Recombinant Desulfovibrio vulgaris Acyl carrier protein (acpP)

What is Acyl Carrier Protein (acpP) in Desulfovibrio vulgaris and why is it significant for research?

Acyl carrier protein (acpP) in Desulfovibrio vulgaris is a small, acidic protein approximately 8-10 kDa in size that functions as a central component of the Type II fatty acid synthesis (FAS) pathway. This protein serves as a shuttle, carrying acyl intermediates between various enzymatic domains during fatty acid biosynthesis. The significance of D. vulgaris acpP stems from its essential role in bacterial metabolism and the unique adaptations of Desulfovibrio species to anaerobic environments. As sulfate-reducing bacteria, Desulfovibrio species like D. vulgaris possess distinctive metabolic pathways that enable them to thrive in oxygen-limited environments and produce hydrogen sulfide (H₂S) . The study of acpP provides insights into these specialized metabolic processes and potential connections to pathogenesis, as Desulfovibrio species have been associated with conditions such as Parkinson's disease .

What expression systems are most effective for producing recombinant D. vulgaris acpP?

For successful expression of recombinant D. vulgaris acpP, Escherichia coli-based expression systems typically yield the best results due to their efficiency and versatility. The most effective approach involves:

• Vector selection: pET expression vectors (particularly pET28a or pET15b) containing T7 promoters provide high-level expression with the added benefit of incorporating histidine tags for simplified purification.

• E. coli strain selection: BL21(DE3) or Rosetta(DE3) strains are preferred, with the latter being advantageous when codon optimization is necessary for the expression of genes from Desulfovibrio species.

• Growth conditions: Culture in LB medium supplemented with appropriate antibiotics at 37°C until reaching OD₆₀₀ of 0.6-0.8, followed by induction with IPTG (typically 0.5-1.0 mM).

• Temperature adjustment: Lowering the temperature to 16-25°C post-induction often enhances proper folding and solubility of the recombinant protein.

For verification of successful transformation, techniques similar to those used for other Desulfovibrio genes can be applied, including PCR products checked by gel electrophoresis, purification, and sequencing as described in protocols for amplifying Desulfovibrio genes .

What purification strategies yield the highest purity recombinant D. vulgaris acpP?

The most effective purification strategy for recombinant D. vulgaris acpP typically involves a multi-step approach:

Table 1: Optimized Purification Protocol for Recombinant D. vulgaris acpP

The purity assessment should involve SDS-PAGE analysis with Coomassie or silver staining, Western blotting using specific anti-acyl carrier protein antibodies , and mass spectrometry for final verification. Detection methods can be adapted from those used for various applications of acyl carrier protein antibodies, including Western blotting techniques that allow specific protein identification .

What are the optimal conditions for long-term storage of purified recombinant D. vulgaris acpP?

For maintaining optimal stability and activity of purified recombinant D. vulgaris acpP during long-term storage, researchers should consider the following evidence-based conditions:

• Buffer composition: 50 mM Tris-HCl or phosphate buffer (pH 7.5-8.0) with 100-150 mM NaCl provides optimal stability. The addition of 1-2 mM DTT or 5 mM β-mercaptoethanol helps maintain reduced states of cysteine residues.

• Storage temperature: Flash-freeze aliquots in liquid nitrogen and store at -80°C for long-term preservation. For working stocks, storage at -20°C is acceptable for up to 1 month.

• Cryoprotectants: Addition of 10% glycerol or 5% trehalose minimizes freeze-thaw damage.

• Concentration considerations: Store the protein at concentrations between 1-5 mg/mL to prevent aggregation while maintaining sufficient concentration for experiments.

• Avoid repeated freeze-thaw cycles: Prepare small aliquots (50-100 μL) to minimize the need for repeated thawing and refreezing.

Stability testing data from comparable proteins suggests that recombinant acpP stored under these conditions typically retains >90% activity for at least 12 months at -80°C.

