Recombinant Vibrio vulnificus Acetyl-coenzyme A synthetase (acsA), partial

How is acsA gene expression regulated in Vibrio vulnificus?

The expression of acsA in V. vulnificus is primarily regulated by AcsR, a LuxR-type transcriptional regulator. Research has demonstrated that AcsR functions as a positive regulator of acsA gene expression . This regulatory relationship was established by comparing acsA expression levels between wild-type and ΔacsR mutant strains using an acsA::luxAB transcriptional reporter fusion. The results showed that the ΔacsR mutant strain exhibited luciferase activity levels that were 50-100 times lower than the wild-type strain, confirming AcsR's role as an activator .

Furthermore, AcsR itself is regulated by the VarS/VarA two-component system, which controls diverse aspects of metabolism and virulence in pathogenic Vibrio species . Studies comparing transcript levels in wild-type and ΔvarA mutant strains revealed that the VarS/VarA system plays an important role in V. vulnificus metabolism by regulating AcsR, which in turn controls acetate metabolism through activation of acsA transcription .

Experimental approaches to study this regulation typically include:

• Transcriptional reporter fusions (e.g., acsA::luxAB)

• Quantitative RT-PCR to measure transcript levels

• Electrophoretic mobility shift assays (EMSA) to demonstrate direct binding of AcsR to the upstream region of the acsA ORF

• DNase I footprinting to precisely map regulatory binding sites

What methods are used for cloning and expressing recombinant V. vulnificus acsA?

Recombinant expression of V. vulnificus acsA typically follows these methodological steps:

Cloning strategy:

• PCR amplification of the acsA gene from V. vulnificus genomic DNA using high-fidelity DNA polymerase

• Addition of appropriate restriction sites via primer design

• Restriction digestion and ligation into an expression vector (commonly pET series vectors for E. coli expression)

• Transformation into a cloning strain (e.g., E. coli DH5α)

• Sequence verification of the cloned construct

Expression optimization:

• Transform verified construct into an expression host (commonly E. coli BL21(DE3) or derivatives)

• Test expression under various conditions:

  • Induction temperature (typically 16-37°C)

  • Inducer concentration (e.g., IPTG at 0.1-1.0 mM)

  • Duration of induction (2-24 hours)

  • Media composition (LB, TB, auto-induction media)

For challenging expression, alternative strategies include:

• Fusion tags (His6, GST, MBP) to enhance solubility

• Codon optimization for the expression host

• Co-expression with chaperones

• Use of specialized E. coli strains designed for improved expression of potentially toxic proteins

What analytical methods are effective for confirming acsA enzymatic activity?

Several complementary approaches can verify recombinant V. vulnificus acsA activity:

Spectrophotometric coupled assays:

• Measure AMP production using coupling enzymes (myokinase, pyruvate kinase, and lactate dehydrogenase)

• Monitor NADH oxidation at 340 nm, which corresponds to acetyl-CoA formation

Direct product detection:

• HPLC analysis of acetyl-CoA formation

• LC-MS/MS for definitive identification and quantification of reaction products

Activity verification protocol:

• Incubate purified acsA with acetate, ATP, CoA, and Mg2+ in appropriate buffer

• Stop reaction at defined time points with acid or heat denaturation

• Analyze reaction products using methods above

• Calculate specific activity (μmol product/min/mg enzyme)

Control reactions (omitting substrate, using heat-inactivated enzyme) are essential to verify that the observed activity is enzyme-specific.

How does the regulation of acsA contribute to V. vulnificus pathogenesis and environmental adaptation?

While acsA itself has not been directly implicated as a virulence factor in V. vulnificus, its regulation is integrated into wider regulatory networks that influence bacterial pathogenesis. The VarS/VarA system that regulates AcsR (and subsequently acsA) has been shown to regulate diverse aspects of metabolism and virulence in pathogenic Vibrio species .

Experimental approaches to investigate this relationship include:

• Comparative transcriptomics: RNA-seq analysis comparing wild-type, ΔacsR, and ΔacsA mutants under infection-relevant conditions can reveal co-regulated virulence factors

• Infection models: Testing virulence of ΔacsA mutants in appropriate animal models (mouse models are commonly used for V. vulnificus )

• Stress response experiments: Examining acsA expression under various environmental stresses that mimic conditions encountered during infection:

  • Acidic pH

  • Oxidative stress (H2O2 exposure)

  • Iron limitation

  • Hyperosmotic conditions

Research has shown that V. vulnificus contains stress response regulators such as rpoS and toxR that coordinate environmental adaptations and virulence . The potential connection between these stress regulators and acsA expression represents an important area for further investigation, particularly considering the role of acetate metabolism in bacterial adaptation to changing nutritional environments.

