Recombinant Vibrio vulnificus Fatty acid oxidation complex subunit alpha (fadJ), partial

What is the biochemical function of fadJ in Vibrio vulnificus metabolism?

FadJ is a multifunctional enzyme in the fatty acid oxidation complex that plays a critical role in long-chain-fatty-acid (LCFA) metabolism, a fundamental cellular process involved in lipid homeostasis and energy production in Vibrio vulnificus . The protein contains multiple enzymatic activities, including:

• Enoyl-CoA hydratase (EC 4.2.1.17)

• 3-hydroxybutyryl-CoA epimerase (EC 5.1.2.3)

• 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35)

These combined activities enable the processing of fatty acids through β-oxidation, converting them to acetyl-CoA units that can enter the TCA cycle for energy production .

What expression systems are recommended for recombinant fadJ production?

Multiple expression systems have been validated for fadJ production, each with specific advantages:

The choice should be guided by your experimental requirements. For structural studies, E. coli systems typically provide sufficient quantities, while mammalian expression may be preferred for functional assays where proper folding is critical .

What purification strategies yield the highest purity of recombinant fadJ?

A multistep purification approach is recommended for obtaining >85% pure recombinant fadJ protein :

• Initial capture: Choose based on your affinity tag:

  • His-tagged fadJ: IMAC (Immobilized Metal Affinity Chromatography)

  • Strep-tagged fadJ: Strep-Tactin® affinity chromatography

  • GST-tagged fadJ: Glutathione Sepharose chromatography

• Intermediate purification:

  • Ion exchange chromatography (typically anion exchange at pH 8.0)

  • Use of buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 10% glycerol has shown optimal results

• Polishing step:

  • Size exclusion chromatography to remove aggregates and ensure homogeneity

For maximum stability during purification, include 1 mM DTT and protease inhibitors in all buffers. The final product typically requires concentration to 0.1-1.0 mg/mL and storage with 5-50% glycerol at -20°C/-80°C .

How should recombinant fadJ be stored to maintain activity?

Based on protein stability studies, the following storage conditions are recommended :

• Short-term storage (1-2 weeks): 4°C in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, and 10% glycerol

• Long-term storage: -20°C/-80°C in aliquots with 50% glycerol as cryoprotectant

• Lyophilization: Viable alternative when reconstituted in deionized sterile water to 0.1-1.0 mg/mL

Activity assays show that recombinant fadJ typically retains >90% activity when stored properly at -80°C for up to 6 months. Avoid repeated freeze-thaw cycles, which can reduce activity by approximately 10-15% per cycle.

How is fadJ connected to Vibrio vulnificus virulence mechanisms?

Recent research has revealed a complex relationship between fatty acid metabolism and virulence in V. vulnificus:

• LCFA homeostasis and motility: FadJ participates in maintaining the proper LCFA composition in the bacterial membrane, which directly affects flagellar motility through the activation of phase variation . This motility is critical for initial colonization during infection.

• Membrane composition and virulence: Studies have shown that disruption of fatty acid oxidation pathway genes (including fadJ) leads to altered lipid profiles in V. vulnificus membranes. The ratio of palmitic/oleic acid to palmitic/palmitoleic acid in phosphatidylethanolamine is significantly affected, which correlates with reduced virulence .

• Relationship to other virulence factors: While the MARTX (multifunctional-autoprocessing RTX) toxin is a primary virulence factor in V. vulnificus , the fatty acid metabolism pathway interacts with toxin production and delivery systems. FadR (a regulator in the pathway involving fadJ) indirectly activates fljB expression, which affects smooth swimming and gut colonization .

Researchers should consider these connections when designing experiments to investigate V. vulnificus pathogenicity mechanisms.

What mutagenesis approaches have been successful for functional studies of fadJ?

Several mutagenesis strategies have been employed to study fadJ function:

When designing mutagenesis experiments, it's crucial to consider that V. vulnificus undergoes natural recombination events that can complicate interpretation of results, particularly in long-term studies .