How does D. vulgaris acpP function differ from acyl carrier proteins in other bacterial species?

D. vulgaris acpP exhibits several distinctive functional characteristics compared to acyl carrier proteins in other bacterial species:

• Anaerobic adaptation: D. vulgaris acpP has evolved to function optimally in anaerobic environments, potentially affecting its interaction with partner enzymes in the fatty acid synthesis pathway. This adaptation is critical given the strict anaerobic nature of Desulfovibrio species, which are sulfate-reducing bacteria that produce H₂S gas .

• Post-translational modifications: The 4'-phosphopantetheine prosthetic group attachment site in D. vulgaris acpP may have subtle differences in surrounding amino acid sequences that influence the efficiency of this critical modification by phosphopantetheinyl transferases.

• Protein-protein interactions: D. vulgaris acpP likely has specialized interaction surfaces for engaging with sulfate reduction pathway enzymes, potentially creating unique metabolic connections between fatty acid synthesis and energy metabolism in these organisms.

• Temperature and pH optima: Given Desulfovibrio's environmental niche, its acpP typically demonstrates optimal activity at neutral to slightly alkaline pH (7.0-8.5) and moderate temperatures (25-37°C), which mirrors the conditions used in culturing these bacteria in specialized media like Postgate B .

• Metal ion interactions: D. vulgaris acpP may have evolved specific interactions with iron and other metal ions that are abundant in their natural environments and critical for their metabolism, particularly given that iron-containing media are used for Desulfovibrio culture .

These functional differences likely contribute to the specialized metabolic capabilities of Desulfovibrio species in their ecological niches and potentially to their role in human health conditions as suggested by recent research linking these bacteria to Parkinson's disease .

What methodological approaches effectively characterize the structural properties of recombinant D. vulgaris acpP?

Comprehensive structural characterization of recombinant D. vulgaris acpP requires a multi-technique approach:

Table 2: Structural Characterization Methods for Recombinant D. vulgaris acpP

For functional state verification, activity assays should be conducted in parallel with structural studies. The integration of these methodologies provides comprehensive insights into the structure-function relationship of D. vulgaris acpP, enabling researchers to understand its role in bacterial metabolism and potential implications in pathogenesis observed in studies linking Desulfovibrio to conditions like Parkinson's disease .

How can recombinant D. vulgaris acpP be used to study the link between Desulfovibrio and Parkinson's disease?

Recent research has established a significant association between Desulfovibrio species and Parkinson's disease (PD), with studies showing these bacteria are present in 80% of PD patients compared to 40% of healthy controls . Recombinant D. vulgaris acpP can serve as a valuable tool in investigating this connection through several methodological approaches:

• Biomarker development: Recombinant D. vulgaris acpP can be used to develop and standardize antibody-based detection systems for monitoring Desulfovibrio presence in patient samples. This approach leverages techniques similar to those used for other antibody applications such as Western blotting, immunohistochemistry, and ELISA .

• Host-pathogen interaction studies: By labeling recombinant acpP or creating fluorescent fusion proteins, researchers can track its cellular localization during infection models to determine whether it plays a role in neural cell interactions or blood-brain barrier penetration.

• Inflammatory response investigation: Using purified recombinant acpP in cell culture models (particularly with microglial cells), researchers can measure inflammatory cytokine production, microglial activation, and oxidative stress markers to determine if this protein contributes to the neuroinflammation characteristic of PD.

• Metabolomic analysis: Recombinant acpP can be employed in enzymatic assays to identify specific fatty acid products that might act as mediators in neuroinflammation or neurotoxicity, building on observations of altered sulfur metabolism in PD patients with Desulfovibrio presence .

• Animal model development: Recombinant acpP can be administered to animal models to determine if it contributes to PD-like symptoms or pathology, potentially establishing a more direct causal relationship between Desulfovibrio components and PD progression.

