Recombinant Acidovorax ebreus Acyl carrier protein (acpP)

Basic Research Questions

• What is the structure and function of Acidovorax ebreus acpP in fatty acid biosynthesis?

Acidovorax ebreus acpP functions as a scaffold for fatty acid biosynthesis, similar to other bacterial ACPs. The protein contains a highly conserved Asp-Ser-Leu-Asp amino acid sequence to which acyl groups attach during fatty acid synthesis. This serves as the attachment point for growing fatty acid chains during biosynthesis .

As a member of the type II fatty acid synthase systems found in bacteria and plants, Acidovorax acpP likely carries acyl intermediates between different enzymatic domains during fatty acid elongation. This includes interactions with 3-hydroxyacyl-ACPs and 2,3-trans-enoyl-ACPs as seen in other bacterial systems . The function of Acidovorax acpP is likely critical to bacterial survival, as the fatty acid synthesis pathway is essential for membrane formation and cellular function.

Methodology for function determination:

• Gene knockout studies comparing wild-type and acpP-deficient strains

• Protein-protein interaction studies with other components of the fatty acid synthesis machinery

• In vitro reconstitution of fatty acid synthesis using purified components

• What expression systems are optimal for producing recombinant Acidovorax ebreus acpP?

Based on approaches used for other recombinant proteins, several expression systems can be considered:

For optimal expression in E. coli:

• Use BL21(DE3) strain to minimize protease activity

• Express with a fusion tag (His6, GST, or MBP) for easier purification

• Optimize induction conditions (0.1-1.0 mM IPTG, 16-25°C)

• Consider codon optimization for rare codons in Acidovorax genes

An expression method similar to that used for human ACPP can be adapted, using a histidine tag for purification via immobilized metal affinity chromatography . The baculovirus expression system might be advantageous when high purity and proper folding are essential.

• What purification strategies work best for recombinant Acidovorax ebreus acpP?

A multi-step purification strategy is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) for His-tagged protein

• Intermediate purification: Ion exchange chromatography (IEX)

• Polishing: Size exclusion chromatography (SEC)

Quality control testing should include:

• SDS-PAGE with Coomassie Blue staining to verify >95% purity

• Western blotting with anti-ACP antibodies

• Mass spectrometry to confirm molecular weight

• Activity assays to verify functional integrity

Buffer recommendations:

• Purification buffer: PBS pH 7.4 with 10% glycerol for stability

• Storage: Aliquot and store at -80°C to avoid repeated freeze-thaw cycles

Advanced Research Questions

• How can researchers accurately quantify acyl-ACP intermediates in Acidovorax ebreus?

Quantification of acyl-ACP intermediates requires sophisticated analytical techniques. A robust method developed for other bacterial systems can be adapted for Acidovorax ebreus:

• Sample preparation:

  • Extract total protein under acidic conditions to preserve acyl-ACP linkages

  • Enrich ACPs using ammonium sulfate precipitation

• LC-MS/MS analysis:

  • Use liquid chromatography coupled with tandem mass spectrometry

  • Develop selective reaction monitoring (SRM) methods for each acyl-ACP species

  • Utilize the highly conserved Asp-Ser-Leu-Asp amino acid sequence in ACPs for selective detection

• Standard preparation:

  • Generate de novo standards for accurate quantification

  • Implement isotopic dilution strategy using stable isotope-labeled standards

This approach allows sensitive quantification to the picogram level and identification of various acyl-ACP intermediates, including unexpected medium-chain (C10:1, C14:1) and polyunsaturated long-chain (C16:3) acyl-ACPs .

• How might Acidovorax ebreus acpP contribute to plant growth promotion characteristics?

Acidovorax species demonstrate varying plant growth promotion capabilities. The role of acpP may be significant based on the following considerations:

• Contribution to fatty acid synthesis:

  • Proper membrane composition affects colonization efficiency

  • Lipid-derived signaling molecules may mediate plant-microbe interactions

• Connection to secondary metabolite production:

  • Acyl-ACP serves as precursor for antimicrobial compounds that may suppress plant pathogens

  • Specific Acidovorax strains produce compounds with antimicrobial properties

• Relationship with phytohormone synthesis:

  • Some plant growth-promoting Acidovorax strains show enhanced capability for phytohormone production

  • Fatty acid derivatives can be precursors for certain plant hormone mimics

Experimental approach:

• Compare acpP sequence and expression between growth-promoting and non-promoting Acidovorax strains

• Conduct plant inoculation studies with wild-type and acpP-modified strains

• Analyze lipid profiles in relation to plant colonization efficiency

Protein families linked to sensing and transport of organic acids, phytohormone production, and antimicrobial compound production/resistance differ between plant-growth promoting and non-promoting Acidovorax strains .