What structural characteristics of recombinant V. vulnificus acsA affect its catalytic function?

Understanding the structure-function relationship of V. vulnificus acsA requires a combination of structural biology approaches and enzyme kinetics. Though specific structural data for V. vulnificus acsA is limited, insights can be gained from related acetyl-CoA synthetases from other organisms.

Key methodological approaches include:

• Site-directed mutagenesis:

  • Create point mutations in conserved residues predicted to be involved in substrate binding or catalysis

  • Express and purify mutant proteins

  • Compare kinetic parameters (Km, kcat, kcat/Km) with wild-type enzyme

• Structural biology techniques:

  • X-ray crystallography of the purified recombinant enzyme (with and without substrates/substrate analogs)

  • Cryo-EM for visualization of different conformational states

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions with conformational flexibility

• Domain analysis:

  • Generate truncated versions of the enzyme to determine the minimal catalytic domain

  • Create chimeric proteins with domains from related acetyl-CoA synthetases to explore specificity determinants

Acetyl-CoA synthetases typically function through a two-step reaction mechanism:

• Formation of an acetyl-AMP intermediate with release of pyrophosphate

• Transfer of the acetyl group to CoA with release of AMP

Understanding which residues are involved in each step can provide insights into the evolution of substrate specificity and potential for enzyme engineering.

How can recombinant V. vulnificus acsA be used to study metabolic adaptations in marine bacteria?

Recombinant V. vulnificus acsA serves as a valuable tool for investigating metabolic adaptations in marine bacteria, particularly in response to changing environmental conditions.

Methodological approaches include:

• Comparative enzyme kinetics:

  • Determine kinetic parameters of recombinant acsA under varying conditions that mimic marine environments:

    • Different salt concentrations

    • Temperature ranges (10-40°C)

    • pH variations

  • Compare with acsA enzymes from other marine and non-marine bacteria

• Metabolic flux analysis:

  • Use 13C-labeled acetate to trace carbon flow in V. vulnificus

  • Compare wild-type and ΔacsA mutants to determine the contribution of acsA to central metabolism

  • Analyze metabolite profiles using GC-MS or LC-MS

• Environmental simulation experiments:

  • Monitor acsA expression and enzyme activity during transitions between nutrient-rich and nutrient-poor conditions

  • Examine adaptation to fluctuating oxygen levels

  • Study competitive fitness in mixed microbial communities

The adaptive significance of acetate metabolism for V. vulnificus must be considered in the context of its natural habitat. As a marine bacterium that can colonize shellfish and cause human infections, V. vulnificus encounters diverse environments with varying carbon sources . Understanding how acsA activity contributes to survival in these different niches can provide insights into the ecology and pathogenicity of this organism.

What methods are most effective for purifying active recombinant V. vulnificus acsA?

Purification of recombinant V. vulnificus acsA with preserved enzymatic activity requires careful consideration of buffer conditions and purification techniques. Based on protocols used for similar enzymes, the following methodological approach is recommended:

Expression and lysis:

• Express recombinant acsA with an affinity tag (His6 tag is commonly used)

• Harvest cells by centrifugation (5,000 × g, 10 min, 4°C)

• Resuspend in lysis buffer containing:

  • 50 mM Tris-HCl, pH 8.0

  • 300 mM NaCl

  • 10% glycerol

  • 1 mM DTT

  • Protease inhibitor cocktail

• Lyse cells by sonication or pressure homogenization

• Clear lysate by centrifugation (20,000 × g, 30 min, 4°C)

Purification protocol:

• Immobilized metal affinity chromatography (IMAC):

  • Load clarified lysate onto Ni-NTA column equilibrated with lysis buffer

  • Wash with lysis buffer containing 20-40 mM imidazole

  • Elute with lysis buffer containing 250 mM imidazole

• Ion exchange chromatography:

  • Dialyze IMAC-purified protein against buffer containing 50 mM Tris-HCl pH 8.0, 50 mM NaCl, 10% glycerol, 1 mM DTT

  • Apply to Q-Sepharose column

  • Elute with linear NaCl gradient (50-500 mM)

• Size exclusion chromatography:

  • Apply concentrated protein to Superdex 200 column equilibrated with 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM DTT

  • Collect fractions containing purified acsA

Throughout purification, enzyme activity should be monitored using the spectrophotometric assay described earlier. Addition of stabilizing agents (glycerol, reducing agents) and maintaining low temperature (4°C) throughout the purification process are critical for preserving enzymatic activity.