These approaches leverage the strong correlation observed between Desulfovibrio presence (detected through species-specific PCR and hydA gene identification) and PD, offering mechanistic insights beyond the epidemiological association .

What challenges exist in expressing post-translationally modified forms of D. vulgaris acpP and how can they be overcome?

Expressing functionally active D. vulgaris acpP presents several challenges, particularly regarding its critical 4'-phosphopantetheine post-translational modification (PTM), which converts the inactive apo-ACP to the active holo-ACP form. The following methodological solutions address these challenges:

Table 3: Challenges and Solutions for Expressing Modified D. vulgaris acpP

These challenges parallel the difficulties encountered in isolation and culture of Desulfovibrio species, which require specialized conditions like anaerobic environments and specific media compositions (such as Postgate B medium supplemented with yeast extract and MgSO₄) . The solutions draw upon methodologies adapted from both protein expression techniques and the careful environmental control needed when working with these fastidious anaerobic bacteria.

How can bacteriophage-based approaches be utilized to study D. vulgaris acpP expression and function?

Bacteriophage-based approaches offer innovative methods for studying D. vulgaris acpP expression and function, building on recent advances in phage-based techniques for Desulfovibrio research:

• Phage display technology: By incorporating the acpP gene or portions thereof into phage display systems, researchers can identify interaction partners and binding epitopes. This approach can reveal previously unknown protein-protein interactions within Desulfovibrio metabolic networks.

• Reporter phage systems: Engineered bacteriophages carrying reporter genes (e.g., luciferase) linked to acpP promoters can serve as biosensors for acpP expression under various environmental conditions. This builds upon established techniques for bacteriophage isolation from Desulfovibrio cultures using methods such as the agar spot test on specialized media .

• CRISPR-Cas delivery via phages: Temperate phages specific to Desulfovibrio can be engineered to deliver CRISPR-Cas systems for precise genome editing of the acpP gene, enabling functional studies through site-directed mutagenesis in the native context.

• Phage-mediated protein delivery: Bacteriophage capsids can be engineered to deliver modified versions of acpP protein directly into Desulfovibrio cells, allowing for complementation studies or dominant-negative approaches to investigate function.

• High-throughput screening platforms: Phage libraries displaying acpP variants can be used to screen for mutations affecting function, stability, or interactions, accelerating the identification of critical residues.

These approaches leverage the natural host-phage relationships observed in Desulfovibrio species, where specific bacteriophages have demonstrated the ability to infect strains like 11X with visible effects after 24-48 hours . Research has shown that different Desulfovibrio strains exhibit varying susceptibility to phage infection, with strains 10D and 11X being most susceptible, indicating the potential for strain-specific phage-based tools .

What analytical techniques are most sensitive for detecting and quantifying D. vulgaris acpP in complex biological samples?

For detecting and quantifying D. vulgaris acpP in complex biological samples such as gut microbiome extracts or environmental samples, researchers should employ a strategic combination of techniques:

• Quantitative PCR (qPCR): This highly sensitive method can detect the acpP gene with a detection limit of approximately 10³ copies per sample. The approach parallels methods used for Desulfovibrio detection in clinical samples, where qPCR with SYBR Green has successfully quantified Desulfovibrio from fecal samples with standard curves ranging from 2×10³ to 2×10⁷ copies . For acpP-specific detection:

  • Design primers targeting conserved regions of the D. vulgaris acpP gene

  • Create standard curves using plasmids containing the acpP gene

  • Include appropriate negative controls from Desulfovibrio-negative samples

• Immunoassays: Development of acpP-specific antibodies enables detection through:

  • Western blotting with a detection limit of ~1 ng protein

  • ELISA with potential detection limits of 10-100 pg/mL

  • Immunohistochemistry for localization in tissue samples
    These approaches can be adapted from established antibody-based methods for acyl carrier protein detection .

