Recombinant Xylella fastidiosa Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is the biochemical function of Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) in Xylella fastidiosa?

Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) is a critical enzyme in the citric acid cycle (also known as the Krebs cycle or tricarboxylic acid cycle) of Xylella fastidiosa. This enzyme specifically catalyzes the reversible conversion of succinyl-CoA to succinate, coupled with the formation of ATP from ADP and inorganic phosphate. The reaction can be represented as:

Succinyl-CoA + Pi + ADP → Succinate + CoA + ATP

This reaction represents one of the substrate-level phosphorylation steps in the citric acid cycle, making it essential for energy production in X. fastidiosa. The ADP-forming subunit beta (sucC) specifically participates in the formation of ATP, distinguishing it from GDP-forming variants of the enzyme found in some other organisms.

How does Xylella fastidiosa's metabolic activity relate to its pathogenicity in plants?

Xylella fastidiosa is a xylem-limited bacterium responsible for economically significant plant diseases, including Pierce's disease in grapevines and citrus variegated chlorosis in citrus trees . Its metabolic activities are directly connected to its pathogenicity through several mechanisms:

• Adaptation to xylem environment: X. fastidiosa has evolved metabolic pathways to thrive in the nutrient-poor xylem environment, including specialized energy production systems like the citric acid cycle in which sucC participates .

• Biofilm formation: Metabolic activity influences biofilm formation, which is critical for virulence. Transcriptome studies show differential gene expression between growth media that resemble xylem fluid (3G10-R) versus complex media (PW), with genes upregulated in 3G10-R often being important for plant colonization and virulence .

• Energy homeostasis: Proteins like Succinyl-CoA ligase are essential for maintaining energy homeostasis within cells, enabling the bacterium to persist and multiply within the hostile plant environment.

Researchers studying the pathogen's metabolism should consider these connections when designing experiments to investigate virulence factors or potential control methods.

What are the optimal conditions for assaying Recombinant Xylella fastidiosa Succinyl-CoA ligase activity?

Based on available enzymatic data for prokaryotic Succinyl-CoA synthetase, the following conditions are generally optimal for assaying activity:

When designing your assay, remember that enzymatic activity can be measured in both forward and reverse directions. For the forward reaction (succinate to succinyl-CoA), you would monitor ATP consumption, while for the reverse reaction, you would track ATP production. Spectrophotometric methods coupling the reaction to NADH oxidation or reduction are commonly used for continuous monitoring of activity .

How can experimental designs utilizing natural competence in X. fastidiosa be applied to study sucC function?

Xylella fastidiosa exhibits natural competence—the ability to take up and incorporate exogenous DNA into its genome through homologous recombination . This characteristic provides a powerful tool for studying gene function, including sucC. A comprehensive experimental design would include:

Methodology for utilizing natural competence to study sucC:

• Construct a donor DNA template: Design a DNA construct containing an antibiotic resistance marker flanked by homologous regions to the sucC gene. This can be used for gene knockout, modification, or complementation studies .

• Optimize recombination conditions: Natural competence rates vary significantly among X. fastidiosa strains, with frequencies ranging from below detection limit to 0.024 per recipient cell . Select a strain with high recombination frequency for initial studies, such as subspecies fastidiosa strains which generally demonstrate higher rates than subspecies multiplex .

• Use microfluidic chambers for transformation: Recombination frequencies are significantly higher under flow conditions in microfluidic chambers (MCs) than in batch culture conditions. These chambers better mimic the natural xylem vessel environment .

• Media selection: PD3 medium yields the highest recombination frequency compared to XFM or PW media. Avoid media components containing bovine serum albumin, which inhibits recombination by affecting twitching motility .

• Verification of recombinants: Confirm successful gene modifications using PCR, DNA sequencing of flanking regions, and phenotypic analysis .

This approach allows for precise genetic manipulation of sucC to study its role in metabolism, pathogenicity, and adaptation to different plant hosts.

What techniques are effective for analyzing the role of sucC in X. fastidiosa's adaptation to different plant hosts?

Analyzing the role of sucC in X. fastidiosa's adaptation to different plant hosts requires a multi-faceted approach combining transcriptomics, comparative genomics, and functional studies:

• Transcriptome analysis under host-mimicking conditions:

  • Compare gene expression of sucC in media that resemble different host xylem compositions (e.g., grape sap vs. citrus sap) .