How can protein-protein interaction studies with fadJ inform our understanding of fatty acid metabolism in Vibrio vulnificus?

To elucidate the role of fadJ in the fatty acid metabolism network:

• Recommended methodologies:

  • Pull-down assays using tagged recombinant fadJ

  • Bacterial two-hybrid systems

  • Co-immunoprecipitation followed by mass spectrometry

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for dynamic interaction studies

• Key interaction partners to investigate:

  • FadR (transcriptional regulator affecting motility and colonization)

  • Other components of the fatty acid oxidation complex

  • Proteins involved in membrane lipid composition maintenance

  • Potential links to virulence factor secretion systems

• Functional validation approaches:

  • In vitro reconstitution of activity with purified components

  • Mutagenesis of interaction interfaces followed by phenotypic assessment

  • Correlation of interaction strength with virulence in animal models

Recent studies suggest that fadJ may function within a larger metabolic complex that influences membrane composition, which in turn affects other virulence determinants .

What detection methods are most sensitive for recombinant fadJ in experimental systems?

Several detection methods have been developed with varying sensitivity levels:

For most laboratory applications, a combination of Western blotting for detection and activity assays for functional validation provides the most comprehensive analysis.

How does recombinant fadJ compare structurally and functionally to the native protein?

Comparative studies between recombinant and native fadJ have revealed important considerations:

• Structural comparisons:

  • Circular dichroism spectroscopy shows similar secondary structure profiles

  • Thermal stability may differ depending on expression system used

  • Post-translational modifications present in native but not E. coli-expressed protein

• Functional parameters comparison:

  • Kinetic parameters (Km, kcat) usually within 2-fold range between native and recombinant

  • Temperature and pH optima generally preserved

  • Substrate specificity preserved for primary substrates

• Expression system effects:

  • E. coli-expressed protein: Highest yield, potentially missing modifications

  • Yeast-expressed protein: Closer to native in glycosylation pattern

  • Mammalian-expressed protein: Most similar to native but lowest yield

For studies requiring precise replication of native function, consider using H. polymorpha or other eukaryotic expression systems that provide better post-translational modifications .

How can fadJ be utilized for developing detection methods for Vibrio vulnificus?

Building on recent advances in rapid detection technologies:

Research shows that recombinant V. vulnificus proteins can be used to develop detection systems with high specificity. While vvh (hemolysin) and empV (metalloprotease) genes have been targeted in published detection methods , fadJ represents an alternative target with distinct advantages:

• Advantages of fadJ-based detection:

  • Present in all V. vulnificus strains

  • Relatively conserved sequence compared to toxin genes

  • Less likely to be affected by horizontal gene transfer

• Potential methodologies:

  • Real-time Recombinase Polymerase Amplification (RPA) targeting fadJ

  • Lateral flow dipstick (LFD) systems using anti-fadJ antibodies

  • CRISPR-Cas12a detection systems with fadJ-specific guides

Current RPA-based methods for other V. vulnificus genes show detection limits of approximately 17 gene copies or 1 CFU per reaction within 2-14 minutes , suggesting similar performance could be achieved with fadJ-targeted systems.

What role might fadJ play in the adaptation of Vibrio vulnificus to different environmental conditions?

Recent findings suggest fatty acid metabolism is central to environmental adaptation:

• Temperature adaptation:

  • V. vulnificus alters membrane fatty acid composition at different temperatures

  • FadJ likely plays a role in remodeling fatty acids during temperature shifts

  • This adaptation may contribute to seasonal patterns of infection

• Salinity response:

  • Growth in brackish waters (preferred environment) vs. higher salinity environments

  • Membrane composition changes mediated by fatty acid metabolism

  • FadJ activity may be regulated in response to osmotic changes

• Experimental approaches to investigate:

  • Transcriptomics and proteomics of V. vulnificus under varying conditions

  • fadJ knockout studies with environmental challenge tests

  • Lipidomics analysis of membrane composition changes

Understanding these adaptations could help predict environmental conditions that promote virulence, potentially leading to improved public health monitoring strategies for V. vulnificus .