Recombinant Xylella fastidiosa Acetyl-coenzyme A synthetase (acsA), partial

What is Acetyl-coenzyme A synthetase (acsA) in Xylella fastidiosa and what is its biochemical function?

Acetyl-coenzyme A synthetase (acsA) in Xylella fastidiosa is an enzyme that catalyzes the reversible conversion of acetate to acetyl-coenzyme A (Acetyl-CoA). This reaction is a fundamental part of central metabolism in bacteria. Biochemically, the enzyme operates via a two-step mechanism: first, it forms an acetyl-adenylate intermediate from acetate and ATP, and then it transfers the acetyl group to the thiol group of coenzyme A, releasing AMP .

The enzyme has the EC classification 6.2.1.1 and represents a high-affinity pathway for acetate utilization. In many bacteria including Xylella fastidiosa, acsA functions primarily in acetate catabolism, while the Ack-Pta pathway (acetate kinase/phosphotransacetylase) typically operates in acetate excretion .

How does the regulation of acsA differ in Xylella fastidiosa compared to other bacterial species?

• In Salmonella enterica, acsA activity is modulated through acetylation of a single lysyl residue (Lys609) by the protein acetyltransferase Pat, and deacetylation by the NAD+-dependent CobB sirtuin deacetylase .

• In Bacillus subtilis, as a comparative model, the acuABC operon encodes a protein acetyltransferase (AcuA) and a protein deacetylase (AcuC) that control acsA activity. Unlike the Salmonella system, the B. subtilis AcuC deacetylase does not require NAD+ as a cosubstrate .

• While not explicitly documented in the search results for Xylella fastidiosa, genomic analyses suggest that regulatory mechanisms similar to those in other Gram-negative bacteria may be present, potentially involving ECF sigma factors for transcriptional regulation .

What structural features characterize Xylella fastidiosa acsA and how do they relate to its function?

Xylella fastidiosa acsA contains several key structural domains that are critical for its enzymatic function:

• Active site domain: Contains residues involved in acetate binding and activation.

• Acetyl-CoA binding pocket: Specifically structured to accommodate the coenzyme A molecule.

• Acetylation site: Based on studies in other bacteria, acsA likely contains a conserved lysine residue (analogous to Lys549 identified in Bacillus subtilis AcsA) that serves as the site for regulatory acetylation .

The enzyme's three-dimensional structure facilitates its sequential reaction mechanism. The acetate molecule first binds to the active site where it is activated by ATP to form acetyl-AMP. This intermediate remains bound to the enzyme while coenzyme A binds, allowing for the transfer of the acetyl group to form acetyl-CoA.

What are the optimal conditions for expressing and purifying recombinant Xylella fastidiosa acsA?

Based on available data from recombinant protein expression systems for Xylella fastidiosa proteins:

Expression System:

• Yeast expression systems have been successfully used for Xylella fastidiosa proteins, as indicated by commercial recombinant protein suppliers .

• E. coli expression systems may also be suitable, using vectors such as pTYB12 for N-terminal chitin purification tags or pET42a for GST fusion proteins, as demonstrated for analogous bacterial proteins .

Purification Protocol:

• Cell lysis under native conditions using buffer containing 0.05 M HEPES (pH 7.5) and reducing agents such as TCEP (200 μM) .

• Affinity chromatography using appropriate tags (His-tag, GST, or chitin-binding domain).

• Size exclusion chromatography for higher purity.

• Final purity should exceed 85% as assessed by SDS-PAGE .

Storage Conditions:

• Store at -20°C for short-term or -80°C for extended storage.

• Add glycerol to a final concentration of 5-50% before freezing.

• Avoid repeated freeze-thaw cycles; store working aliquots at 4°C for up to one week .

How can the enzymatic activity of recombinant Xylella fastidiosa acsA be accurately measured?

Standard Enzymatic Assay:

• Forward Reaction (Acetyl-CoA formation):

  • Measure the production of acetyl-CoA spectrophotometrically at 340 nm by coupling the reaction to the reduction of NAD+ to NADH.