  • Use microarray or RNA-Seq to identify co-regulated genes that might form functional networks with sucC .

  • Example from research: Transcriptome analysis of X. fastidiosa grown in 3G10-R (grape sap-like medium) versus PW (complex medium) revealed 299 differentially expressed transcripts, providing insights into genes important for plant colonization .

• Comparative analysis across strains with different host ranges:

  • Examine sequence conservation and expression patterns of sucC across strains that infect different hosts.

  • Analyze strain-specific gene regulation networks involving sucC.

  • Utilize natural competence and recombination to study intersubspecific gene transfer .

• In planta studies with genetically modified strains:

  • Create sucC knockdown/knockout mutants using natural competence methodologies .

  • Assess colonization ability, virulence, and metabolic adaptation in different host plants.

  • Use microfluidic chambers with host plant xylem sap to simulate in planta conditions .

• Metabolic profiling:

  • Compare metabolite production in wild-type versus sucC-modified strains.

  • Assess energy production efficiency under conditions mimicking different host environments.

This integrated approach can reveal how sucC contributes to the metabolic adaptations required for X. fastidiosa to colonize and cause disease in different plant species.

What are the methodological approaches for investigating potential interactions between sucC and X. fastidiosa virulence factors?

Investigating interactions between sucC and virulence factors requires connecting central metabolism to pathogenicity mechanisms. Here are methodological approaches to explore these connections:

• Protein-protein interaction studies:

  • Implement bacterial two-hybrid systems to screen for physical interactions between sucC and known virulence factors.

  • Use co-immunoprecipitation followed by mass spectrometry to identify interaction partners in vivo.

  • Apply proximity-dependent biotin labeling (BioID) to detect transient or weak interactions in the native cellular environment.

• Gene co-expression network analysis:

  • Perform RNA-Seq under various conditions that induce virulence (e.g., biofilm formation, plant extract exposure).

  • Construct co-expression networks to identify genes whose expression patterns correlate with sucC.

  • Compare with known virulence regulons to identify potential regulatory connections.

• Genetic interaction mapping:

  • Create a sucC conditional expression strain where enzyme levels can be modulated.

  • Combine with mutations in virulence factors to identify synthetic phenotypes that suggest functional relationships.

  • Utilize the natural competence of X. fastidiosa for genetic manipulations .

• Metabolic flux analysis:

  • Trace carbon flow through central metabolism using isotope-labeled substrates.

  • Compare metabolic flux distributions between wild-type and virulence factor mutants.

  • Identify altered flux through the sucC-catalyzed reaction that may connect to virulence mechanisms.

• Biofilm formation assessment:

  • Evaluate how sucC expression affects biofilm development, a critical virulence determinant.

  • Similar to studies with the cold shock protein homolog (Csp1) that influenced biofilm formation and pili production .

  • Utilize microscopy and crystal violet assays to quantify biofilm changes.

Implementation of these complementary approaches can reveal whether and how sucC contributes to virulence beyond its canonical metabolic role.

How do environmental conditions in plant xylem vessels affect the expression and function of Succinyl-CoA ligase in X. fastidiosa?

The unique environment of plant xylem vessels significantly impacts gene expression and protein function in X. fastidiosa, including Succinyl-CoA ligase. Understanding these environmental effects requires specialized experimental approaches:

• Microfluidic chamber systems for environmental simulation:

  • Utilize microfluidic chambers (MCs) to mimic the liquid flow conditions of xylem vessels .

  • Incorporate actual plant xylem sap from different host species (e.g., grapevine varieties like Chardonnay or Blanc Du Bois) into growth media .

  • Monitor gene expression and protein activity under these flow conditions versus static cultures.

• Transcriptomic profiling under xylem-like conditions:

  • Compare sucC expression in media resembling xylem fluid (e.g., 3G10-R for grape sap) versus standard complex media (PW) .

  • Research has shown that X. fastidiosa cells grown in xylem-like media express different genes than those in complex media, with xylem-induced genes often related to plant colonization and virulence .

• Nutrient limitation studies:

  • Systematically vary concentrations of key nutrients found in xylem (nitrogen sources, minerals, carbon compounds) to determine their effects on sucC expression.

  • Correlate expression changes with metabolic shifts and energy production.

• Host specificity analysis:

  • Compare sucC expression and enzyme activity in response to sap from different plant hosts.