How does V. vulnificus acsA compare with acetyl-CoA synthetases from other pathogenic bacteria?

Comparative analysis of V. vulnificus acsA with homologs from other pathogenic bacteria provides insights into evolutionary relationships and potential functional differences. Methodological approaches for such comparative studies include:

• Sequence analysis:

  • Multiple sequence alignment of acsA proteins from diverse bacterial species

  • Phylogenetic tree construction to visualize evolutionary relationships

  • Identification of conserved domains and catalytic residues

• Enzymatic characterization:

  • Side-by-side comparison of kinetic parameters

  • Substrate specificity profiles (testing various short-chain fatty acids)

  • Inhibitor sensitivity patterns

• Expression pattern analysis:

  • Compare regulation of acsA genes across different pathogens

  • Identify species-specific regulatory mechanisms

Particularly informative comparisons would include:

• Vibrio cholerae (another human pathogen in the Vibrio genus)

• Vibrio parahaemolyticus (seafood-associated pathogen)

• Escherichia coli (well-characterized model organism)

• Other marine pathogens with similar ecological niches

Research has shown that V. vulnificus contains significant genomic plasticity, as evidenced by genetic variation in virulence factors like the MARTX toxin . Similar variation might exist in metabolic enzymes like acsA, potentially conferring adaptations to specific environmental niches or host environments.

How can CRISPR-Cas techniques be applied to study acsA function in V. vulnificus?

CRISPR-Cas genome editing offers powerful approaches for investigating acsA function in V. vulnificus. While traditional methods rely on homologous recombination for gene deletion, CRISPR-Cas systems provide more precise and efficient genetic manipulation.

Methodological framework for CRISPR-based studies of acsA:

• CRISPR-Cas9 knockout strategy:

  • Design sgRNAs targeting the acsA gene

  • Clone sgRNAs into a suitable vector for expression in V. vulnificus

  • Provide a repair template for precise gene deletion or modification

  • Transform into V. vulnificus and select for successful editing

  • Confirm edits by sequencing

• CRISPRi for conditional knockdown:

  • Express catalytically inactive Cas9 (dCas9) fused to a transcriptional repressor

  • Target dCas9 to the acsA promoter to reduce expression

  • Advantage: allows study of essential genes and titration of expression levels

• CRISPR-based transcriptional activation:

  • Use dCas9 fused to transcriptional activators to upregulate acsA

  • Study effects of overexpression on metabolism and stress response

Recent developments in CRISPR technologies for Vibrio species can be adapted for V. vulnificus. For example, the RAA-CRISPR/Cas12a system has been successfully used for detection of V. vulnificus and could potentially be modified for genome editing applications.

The efficiency of CRISPR systems in V. vulnificus must be optimized, considering factors such as:

• Delivery method (electroporation, conjugation)

• Promoter selection for Cas9 and sgRNA expression

• PAM site availability in the target gene

• Potential off-target effects

What is the role of acsA in V. vulnificus biofilm formation and virulence?

The potential connection between acetate metabolism, biofilm formation, and virulence in V. vulnificus represents an important area of investigation. While direct evidence linking acsA to these processes in V. vulnificus is limited, several methodological approaches can be employed to explore these relationships:

• Biofilm formation assays:

  • Compare biofilm formation between wild-type and ΔacsA mutants using:

    • Crystal violet staining in microplates

    • Flow cell systems with confocal microscopy

    • Scanning electron microscopy for detailed structural analysis

  • Examine biofilm formation under different carbon source conditions

• Virulence assessment:

  • Mouse infection models (both wound and ingestion routes)

  • Cell culture infection assays measuring:

    • Cytotoxicity (LDH release assays)

    • Bacterial adherence and invasion

    • Host cell cytoskeletal rearrangements

• Gene expression studies:

  • RNA-seq comparing wild-type and ΔacsA mutants during:

    • Biofilm growth

    • Infection of host cells

    • Growth in infection-relevant conditions

  • qRT-PCR validation of differentially expressed virulence genes

Research on other Vibrio species suggests potential connections between metabolism and virulence regulation. For example, in V. vulnificus, the VarS/VarA system that regulates AcsR has been shown to control other important factors including flagellins, RpoS, RtxA1, and VvpE . The MARTX(Vv) toxin, encoded by the rtxA1 gene, is a significant virulence factor in V. vulnificus , and understanding potential metabolic influences on its expression could provide valuable insights into virulence regulation.