  • Reaction mixture: acetate, ATP, coenzyme A, MgCl₂, and purified acsA enzyme in appropriate buffer (e.g., 50 mM HEPES, pH 7.5).

• Reverse Reaction (Acetate formation):

  • Quantify acetate production using gas chromatography or HPLC.

• Isotope-Based Assays:

  • Use [1-¹⁴C]acetate as a substrate and measure incorporation into acetyl-CoA .

Activity Control Factors:

• Temperature: Optimal activity typically at 25-37°C.

• pH: Generally optimal at pH 7.0-7.5.

• Divalent cations: Mg²⁺ is essential for activity.

• Reducing environment: Include DTT or TCEP to maintain thiol groups.

What methods are available for studying the acetylation status of Xylella fastidiosa acsA?

Several analytical methods can be employed to investigate the acetylation status of acsA:

• Mass Spectrometry:

  • Tryptic digest followed by LC-MS/MS analysis can identify acetylated lysine residues.

  • This approach successfully identified Lys549 as the acetylation site in B. subtilis AcsA .

• Western Blot Analysis:

  • Use anti-acetyl-lysine antibodies to detect acetylated forms of the protein.

  • Quantify relative acetylation levels through densitometry.

• In Vitro Acetylation/Deacetylation Assays:

  • Incubate purified acsA with acetyltransferase (e.g., AcuA homolog) and [1-¹⁴C]acetyl-CoA.

  • Measure incorporation of radioactive acetyl groups .

• Complementation Assays:

  • Express Xylella fastidiosa acsA in model systems like Salmonella enterica strains lacking CobB sirtuin deacetylase activity.

  • Assess growth on acetate medium to determine functional impact of acetylation .

How can acsA be used in differentiation and typing of Xylella fastidiosa strains?

acsA can serve as a valuable marker for genetic differentiation of Xylella fastidiosa strains:

• Multilocus Sequence Typing (MLST):

  • While acsA is not typically included in standard MLST schemes for Xylella fastidiosa, it could be incorporated as an additional marker for enhanced strain discrimination.

  • Current MLST approaches for Xylella fastidiosa typically use seven housekeeping genes, but including environmentally mediated genes like acsA could improve resolution .

• Sequence Variation Analysis:

  • The dN/dS ratio (rate of nonsynonymous to synonymous substitutions) for acsA can indicate selective pressure on the gene.

  • Higher dN/dS ratios suggest positive selection, making the gene potentially more informative for strain differentiation .

• Recombination Detection:

  • acsA sequences can be analyzed for evidence of horizontal gene transfer or recombination events between subspecies.

  • Recombination has been shown to play a significant role in Xylella fastidiosa evolution and host adaptation .

Data from studies of genomic diversity in Xylella fastidiosa have shown that:

• Environmentally mediated genes often show greater sequence variation than housekeeping genes.

• Including such genes in phylogenetic analyses can increase resolution between closely related isolates, particularly those infecting the same plant host .

What is known about recombination events affecting acsA in different Xylella fastidiosa subspecies?

While specific recombination events in the acsA gene were not directly reported in the search results, the general patterns of recombination in Xylella fastidiosa provide insights:

• Inter-subspecies Recombination:

  • Extensive homologous recombination has been documented between Xylella fastidiosa subspecies.

  • Notable examples include the recombination between subspecies fastidiosa and multiplex to create the chimeric genome of subspecies morus .

  • Similar genetic exchanges have been observed between subspecies multiplex and pauca in South America .

• Population Genetic Structure:

  • Recent studies in Taiwan have shown that recombination significantly contributes to genetic differentiation of subspecies fastidiosa isolates after introduction to new regions .

  • Core-genome recombination can dramatically alter phylogenetic relationships and haplotype networks .

• Functional Consequences:

  • Recombination events introducing new allelic diversity can potentially affect host specificity and pathogenicity.

  • The introduction of novel allelic variants into regions where Xylella fastidiosa is already present poses a significant risk for disease emergence .