  • Research has shown that recombination frequencies differ between media supplemented with sap from different grapevine varieties (e.g., higher in Blanc Du Bois compared to Chardonnay sap) , suggesting host-specific factors affect bacterial physiology.

• In planta gene expression:

  • Extract bacteria directly from infected plants to analyze native gene expression patterns.

  • Compare with in vitro expression to identify environmentally responsive regulation.

These methodological approaches can help researchers understand how the specialized xylem environment shapes X. fastidiosa metabolism through effects on Succinyl-CoA ligase expression and function.

What experimental designs are most effective for studying the relationship between sucC activity and bacterial fitness during different growth phases?

To effectively study the relationship between sucC activity and bacterial fitness across growth phases, researchers should implement a multi-faceted experimental design that integrates temporal analysis with functional assessments:

This comprehensive experimental approach will provide insights into how sucC activity is regulated throughout bacterial growth and how it contributes to fitness under different physiological states.

What are the most effective protocols for expressing and purifying Recombinant X. fastidiosa Succinyl-CoA ligase for structural studies?

For structural studies of Recombinant X. fastidiosa Succinyl-CoA ligase, high-quality protein with proper folding and activity is essential. Here is a methodological approach for expression and purification:

• Expression system selection:

  • Bacterial expression: E. coli BL21(DE3) is recommended for initial trials due to its reduced protease activity and high expression yields.

  • Vector design: Construct a pET-based vector containing the sucC gene with a C-terminal 6xHis-tag to facilitate purification while minimizing interference with enzyme activity.

  • Codon optimization: Consider codon optimization for E. coli if initial expression yields are low, as X. fastidiosa has different codon usage patterns.

• Expression optimization:

  • Temperature: Test expression at 16°C, 25°C, and 37°C; lower temperatures often improve folding of complex proteins.

  • Induction conditions: Compare IPTG concentrations (0.1-1.0 mM) and induction times (4h vs. overnight).

  • Media composition: Test standard LB versus enriched media like Terrific Broth for higher yields.

  • Co-expression strategies: Consider co-expressing with chaperones if solubility is an issue.

• Purification protocol:

  • Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin.

  • Secondary purification: Ion exchange chromatography (IEX) to remove contaminants with similar affinity for IMAC.

  • Polishing step: Size exclusion chromatography (SEC) to achieve high purity and remove aggregates.

  • Buffer optimization: Include stabilizing agents like glycerol (10-20%) and reducing agents (1-5 mM DTT or 2-ME) in purification buffers.

• Quality control assessments:

  • Purity analysis: SDS-PAGE with Coomassie staining (aim for >95% purity).

  • Activity assay: Verify enzyme activity using spectrophotometric methods .

  • Thermal stability: Differential scanning fluorimetry (DSF) to identify optimal buffer conditions.

  • Oligomeric state analysis: Analytical SEC or dynamic light scattering (DLS).

• Structural biology preparation:

  • Crystallization trials: Screen various precipitants, buffers, and additives using commercial crystallization kits.

  • Cryoprotection optimization: Test glycerol, PEG, sucrose, or other cryoprotectants.

  • Alternative approaches: Consider small-angle X-ray scattering (SAXS) or cryo-electron microscopy if crystallization proves challenging.

Following this systematic approach should yield protein suitable for high-resolution structural studies, providing insights into the molecular mechanisms of this important metabolic enzyme.

How can researchers design experiments to distinguish between the metabolic and potential regulatory roles of sucC in X. fastidiosa?

Distinguishing between metabolic and regulatory roles of sucC requires experimental designs that can separate these functions. Here's a methodological framework:

• Catalytic versus structural mutant analysis:

  • Create point mutations that specifically disrupt the catalytic activity while maintaining protein structure.

  • Compare with complete gene deletion mutants.

  • Assess whether phenotypic differences exist between catalytically inactive protein versus absence of protein, which would suggest non-catalytic roles.

• Protein-protein interaction network mapping:

  • Implement affinity purification coupled with mass spectrometry (AP-MS) to identify the interactome of Succinyl-CoA ligase.

  • Use bacterial two-hybrid screening to detect binary interactions.

  • Analyze interaction partners for enrichment of regulatory proteins versus metabolic enzymes.