• Mass spectrometry-based proteomics: For unambiguous identification and quantification:

  • Selected reaction monitoring (SRM) targeting unique peptides from D. vulgaris acpP

  • Parallel reaction monitoring (PRM) for improved specificity

  • Absolute quantification using isotopically labeled peptide standards

Table 4: Comparative Analysis of Detection Methods for D. vulgaris acpP

The combined application of these techniques provides comprehensive data on both the presence of the acpP gene and its expression levels in complex samples, similar to the multi-technique approach used in studies examining Desulfovibrio presence in clinical samples .

What are the most effective methods for studying interactions between D. vulgaris acpP and partner proteins in fatty acid synthesis?

Investigating protein-protein interactions involving D. vulgaris acpP requires sophisticated methodological approaches that capture both stable and transient interactions within the fatty acid synthesis pathway:

• Pull-down assays and co-immunoprecipitation:

  • Express recombinant D. vulgaris acpP with affinity tags (His-tag or GST)

  • Immobilize on appropriate resin and incubate with cell lysates

  • Identify binding partners using mass spectrometry

  • Validate with reciprocal pull-downs using antibodies specific to acyl carrier protein

• Surface Plasmon Resonance (SPR) and Bio-Layer Interferometry (BLI):

  • Immobilize purified acpP on sensor chips/tips

  • Measure real-time binding kinetics with purified partner proteins

  • Determine association/dissociation rate constants and equilibrium dissociation constants

  • Compare binding parameters across different conditions (pH, temperature, ionic strength)

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Monitor changes in hydrogen-deuterium exchange rates upon complex formation

  • Map interaction interfaces at peptide-level resolution

  • Identify conformational changes induced by binding

• Förster Resonance Energy Transfer (FRET):

  • Generate fluorescent protein fusions with acpP and potential partners

  • Measure energy transfer as indicator of protein proximity in vitro or in vivo

  • Use time-resolved FRET for dynamic interaction studies

• Crosslinking Mass Spectrometry (XL-MS):

  • Apply chemical crosslinkers to stabilize transient interactions

  • Digest complexes and identify crosslinked peptides by mass spectrometry

  • Generate distance constraints for computational modeling

• Bacterial Two-Hybrid Systems:

  • Adapt for anaerobic conditions to mimic native D. vulgaris environment

  • Screen for interactions in conditions mimicking the sulfate-reducing bacterial environment

  • Validate with controls specific to anaerobic metabolic pathways

These methodologies can be integrated with approaches used to study Desulfovibrio in various contexts, including techniques adapted from research on Desulfovibrio's role in health conditions like Parkinson's disease and methods for cultivating these bacteria in specialized media under appropriate conditions .

How can recombinant D. vulgaris acpP be utilized to develop inhibitors targeting Desulfovibrio in microbiome-associated diseases?

The strong association between Desulfovibrio species and Parkinson's disease (PD), with 80% of PD patients showing Desulfovibrio presence compared to 40% of healthy controls , highlights the potential of D. vulgaris acpP as a therapeutic target. A systematic approach to developing inhibitors includes:

• Structure-based drug design:

  • Solve the high-resolution crystal structure of D. vulgaris acpP, focusing on the 4'-phosphopantetheine attachment site and protein interaction surfaces

  • Perform in silico molecular docking with virtual compound libraries

  • Identify potential binding pockets unique to D. vulgaris acpP compared to human homologs

  • Design competitive inhibitors that prevent acpP interaction with partner enzymes

• Fragment-based screening approach:

  • Use nuclear magnetic resonance (NMR) or surface plasmon resonance (SPR) to screen fragment libraries against purified recombinant acpP

  • Identify fragment hits that bind to functionally important regions

  • Elaborate fragments through medicinal chemistry to improve affinity and specificity

  • Combine fragments that bind to adjacent sites to create more potent inhibitors

• High-throughput enzymatic assays:

  • Develop assays monitoring acpP-dependent fatty acid synthesis activities

  • Screen compound libraries for inhibition of these activities

  • Prioritize compounds with selective activity against D. vulgaris acpP versus human fatty acid synthesis proteins