How is the acsA gene organized in the Xylella fastidiosa genome and what is known about its transcriptional regulation?

The genomic context and transcriptional regulation of acsA in Xylella fastidiosa can be inferred from general patterns of gene organization and regulation in this bacterium:

• Genomic Organization:

  • The Xylella fastidiosa genome has a predicted origin of replication identified by the location of the dnaA gene, clusters of DnaA boxes, and inversion of G/C and A/T skews .

  • Approximately 59-60% of genes are encoded on the leading replication strands .

  • While the specific location of acsA was not detailed in the search results, metabolic genes are typically distributed throughout the chromosome.

• Transcriptional Regulation:

  • Xylella fastidiosa possesses several sigma factors that could potentially regulate acsA expression, including:

    • rpoD (σ70): The principal sigma factor

    • rpoH (σ32): The heat shock sigma factor

    • rpoE (σE): A single ECF sigma factor

    • rpoN (σ54): Belongs to the sigma 54 family

  • The ECF sigma factor (σE) in Xylella fastidiosa regulates genes involved in protein folding and degradation, signal transduction, and DNA restriction modification , suggesting potential involvement in metabolic gene regulation like acsA.

• Promoter Structure:

  • Based on mapped promoters of σE-dependent genes in Xylella fastidiosa, a consensus sequence of GAACnn-[N]16-17-GTCnnA has been identified .

  • This consensus is similar to E. coli σE and P. aeruginosa AlgU binding sites.

What is the role of acsA in Xylella fastidiosa virulence and host colonization?

While the search results do not directly address the specific role of acsA in Xylella fastidiosa virulence, we can infer its potential importance based on general metabolic functions and host-pathogen interactions:

• Carbon Metabolism During Infection:

  • As Xylella fastidiosa colonizes the nutrient-poor environment of plant xylem, efficient utilization of available carbon sources, including acetate, is likely critical for successful infection.

  • acsA would enable the utilization of acetate as a carbon source during colonization of plant hosts.

• Biofilm Formation:

  • Xylella fastidiosa produces exopolysaccharides (EPS) and forms robust biofilms in both plant hosts and insect vectors .

  • Central metabolic pathways, potentially involving acetyl-CoA produced by acsA, contribute to the synthesis of biofilm components.

  • Interestingly, biofilm formation in Xylella fastidiosa appears to attenuate movement in the xylem, which can slow disease progression .

• Adaptation to Different Hosts:

  • Xylella fastidiosa can infect over 600 plant species, but behaves as a benign commensal in most and as a pathogen in only some hosts like grapevines .

  • Metabolic flexibility, potentially involving acsA-mediated acetate utilization, may contribute to this host adaptation capacity.

How do experimental conditions affect the detection of Xylella fastidiosa acsA expression in infected plant samples?

Detection of Xylella fastidiosa acsA expression in plant samples requires consideration of several experimental factors:

• Sample Collection and Storage:

  • Studies have shown that bacterial leaf scorch suspect samples can remain at ambient temperature for up to six days after collection without adversely affecting detectability of Xylella fastidiosa .

  • This suggests that RNA stability for gene expression studies may be similarly maintained over short periods.

• Non-uniform Distribution:

  • Xylella fastidiosa is non-uniformly distributed in host plants, which can affect detection success .

  • Multiple sampling from different plant tissues is recommended to increase detection probability.

• Extraction Methods:

  • Studies have shown that DNA extracted from ELISA preparations can be successfully used for PCR detection .

  • For RNA extraction and gene expression analysis, rapid processing in RNase-free conditions is essential.

• Detection Techniques:

  • Real-time PCR (qPCR) has been successfully used for Xylella fastidiosa detection regardless of sample storage conditions .

  • For gene expression studies, RT-qPCR with appropriate reference genes would be recommended.

  • RNA-Seq approaches can provide comprehensive expression profiles.

How can recombinant Xylella fastidiosa acsA be utilized in developing control strategies for Xylella-associated diseases?

