromised and immunocompetent patients .

The Acyl carrier protein (acpP) in D. acidovorans, like other bacterial acpP proteins, likely plays a crucial role in fatty acid biosynthesis, serving as a shuttle that carries growing fatty acid chains between enzymatic domains. This protein would be particularly interesting for researchers studying bacterial metabolism, biotransformation capabilities, and potential applications in biodegradation.

D. acidovorans has gained scientific interest due to its remarkable metabolic versatility, including its ability to:

• Produce gold nanoparticles from gold ions via delftibactin production

• Degrade various environmental pollutants, including dichlorprop through the activity of enantiospecific α-ketoglutarate dependent dioxygenases

• Form biofilms with other bacteria like Cupriavidis metallidurans on gold nuggets

• Promote plant growth in rhizospheres

How does the structure of Delftia acidovorans acpP compare to acyl carrier proteins from other bacterial species?

While the specific structural details of D. acidovorans acpP are not directly presented in the provided sources, we can infer structural characteristics based on general bacterial acyl carrier protein biology.

Bacterial acyl carrier proteins typically:

• Are small, acidic proteins (approximately 8-10 kDa)

• Contain a conserved serine residue that becomes post-translationally modified with a 4'-phosphopantetheine prosthetic group

• Possess a four-helix bundle structure with the modified serine positioned in a flexible loop region

The structural conservation of acpP across bacterial species suggests that D. acidovorans acpP likely maintains these core features, though species-specific variations may exist that could influence its interaction with other enzymes in the fatty acid synthase complex or alternative metabolic pathways specific to D. acidovorans.

What expression systems are most effective for producing recombinant Delftia acidovorans acpP?

For researchers seeking to express recombinant D. acidovorans acpP, several expression systems may be considered:

E. coli-based expression systems:

• BL21(DE3) strains are typically preferred for their reduced protease activity

• Consider using pET vector systems with T7 promoters for high-yield expression

• Fusion tags such as His6, GST, or MBP can facilitate purification and may enhance solubility

Optimization considerations:

• Lower temperatures (16-25°C) during induction may improve protein folding

• IPTG concentration optimization (typically 0.1-1.0 mM) is recommended

• Testing multiple growth media (LB, TB, or defined media) can impact yield and quality

• Consider codon optimization if expression levels are low

What are the critical purification steps to obtain highly pure and active Delftia acidovorans acpP?

A robust purification strategy for D. acidovorans acpP would typically involve:

• Initial capture: Affinity chromatography

  • His-tag purification using Ni-NTA resin (if His-tagged construct)

  • GST-affinity chromatography (if GST-fusion is used)

• Intermediate purification:

  • Ion exchange chromatography (likely cation exchange given acpP's acidic nature)

  • Optional tag cleavage step using appropriate protease (TEV, thrombin, etc.)

• Polishing step:

  • Size exclusion chromatography to achieve high purity and remove aggregates

  • Buffer optimization to ensure stability (typically pH 7-8 with reducing agents)

• Quality control assessments:

  • SDS-PAGE for purity evaluation (>95% purity target)

  • Mass spectrometry to confirm protein identity and integrity

  • Circular dichroism to assess secondary structure

  • Activity assays to confirm functionality (phosphopantetheinylation capacity)

How can researchers verify the post-translational modification status of recombinant Delftia acidovorans acpP?

Verification of 4'-phosphopantetheine attachment to the acpP is crucial for functional studies. Recommended methodologies include:

Mass spectrometry approaches:

• MALDI-TOF MS to detect the mass shift (~340 Da) between apo- and holo-forms

• LC-MS/MS analysis following tryptic digestion to identify the modified peptide

• Top-down proteomics for intact protein analysis

Biochemical verification:

• Conformational gel shift assays (apo- vs. holo-forms often migrate differently)

• Phosphopantetheinyl transferase (PPTase) in vitro modification assays

• Radioactive labeling using [³H]-β-alanine or [¹⁴C]-pantothenic acid incorporation

Table 1: Comparison of Methods for acpP Post-translational Modification Analysis

What approaches can be used to study protein-protein interactions involving Delftia acidovorans acpP in fatty acid synthesis pathways?

To investigate the interactions between D. acidovorans acpP and other fatty acid synthesis enzymes, researchers can employ:

In vitro approaches:

• Pull-down assays using tagged acpP as bait

• Surface plasmon resonance (SPR) for real-time binding kinetics

• Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Crosslinking studies followed by mass spectrometry (XL-MS)

• Fluorescence resonance energy transfer (FRET) using fluorescently labeled proteins

In vivo approaches:

• Bacterial two-hybrid systems

• Co-immunoprecipitation followed by proteomics

• FRET or BRET in cellular contexts

• Proximity ligation assays

Structural biology approaches:

• X-ray crystallography of acpP with partner proteins

• NMR studies to map interaction interfaces

• Cryo-electron microscopy for larger complexes

How might Delftia acidovorans acpP be involved in the organism's unique metabolic capabilities, such as gold nanoparticle formation or xenobiotic degradation?