• What are the optimal conditions for assessing the enzymatic activity of Acidovorax ebreus acpP?

Enzymatic activity assessment for acpP requires careful consideration of its functional context:

• Activity assay development:

  • Measure acpP's ability to accept acyl groups from acyl-ACP synthetase

  • Monitor phosphopantetheinylation efficiency with purified phosphopantetheinyl transferase

  • Assess integration into fatty acid synthesis pathways using reconstituted systems

• Assay conditions optimization:

  • pH optimization: Test range from pH 5.0-8.0

  • Temperature range: 25-37°C based on Acidovorax growth preferences

  • Buffer composition: Evaluate effects of divalent cations (Mg²⁺, Mn²⁺)

  • Substrate specificity: Test various fatty acyl-CoA chain lengths

• Activity quantification:

  • Specific activity can be defined using appropriate substrates, similar to how phosphatase activity is measured with p-nitrophenyl phosphate (pNPP)

  • Use LC-MS/MS to monitor acyl-chain attachment and transfer

Validation approach:

• Compare wild-type versus site-directed mutants

• Assess activity in presence of known ACP inhibitors

• Confirm functionality within reconstituted fatty acid synthesis systems

• How does Acidovorax ebreus acpP compare functionally with homologs from other bacterial species?

Comparative analysis of bacterial acpPs reveals important functional insights:

• Phylogenetic considerations:

  • Acidovorax belongs to beta-proteobacteria, allowing comparison with related genera

  • Some bacteria like Ralstonia solanacearum contain multiple ACP proteins, but only one (AcpP1) is functionally essential

• Structural comparisons:

  • Conservation of the critical phosphopantetheine attachment site

  • Variations in surface charge distribution affecting protein-protein interactions

  • Differences in substrate-binding pocket dimensions influencing acyl chain specificity

• Functional complementation:

  • Test if Acidovorax ebreus acpP can functionally replace acpPs in other bacteria

  • Identify unique features that might contribute to Acidovorax-specific metabolism

Experimental design should include:

• Heterologous complementation assays in ACP-deficient strains

• In vitro reconstitution with fatty acid synthesis enzymes from different species

• Site-directed mutagenesis targeting conserved versus variable residues

• What role might Acidovorax ebreus acpP play in bacterial adaptation to different environmental conditions?

Environmental adaptation likely involves acpP-dependent mechanisms:

• Temperature adaptation:

  • Changes in membrane fluidity require modified fatty acid composition

  • ACP must accommodate different acyl intermediates under various temperature regimes

• Stress response:

  • Oxidative stress may require modified lipid compositions

  • Nutrient limitation can trigger changes in fatty acid metabolism

• Host interaction:

  • Plant-associated Acidovorax strains may modify acyl-ACP pools during colonization

  • Different lipid compositions may help evade host defense responses

Research methodology:

• Compare acpP expression levels under various environmental conditions

• Analyze acyl-ACP profiles in response to stress using the LC-MS/MS method

• Examine acpP mutants for altered environmental fitness

• How can researchers develop effective mutational analysis strategies for Acidovorax ebreus acpP?

A comprehensive mutational analysis should follow these steps:

• Target selection:

  • Phosphopantetheine attachment site (conserved serine)

  • Helix II residues involved in enzyme recognition

  • Surface residues potentially involved in protein-protein interactions

• Mutagenesis strategy:

  • Alanine scanning to identify essential residues

  • Conservative substitutions to probe functional requirements

  • Domain swapping with other bacterial ACPs to identify specificity determinants

• Phenotypic analysis:

  • Growth assays under various conditions

  • Fatty acid profiling of mutant strains

  • Protein-protein interaction assays with fatty acid synthesis partners

• Structural validation:

  • Circular dichroism to assess secondary structure integrity

  • Thermal stability assays to evaluate folding

  • Solution NMR for structural perturbations of mutants

This approach can help identify residues critical for Acidovorax ebreus acpP function and inform the development of specific inhibitors or engineering strategies.

• What potential applications exist for recombinant Acidovorax ebreus acpP in agricultural biotechnology?

Recombinant Acidovorax ebreus acpP has several potential applications:

• Biocontrol development:

  • Engineering enhanced antimicrobial compound production

  • Modification of plant-microbe signaling pathways

  • Development of ACP-targeting phage therapy against pathogenic Acidovorax

• Plant growth promotion:

  • Engineering enhanced colonization capabilities

  • Improving stress tolerance in beneficial Acidovorax strains

  • Optimizing lipid-mediated signaling with host plants

• Biosensing applications:

  • Development of ACP-based biosensors for fatty acid metabolism

  • Detection systems for Acidovorax pathogens in agricultural settings

These applications require thorough characterization of wild-type acpP function and careful engineering to enhance desired properties while maintaining essential activities.