Recombinant Xylella fastidiosa acsA could be leveraged in several control strategies:

• Target-based Antimicrobial Development:

  • Understanding the structure and function of acsA could aid in the design of specific inhibitors that disrupt acetate metabolism in Xylella fastidiosa.

  • Such inhibitors could potentially reduce bacterial survival in plant xylem.

• Diagnostic Tool Development:

  • Antibodies raised against recombinant acsA could be used in immunodetection assays for early diagnosis of Xylella fastidiosa infections.

  • Species-specific epitopes could enable differentiation between Xylella subspecies.

• Resistance Screening:

  • Recombinant acsA could be used to screen for plant compounds that inhibit its activity.

  • Plants producing such inhibitors may have enhanced resistance to Xylella fastidiosa.

• Vaccination Strategies:

  • While not conventional for plant diseases, recombinant acsA could potentially be used to develop strategies that prime plant immune responses against Xylella fastidiosa.

How can protein-protein interaction studies with recombinant acsA enhance our understanding of Xylella fastidiosa metabolism?

Investigating protein-protein interactions involving acsA can provide insights into metabolic networks:

• Identification of Interaction Partners:

  • Pull-down assays using tagged recombinant acsA as bait

  • Yeast two-hybrid screening

  • Proximity-based labeling approaches (BioID, APEX)

• Regulatory Interactions:

  • Based on B. subtilis and S. enterica models, acsA likely interacts with:

    • Acetyltransferases (analogous to AcuA)

    • Deacetylases (analogous to AcuC or CobB)

    • These interactions regulate acsA activity through post-translational acetylation

• Metabolic Complexes:

  • acsA may participate in multi-enzyme complexes involving other acetate metabolism enzymes

  • Such complexes could facilitate substrate channeling and metabolic efficiency

• Methods for Interaction Validation:

  • Co-immunoprecipitation

  • Surface plasmon resonance

  • Isothermal titration calorimetry

  • Fluorescence resonance energy transfer (FRET)

What are the comparative differences between recombinant acsA from different Xylella fastidiosa subspecies?

Comparative analysis of acsA from different Xylella fastidiosa subspecies could reveal:

• Sequence Variation:

  • Multilocus sequence typing (MLST) studies have shown genetic differences between Xylella fastidiosa subspecies .

  • These differences likely extend to metabolic genes like acsA.

• Functional Differences:

  • Substrate affinity (Km for acetate)

  • Catalytic efficiency (kcat/Km)

  • Thermal stability

  • pH optima

  • Susceptibility to acetylation regulation

• Structural Variations:

  • X-ray crystallography or cryo-EM studies could reveal structural differences

  • Molecular dynamics simulations could predict how sequence variations affect protein dynamics

• Experimental Approach:

  • Express and purify recombinant acsA from multiple subspecies (fastidiosa, pauca, multiplex, morus)

  • Perform comparative biochemical characterization

  • Correlate differences with host specificity and virulence characteristics

What are the challenges in developing acsA-targeted therapeutic approaches for controlling Xylella fastidiosa infections?

Development of acsA-targeted therapeutics faces several challenges:

• Target Specificity:

  • acsA is conserved across many bacterial species and has homologs in plants

  • Achieving specificity for Xylella fastidiosa acsA requires detailed structural understanding

  • In silico screening can identify compounds that bind specifically to unique regions of Xylella fastidiosa acsA

• Delivery Methods:

  • Xylella fastidiosa resides in plant xylem, requiring compounds with appropriate physicochemical properties for xylem mobility

  • Systemic delivery through roots or stems would be necessary

  • Compounds must be stable in planta and resist plant metabolism

• Resistance Development:

  • Single-target approaches risk rapid development of resistance

  • Combination strategies targeting multiple metabolic enzymes may be more effective

  • Understanding the genetic diversity and recombination potential of acsA is crucial for predicting resistance emergence

• Regulatory Approval:

  • Novel antimicrobials for plant pathogens require extensive testing for:

    • Efficacy against various Xylella fastidiosa subspecies

    • Safety for non-target organisms

    • Environmental impact assessment

    • Residue analysis in agricultural products

How can contamination issues be addressed when working with recombinant Xylella fastidiosa acsA?