• Differential expression analysis with metabolic uncoupling:

  • Apply metabolic inhibitors that block TCA cycle but preserve cellular ATP levels through alternative pathways.

  • Assess whether sucC expression responds to signals beyond metabolic needs.

  • Compare with known regulatory networks, such as those involving the cold shock protein Csp1 that affects gene expression and biofilm formation .

• Domain function analysis:

  • Create chimeric proteins with domains from other bacterial species.

  • Assess whether specific domains contribute to functions beyond catalysis.

  • Use domain deletion constructs to map regions responsible for potential regulatory interactions.

• Temporal resolution studies:

  • Implement time-course experiments following environmental shifts.

  • Compare the kinetics of sucC expression changes with metabolic adjustments and expression of potential target genes.

  • Use mathematical modeling to distinguish direct regulatory effects from indirect metabolic consequences.

• Heterologous expression complementation:

  • Express sucC homologs from non-pathogenic bacteria in X. fastidiosa sucC mutants.

  • Determine whether metabolic function is restored without regulatory effects.

  • This approach can separate conserved catalytic functions from species-specific regulatory roles.

By implementing these complementary approaches, researchers can build evidence for potential regulatory roles of sucC that extend beyond its well-established metabolic function in the TCA cycle.

What considerations should be taken into account when designing experiments to investigate sucC expression during X. fastidiosa infection in different plant hosts?

Investigating sucC expression during infection across different plant hosts presents unique challenges requiring specialized experimental design considerations:

• Plant host selection and experimental system design:

  • Host diversity: Include both susceptible and resistant varieties of the same plant species (e.g., Chardonnay grapevines versus resistant varieties) .

  • Time-course design: Plan sampling at multiple timepoints post-inoculation to capture different infection stages.

  • Inoculation method standardization: Use consistent inoculation techniques (needle puncture, insect transmission) across experiments for comparability.

  • Controls: Include mock-inoculated plants and in vitro bacterial cultures as reference points.

• Bacterial recovery and RNA preservation methods:

  • Extraction protocols: Optimize protocols for bacterial isolation from plant xylem with minimal contamination from plant material.

  • RNA stabilization: Implement immediate RNA stabilization upon sampling to prevent expression changes during processing.

  • Low biomass considerations: Design approaches for working with low bacterial numbers in early infection stages or resistant hosts.

  • Sample pooling strategies: Consider pooling samples from multiple infection sites to obtain sufficient material while accounting for site-to-site variability.

• Expression analysis approaches:

  • RT-qPCR optimization: Design primers specific to X. fastidiosa sucC that won't amplify plant homologs.

  • Reference gene selection: Validate stable reference genes under in planta conditions rather than assuming stability of standard housekeeping genes.

  • RNA-Seq considerations: Implement rRNA depletion and bacterial enrichment strategies for whole transcriptome analysis .

  • In situ approaches: Consider RNAscope or similar techniques for visualizing gene expression in the context of plant tissues.

• Confounding factor control:

  • Host response variables: Monitor plant defense responses that might indirectly affect bacterial gene expression.

  • Bacterial population density effects: Normalize expression data to account for different bacterial loads across samples.

  • Microenvironmental conditions: Record temperature, humidity, and light conditions that might influence both plant physiology and bacterial responses.

  • Mixed infections: Screen for potential contaminating microorganisms that might be present in the xylem.

• Validation approaches:

  • Reporter systems: Develop fluorescent or luminescent reporters for sucC expression for spatial-temporal visualization in planta.

  • Protein-level confirmation: Use immunodetection or targeted proteomics to confirm that transcriptional changes translate to protein abundance.

  • Microfluidic chambers with xylem sap: Use extracted xylem sap in microfluidic systems as an intermediate validation approach between in vitro and in planta conditions .

These methodological considerations will help researchers obtain reliable data on sucC expression during plant infection, enabling insights into its role in host-pathogen interactions.

How does X. fastidiosa Succinyl-CoA ligase compare structurally and functionally to orthologs in other bacteria, particularly other plant pathogens?

Comparative analysis of X. fastidiosa Succinyl-CoA ligase with orthologs in other bacteria provides evolutionary insights and potential targets for intervention. Here's a methodological approach to this comparative analysis:

• Sequence-based comparative analysis:

  • Perform multiple sequence alignment of sucC sequences from diverse bacteria, particularly focusing on:

    • Other plant pathogens (e.g., Ralstonia, Pseudomonas, Erwinia)

    • Closely related non-pathogens (e.g., Xanthomonas)

    • Model organisms with well-characterized enzymes (e.g., E. coli, B. subtilis)

  • Identify conserved catalytic residues versus variable regions that might confer species-specific properties.