D. acidovorans possesses remarkable metabolic capabilities that may involve acpP either directly or indirectly:

Potential role in delftibactin biosynthesis:
While the delftibactin biosynthetic pathway involves a nonribosomal peptide synthetase (NRPS) cluster (delA-delP), acyl carrier proteins can sometimes interact with or complement NRPS systems . Researchers should investigate whether:

• acpP participates in loading or transferring intermediates within the delftibactin pathway

• The phosphopantetheine arm of acpP could potentially interact with delftibactin intermediates

• acpP expression levels correlate with delftibactin production

Connection to xenobiotic degradation:
D. acidovorans MC1 possesses specialized dioxygenases (RdpA and SdpA) for dichlorprop degradation . Researchers might explore:

• Whether fatty acid metabolism (involving acpP) provides precursors or energy for xenobiotic degradation

• If acpP expression is co-regulated with genes involved in biodegradation pathways

• The impact of acpP knockout or overexpression on biodegradation capabilities

What structural biology approaches are most promising for determining the three-dimensional structure of Delftia acidovorans acpP?

To determine the structure of D. acidovorans acpP, researchers should consider these approaches:

X-ray crystallography:

• Requires high-purity protein (>95%)

• Optimization of crystallization conditions (pH, ionic strength, temperature, precipitants)

• May benefit from surface entropy reduction mutations to promote crystal packing

• Consider apo- vs. holo-form crystallization attempts

NMR spectroscopy:

• Particularly suitable for smaller proteins like acpP

• Requires isotopic labeling (¹⁵N, ¹³C) for structural determination

• Can provide dynamic information not available from crystallography

• May reveal differences between apo- and holo-forms in solution

Cryo-electron microscopy:

• Generally more suitable for larger complexes

• Consider if studying acpP in complex with partner enzymes

• Recent advances make this viable for smaller complexes

Computational approaches:

• Homology modeling based on other bacterial acpP structures

• Molecular dynamics simulations to study conformational dynamics

• AlphaFold2 or similar AI-based structure prediction tools

What strategies can address poor solubility or stability of recombinant Delftia acidovorans acpP?

Researchers facing solubility or stability issues with recombinant D. acidovorans acpP should consider:

Improving solubility:

• Utilize solubility-enhancing fusion partners (MBP, SUMO, TrxA)

• Optimize buffer conditions (pH, salt concentration, additives like glycerol)

• Employ co-expression with chaperones (GroEL/ES, DnaK/J/GrpE)

• Consider refolding from inclusion bodies if necessary

Enhancing stability:

• Include reducing agents (DTT, β-mercaptoethanol) to prevent oxidation of cysteine residues

• Add protease inhibitors during purification

• Optimize storage conditions (temperature, buffer composition)

• Test the addition of stabilizing agents (glycerol, trehalose, sucrose)

Alternative approaches:

• Design truncated constructs if terminal regions contribute to instability

• Introduce stabilizing mutations based on homology to stable acpP proteins

• Consider co-expression with protein partners that may stabilize the structure

How can researchers overcome challenges in achieving the correct post-translational modification of recombinant Delftia acidovorans acpP?

Ensuring proper phosphopantetheinylation of recombinant acpP can be challenging. These approaches may help:

Co-expression strategies:

• Co-express acpP with a compatible phosphopantetheinyl transferase (PPTase)

• Try different PPTases (Sfp, AcpS) if one is ineffective

• Optimize expression conditions to improve PPTase activity

In vitro modification:

• Purify apo-acpP and modify using purified PPTase

• Optimize reaction conditions (Mg²⁺ concentration, pH, incubation time)

• Purify holo-acpP using additional chromatography steps

Advanced approaches:

• Use cell-free protein synthesis systems with PPTase supplementation

• Consider chemical methods for attaching phosphopantetheine analogs

• Explore enzymatic strategies using purified Coenzyme A biosynthetic enzymes

How can transcriptomic and proteomic data inform our understanding of Delftia acidovorans acpP regulation and function?