Researchers working with recombinant Xylella fastidiosa acsA may encounter various contamination challenges:

• Endotoxin Contamination:

  • As a Gram-negative bacterium, Xylella fastidiosa contains lipopolysaccharides that can co-purify with recombinant proteins.

  • Solution: Use endotoxin removal columns or treatments with Triton X-114.

  • Verification: Limulus amebocyte lysate (LAL) assay to confirm endotoxin removal.

• Nucleic Acid Contamination:

  • DNA/RNA can co-purify with proteins, affecting downstream applications.

  • Solution: Treat with Benzonase nuclease during purification.

  • Verification: Measure A260/A280 ratio; values close to 0.6 indicate pure protein.

• Host Protein Contamination:

  • Expression host proteins may co-purify with the target protein.

  • Solution: Implement multiple purification steps (ion exchange, size exclusion chromatography).

  • Verification: SDS-PAGE with silver staining or mass spectrometry analysis.

• Microbial Contamination During Storage:

  • Solution: Add sodium azide (0.02%) to storage buffers for non-enzymatic applications.

  • Solution: Filter-sterilize protein solutions through 0.22 μm filters.

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

What strategies can optimize the yield and activity of recombinant Xylella fastidiosa acsA?

Optimizing recombinant acsA production requires attention to several factors:

• Expression Conditions:

  • Temperature: Lower induction temperatures (16-18°C) often increase soluble protein yield.

  • Induction time: Extended expression periods (overnight) at lower IPTG concentrations.

  • Medium composition: Enriched media (TB, 2xYT) can increase biomass and protein yield.

  • Co-expression with chaperones: Can improve folding of complex proteins.

• Protein Solubility:

  • Fusion tags: Solubility-enhancing tags (MBP, SUMO) can improve yield of soluble protein.

  • Buffer optimization: Screen various buffers, pH values, and salt concentrations.

  • Additives: Glycerol (5-10%), reducing agents (DTT, TCEP), and specific ions may stabilize the protein.

• Purification Strategy:

  • Multi-step approach: Combine affinity chromatography with ion exchange and size exclusion.

  • On-column refolding: For proteins recovered from inclusion bodies.

  • Limited proteolysis: Remove flexible regions that may cause aggregation.

• Reconstitution Protocol:

  • Centrifuge vial before opening to bring contents to the bottom.

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

  • Add glycerol to 5-50% final concentration for long-term storage .

How can researchers distinguish between endogenous and recombinant acsA when studying Xylella fastidiosa?

Distinguishing between endogenous and recombinant acsA requires careful experimental design:

• Epitope Tagging:

  • Incorporate epitope tags (His, FLAG, HA) into recombinant constructs.

  • Use tag-specific antibodies for selective detection.

  • Example detection method: Western blotting with anti-tag antibodies.

• Size Differentiation:

  • Design recombinant constructs with fusion proteins that alter molecular weight.

  • Separate proteins by SDS-PAGE and detect by immunoblotting.

  • Resolution requirement: Polyacrylamide percentage should be optimized to resolve small size differences.

• Genetic Marking:

  • Introduce silent mutations that allow differentiation at the nucleic acid level.

  • Design PCR primers that specifically amplify either endogenous or recombinant sequences.

  • Application: Useful for tracking recombinant gene expression in vivo.

• Mass Spectrometry Approaches:

  • Isotope labeling: Express recombinant protein in media with heavy isotopes.

  • Peptide mass fingerprinting: Identify unique peptides that distinguish variants.

  • Selective reaction monitoring (SRM): Target specific peptides unique to each form.

• Functional Complementation:

  • Express recombinant acsA in mutant strains lacking endogenous acsA activity.

  • Example system: Similar to the Salmonella enterica complementation system used for B. subtilis AcsA studies .