  • Calculate evolutionary rates across different protein domains to identify regions under selection pressure.

• Structural comparison methodology:

  • Develop homology models of X. fastidiosa Succinyl-CoA ligase based on existing crystal structures from other bacteria.

  • Compare substrate binding pockets, active sites, and oligomerization interfaces.

  • Analyze electrostatic surface potentials to identify potential differences in substrate preference or protein-protein interactions.

  • Implement molecular dynamics simulations to compare conformational flexibility.

• Functional comparison approaches:

  • Express recombinant enzymes from multiple species under identical conditions.

  • Compare biochemical parameters (Km, kcat, substrate specificity, pH optima) under standardized assay conditions .

  • Test thermostability and resistance to inhibitors to identify potential functional differences.

  • Perform complementation studies by expressing heterologous sucC genes in X. fastidiosa mutants.

• Genomic context analysis:

  • Compare the organization of TCA cycle genes across species.

  • Identify potential regulatory elements (promoters, terminators, transcription factor binding sites) that differ between species.

  • Assess whether sucC is part of different operons or regulons in different bacteria.

• Host-specific adaptation analysis:

  • Correlate sequence/structural features with host range of different pathogens.

  • Determine whether enzymes from bacteria adapted to similar ecological niches share common features despite phylogenetic distance.

  • Compare with homologs from the related plant symbiont Rhizobium loti to identify potential pathogen-specific characteristics.

This comprehensive comparative approach can reveal how X. fastidiosa Succinyl-CoA ligase has potentially evolved specific features relevant to its lifestyle as a xylem-limited plant pathogen, distinguishing it from orthologs in other bacteria.

What evidence suggests that sucC may have been involved in horizontal gene transfer or recombination events among X. fastidiosa strains?

Analyzing potential horizontal gene transfer (HGT) or recombination events involving sucC requires a systematic approach combining genomic analysis with experimental validation. Here's a methodological framework:

• Phylogenetic incongruence detection:

  • Construct phylogenetic trees based on:

    • sucC gene sequences from multiple X. fastidiosa strains and related species

    • Concatenated sequences of multiple housekeeping genes

    • Whole genome SNP analysis

  • Compare topology of sucC-based trees with species/strain trees

  • Significant incongruence may indicate HGT or recombination events

• Sequence-based recombination detection:

  • Implement multiple recombination detection algorithms (e.g., RDP, GENECONV, MaxChi)

  • Apply sliding window analyses to detect localized regions with aberrant evolutionary histories

  • Calculate linkage disequilibrium patterns around the sucC locus

  • Analyze GC content and codon usage bias for sucC versus genomic average

• Experimental validation using natural competence:

  • Utilize the documented natural competence of X. fastidiosa to test sucC transfer between strains

  • Design experiments based on established protocols showing intersubspecific recombination between subspecies fastidiosa and multiplex

  • Create donor strains with marked sucC genes and monitor transfer frequencies

  • Research has shown recombination frequencies vary greatly among strains (from below detection limit to 0.024 per recipient cell) , so select appropriate strains

• Growth condition optimization for recombination studies:

  • Use PD3 medium which yields highest recombination frequencies compared to XFM or PW media

  • Implement microfluidic chambers that simulate natural xylem vessel environments and increase recombination rates

  • Include conditions that promote twitching motility, which correlates with higher recombination frequency

• Comparative genomic analysis across subspecies:

  • Analyze sucC sequence variation patterns across X. fastidiosa subspecies (fastidiosa, multiplex, pauca)

  • Look for mosaic structures indicating past recombination events

  • Compare with genomic regions known to be involved in recombination events

  • Examine flanking regions for mobile genetic elements that might facilitate HGT

The well-documented natural competence of X. fastidiosa makes sucC a potential candidate for horizontal transfer between strains, potentially contributing to metabolic adaptation as strains evolve to colonize different plant hosts.

How might understanding the role of Succinyl-CoA ligase contribute to developing novel control strategies for X. fastidiosa infections?