  • Validate hits in increasingly complex systems, from purified enzymes to cellular assays

• Bacteriophage-based delivery systems:

  • Engineer bacteriophages that specifically target Desulfovibrio species, building on research showing strain-specific phage susceptibility

  • Use these phages as delivery vehicles for acpP-targeting compounds or genetic elements

  • Develop phage cocktails targeting multiple Desulfovibrio species found in PD patients (D. desulfuricans, D. fairfieldensis, and D. piger)

• Screening for natural product inhibitors:

  • Test compounds from organisms naturally competing with Desulfovibrio in ecological niches

  • Evaluate traditional medicines with reported benefits in conditions now associated with Desulfovibrio

  • Identify active compounds and optimize their properties through medicinal chemistry

This multi-faceted approach leverages recombinant D. vulgaris acpP as both a screening target and a tool for validating compound mechanisms, potentially leading to novel therapeutics for microbiome-associated diseases like Parkinson's disease where Desulfovibrio plays a role .

What experimental designs best evaluate the impact of environmental factors on D. vulgaris acpP expression?

To systematically evaluate how environmental factors affect D. vulgaris acpP expression, researchers should implement comprehensive experimental designs that reflect the complex ecological niches of Desulfovibrio species:

Table 5: Experimental Design for Environmental Factor Assessment on acpP Expression

For each condition, time-course experiments should be conducted to distinguish between immediate responses and long-term adaptations. Statistical analysis should employ ANOVA with post-hoc tests similar to those used in clinical studies of Desulfovibrio, setting significance thresholds at p<0.05 .

The experimental systems should incorporate specialized culturing techniques adapted for Desulfovibrio, such as the double-layer agar method and use of specific media like TCBS or Postgate B that contain essential components such as yeast extract for amino acids and peptides, and appropriate salt concentrations for osmotic balance . Integration of these carefully controlled experimental conditions with sophisticated molecular analyses will provide comprehensive insights into environmental regulation of D. vulgaris acpP expression.

What are the most promising future research directions for D. vulgaris acpP studies?

The study of recombinant Desulfovibrio vulgaris acyl carrier protein (acpP) offers several promising research directions that could significantly impact our understanding of bacterial metabolism, pathogenesis, and potential therapeutic interventions:

• Clinical biomarker development: Building on the strong association between Desulfovibrio species and Parkinson's disease , acpP or its metabolic products could be developed as diagnostic biomarkers. Future research should focus on large-scale clinical validation studies correlating acpP expression levels with disease progression using antibody-based detection methods .

• Microbiome-targeted therapeutics: The development of narrow-spectrum antimicrobials targeting acpP function specifically in Desulfovibrio could provide precision tools for microbiome modulation without the collateral damage associated with broad-spectrum antibiotics. This approach may be particularly valuable given the specificity of bacteriophages for different Desulfovibrio strains, suggesting potential for highly targeted interventions .

• Synthetic biology applications: Engineered variants of D. vulgaris acpP could be developed for biosynthesis of specialized fatty acids and polyketides with industrial or pharmaceutical applications. These systems could potentially operate under anaerobic conditions, offering advantages for certain industrial processes.

• Evolutionary and comparative studies: Comprehensive analysis of acpP across different Desulfovibrio species and other sulfate-reducing bacteria could reveal evolutionary adaptations to various ecological niches and provide insights into the emergence of pathogenic traits.

• Structure-function relationship mapping: High-resolution structural studies of acpP in complex with various partner proteins would enhance our understanding of the unique aspects of fatty acid synthesis in anaerobic bacteria and potentially reveal novel regulatory mechanisms.

• Host-microbe interaction studies: Investigation of how acpP and its metabolic products influence host immune responses and neural function could shed light on the mechanistic link between Desulfovibrio and neurological disorders like Parkinson's disease, where strong statistical associations have been demonstrated (p = 0.022, Phi value = 0.408) .