Systems biology approaches can provide valuable insights into acpP function:

Transcriptomic analyses:

• RNA-Seq to identify co-expressed genes under various conditions

• Compare expression patterns of acpP with fatty acid synthesis genes

• Investigate expression changes during growth on different carbon sources

• Examine transcriptional responses during gold ion exposure or xenobiotic degradation

Proteomic approaches:

• Quantitative proteomics to measure acpP abundance across conditions

• Phosphoproteomics to identify regulatory post-translational modifications

• Protein-protein interaction network mapping using affinity purification-mass spectrometry

• Protein turnover studies to understand acpP stability in vivo

Integration of data:

• Correlation analysis between acpP expression and metabolic pathways

• Pathway enrichment analysis to identify functional associations

• Construction of gene regulatory networks to identify transcriptional regulators

What genetic engineering approaches might be useful for studying the physiological role of acpP in Delftia acidovorans?

To elucidate the physiological importance of acpP, researchers might employ:

Gene knockout/knockdown strategies:

• CRISPR-Cas9 genome editing (if transformation protocols exist for D. acidovorans)

• Antisense RNA expression to reduce acpP levels

• Conditional knockouts using inducible promoters

Complementation and overexpression studies:

• Expression of heterologous acpP proteins to assess functional conservation

• Point mutations of key residues (especially the phosphopantetheine attachment site)

• Domain swapping with acpP from other species

Reporter systems:

• Translational fusions with fluorescent proteins to monitor expression and localization

• Promoter fusions to study transcriptional regulation

• Two-hybrid systems to identify interaction partners in vivo

Table 2: Advantages and Limitations of Genetic Manipulation Approaches for Studying acpP in D. acidovorans

How does Delftia acidovorans acpP function compare with acyl carrier proteins in other bacteria with unique metabolic capabilities?

Comparing D. acidovorans acpP with those from other metabolically versatile bacteria could provide evolutionary and functional insights:

Comparison with related environmental bacteria:

• Cupriavidis metallidurans (metal resistance capabilities)

• Pseudomonas putida (versatile xenobiotic degradation)

• Ralstonia eutropha (polyhydroxyalkanoate production)

Functional considerations:

• Sequence conservation in the phosphopantetheine attachment site

• Differences in surface charge distribution that might affect protein-protein interactions

• Presence of unique structural elements that could enable species-specific interactions

• Conservation of residues involved in acyl-chain binding pocket

Evolutionary analysis:

• Phylogenetic tree construction to understand evolutionary relationships

• Assessment of selective pressure on different regions of the protein

• Identification of horizontally transferred elements

What insights might be gained from studying the integration of acpP in fatty acid synthesis versus specialized metabolite production in Delftia acidovorans?

Understanding how acpP functions across different metabolic contexts could reveal:

Pathway crosstalk mechanisms:

• How intermediates might be shuttled between fatty acid synthesis and specialized metabolite production

• Whether acpP interacts directly with enzymes involved in delftibactin biosynthesis

• If acpP plays roles in providing precursors for biodegradation pathways

Regulatory insights:

• Whether acpP expression is coordinated with both primary and specialized metabolism

• How environmental conditions might shift acpP function between different pathways

• If post-translational modifications modulate acpP's role in different metabolic contexts

Biotechnological applications:

• Potential for engineering acpP to enhance production of valuable metabolites

• Using acpP as a scaffold for introducing novel biosynthetic capabilities

• Harnessing D. acidovorans acpP characteristics for heterologous expression systems

What emerging technologies might advance our understanding of Delftia acidovorans acpP and its role in the organism's metabolism?

Several cutting-edge approaches could provide new insights:

Single-molecule techniques:

• Single-molecule FRET to study conformational dynamics

• Force spectroscopy to examine protein-protein interaction strengths

• Super-resolution microscopy to visualize acpP localization in vivo

Synthetic biology approaches:

• Minimal synthetic pathways incorporating acpP

• Biosensors based on acpP interactions

• Cell-free systems to reconstitute acpP-dependent pathways

Advanced computational methods:

• Molecular dynamics simulations with enhanced sampling

• Machine learning approaches to predict interaction partners

• Systems biology models integrating acpP into metabolic networks

How might understanding Delftia acidovorans acpP contribute to biotechnological applications in bioremediation or biomaterial production?

Knowledge of D. acidovorans acpP could lead to applications in:

Enhanced bioremediation:

• Engineering acpP to improve integration with xenobiotic degradation pathways

• Optimizing fatty acid metabolism to support higher degradation rates

• Creating biosensors for environmental pollutants based on acpP interactions

Biomaterial production:

• Enhancing gold nanoparticle formation by modifying acpP-related pathways

• Engineering hybrid systems combining acpP with delftibactin production

• Developing novel biomaterials by exploiting D. acidovorans metabolic capabilities

Biocatalysis applications:

• Using acpP as a scaffold for designer enzymatic reactions

• Developing bioconversion processes for challenging transformations

• Creating chimeric proteins with acpP domains for novel activities

These research directions could significantly expand our understanding of bacterial metabolism while creating new biotechnological tools for environmental and industrial applications.