Developing control strategies based on Succinyl-CoA ligase requires translating basic research into applied approaches. Here's a methodological framework for this translation:

• Target validation approaches:

  • Create conditional sucC mutants to confirm essentiality under various conditions.

  • Implement tissue-specific or temporal gene regulation systems to determine when and where the enzyme is most critical.

  • Quantify fitness costs of sucC disruption in different environments (in vitro, in planta, in insect vectors).

  • Determine whether partial inhibition is sufficient to prevent disease progression.

• Structure-based inhibitor design methodology:

  • Perform virtual screening of compound libraries against the enzyme's active site.

  • Implement fragment-based drug design approaches to develop novel inhibitors.

  • Assess selectivity by comparing with host plant homologs to ensure minimal off-target effects.

  • Test promising compounds in enzyme assays, followed by cell-based assays and plant infection models.

• Alternative control strategy development:

  • Explore RNA interference (RNAi) approaches targeting sucC mRNA.

  • Design antimicrobial peptides that disrupt protein-protein interactions involving Succinyl-CoA ligase.

  • Develop engineered phages carrying CRISPR-Cas systems to specifically target sucC.

  • Investigate competitive exclusion using non-pathogenic strains with modified sucC.

• Delivery system optimization:

  • Test trunk injection methods for delivering inhibitors into the xylem.

  • Develop nanoparticle formulations for sustained release of inhibitors.

  • Explore grafting of transgenic rootstocks expressing inhibitory molecules.

  • Consider vector management strategies that might reduce transmission efficiency.

• Resistance management planning:

  • Assess the likelihood of resistance development through in vitro evolution experiments.

  • Design multi-target approaches that simultaneously affect sucC and other essential pathways.

  • Model the impact of various intervention strategies on pathogen population dynamics and resistance evolution.

  • Develop diagnostic tools to monitor for the emergence of resistant strains.

By systematically addressing these aspects, researchers can translate fundamental knowledge about Succinyl-CoA ligase into practical control strategies that might help manage devastating diseases caused by X. fastidiosa.

What future research directions would most advance our understanding of the relationship between central metabolism and virulence in X. fastidiosa?

Advancing our understanding of the metabolism-virulence nexus in X. fastidiosa requires integrative approaches spanning multiple disciplines. Here are methodological frameworks for future research:

• Systems biology approaches:

  • Implement genome-scale metabolic modeling to predict metabolic flux distributions under different conditions.

  • Integrate transcriptomic, proteomic, and metabolomic data into multi-omics networks.

  • Develop condition-specific models that account for differences between in vitro growth and in planta environments.

  • Use flux balance analysis to identify metabolic vulnerabilities that could be targeted for disease control.

• Spatiotemporal resolution studies:

  • Develop reporter strains for real-time monitoring of metabolic activity in situ.

  • Implement microfluidic devices that mimic the physical and chemical properties of xylem vessels .

  • Use advanced microscopy techniques to correlate metabolic activity with biofilm formation and virulence factor expression.

  • Track metabolic changes during different stages of plant colonization and disease development.

• Comparative approaches across strains with different virulence profiles:

  • Analyze metabolic network differences between highly virulent and less virulent strains.

  • Perform experimental evolution under different selective pressures followed by metabolic profiling.

  • Utilize natural competence to create recombinant strains with hybrid metabolic capabilities .

  • Compare metabolic adaptation strategies across different subspecies (fastidiosa, multiplex, pauca).

• Host-pathogen metabolic interaction studies:

  • Investigate how host plant metabolites influence X. fastidiosa central metabolism.

  • Examine differences in bacterial metabolic responses to sap from susceptible versus resistant plant varieties.

  • Develop co-culture systems that model the complex microbial communities in plant xylem.

  • Implement isotope labeling studies to track nutrient exchange between host and pathogen.

• Targeted genetic approaches:

  • Create a comprehensive library of central metabolism mutants using natural competence methodologies .

  • Implement CRISPRi for tunable repression of metabolic genes to avoid lethality.

  • Develop synthetic biology tools for rewiring metabolic pathways to test hypotheses about metabolic control of virulence.

  • Use metabolic sensors to isolate bacteria with altered metabolic states directly from infected plants.

These research directions would significantly advance our understanding of how central metabolic enzymes like Succinyl-CoA ligase contribute to X. fastidiosa virulence and adaptation to different plant hosts, potentially revealing new approaches for disease management.