Recombinant Xylella fastidiosa Putative reductase PD_1032 (PD_1032)

What is the genomic context of the PD_1032 gene in Xylella fastidiosa?

PD_1032 is encoded within the genome of Xylella fastidiosa, an important bacterial plant pathogen causing high-consequence diseases in agricultural crops globally. The gene is part of the core genome found across X. fastidiosa strains. While X. fastidiosa demonstrates significant strain variability regarding virulence on specific host plants, PD_1032 appears to be conserved across subspecies . The genomic context may vary slightly between different X. fastidiosa subspecies such as pauca (Xfp) and multiplex (Xfm), which show different host plant preferences . Comparative genomic analyses across the 129 X. fastidiosa genome assemblies representing all known subspecies and 32 sequence types indicate conservation of metabolic functions, including putative reductases like PD_1032 .

What are the structural characteristics and predicted functions of the PD_1032 protein?

The PD_1032 protein is a putative reductase involved in metabolic pathways that may contribute to X. fastidiosa's survival within plant xylem tissue. As a reductase, it likely catalyzes reduction reactions essential for bacterial metabolism, potentially playing a role in the bacterium's adaptation to the xylem environment. Structural predictions suggest typical reductase domains with conserved active sites for cofactor binding, likely including NAD(P)H binding domains characteristic of oxidoreductases. The protein may function in redox homeostasis, particularly important for X. fastidiosa survival within the oxidative stress environment of plant xylem vessels during infection.

How does PD_1032 expression vary across different growth conditions and infection stages?

Expression of PD_1032 varies depending on growth conditions and stages of plant infection. Transcriptomic analyses reveal differential expression patterns between in vitro cultures and in planta conditions, suggesting environmental regulation . During early infection stages, when the bacterium first colonizes xylem vessels, PD_1032 expression may be upregulated to help counter plant defense responses, particularly oxidative stress. Expression patterns can be monitored using RT-qPCR assays targeting PD_1032 transcripts from infected plant tissues, similar to methodologies used for other X. fastidiosa genes like cvaC-1, which demonstrated expression differences between inoculation points and systemic infection sites . The expression patterns may also differ between subspecies, with X. fastidiosa subspecies pauca and X. fastidiosa subspecies multiplex showing distinct expression profiles in various host plants.

What are the optimal expression systems for producing recombinant PD_1032 protein?

The optimal expression system for recombinant PD_1032 depends on research goals and downstream applications. For bacterial expression, E. coli BL21(DE3) remains the most versatile host for initial attempts, offering high yields and simplicity. For proper folding of this putative reductase, consider E. coli strains with enhanced disulfide bond formation capabilities such as Origami or SHuffle strains.

For expression, a pET system with an N-terminal His-tag facilitates purification while maintaining enzymatic activity. Optimize expression conditions with this general protocol:

• Transform expression plasmid into host strain

• Culture in LB medium at 37°C until OD600 reaches 0.6-0.8

• Induce with 0.1-0.5 mM IPTG

• Lower temperature to 16-25°C post-induction

• Continue expression for 12-18 hours

• Harvest cells by centrifugation

Alternative systems include yeast (Pichia pastoris) for glycosylated versions if necessary, though bacterial systems generally suffice for functional studies of bacterial reductases.

What purification strategies yield the highest activity for recombinant PD_1032?

Purification strategies that preserve the enzymatic activity of PD_1032 typically employ gentle conditions maintaining the protein's native conformation. A multi-step purification protocol yielding high activity includes:

• Cell lysis in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitors

• Initial capture using immobilized metal affinity chromatography (IMAC) with Ni-NTA resin for His-tagged protein

• Buffer exchange to remove imidazole using dialysis or gel filtration

• Ion exchange chromatography as a polishing step

• Final size exclusion chromatography in buffer containing reducing agent

Critical considerations include maintaining reducing conditions throughout purification (1-5 mM DTT or 0.5-2 mM β-mercaptoethanol) to preserve cysteine residues often essential for reductase activity. Adding cofactors like NADPH (0.1-0.5 mM) to buffers can enhance stability. For long-term storage, flash-freeze aliquots in buffer containing 20% glycerol and store at -80°C to preserve activity.

How can researchers verify the proper folding and activity of recombinant PD_1032?

Verifying proper folding and activity of recombinant PD_1032 requires multiple complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure elements

  • Intrinsic tryptophan fluorescence to assess tertiary structure

  • Size exclusion chromatography to confirm monomeric state or expected oligomerization

• Functional assays:

  • Spectrophotometric activity assays monitoring NAD(P)H oxidation at 340 nm

  • Substrate-specific assays based on predicted reductase function

  • Thermal shift assays (Thermofluor) to assess stability and cofactor binding

• Cofactor binding verification:

  • UV-visible spectroscopy to detect characteristic absorbance of bound cofactors

  • Isothermal titration calorimetry (ITC) to determine binding constants

A typical activity assay buffer might contain 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1-0.2 mM NAD(P)H, and substrate at appropriate concentration. Monitor absorbance decrease at 340 nm as NAD(P)H is oxidized during the reductase reaction. Calculate specific activity as μmol substrate reduced per minute per mg protein under standardized conditions.

What are the substrate specificity profiles of PD_1032 compared to related reductases?

The substrate specificity profile of PD_1032 can be systematically evaluated against a panel of potential substrates and compared with related reductases from other bacterial species. Research indicates that putative reductases in X. fastidiosa often show activity toward multiple substrates with varying efficiency.

A comparison study of substrate preferences might yield results as follows:

The methodological approach involves:

• Screening diverse substrates in standardized assay conditions

• Determining kinetic parameters (Km, Vmax, kcat/Km) for each viable substrate

• Evaluating cofactor preferences (NADH vs. NADPH)

• Comparing catalytic efficiencies across homologous proteins

Structure-function analyses using site-directed mutagenesis of key residues in the active site can further elucidate the molecular basis for substrate recognition and catalysis, which may differ from related reductases in other bacterial species.

How does PD_1032 activity contribute to X. fastidiosa virulence and survival in host plants?

PD_1032 activity likely contributes to X. fastidiosa virulence through several potential mechanisms:

• Oxidative stress resistance: As a putative reductase, PD_1032 may participate in detoxification of reactive oxygen species (ROS) generated during plant defense responses. X. fastidiosa must counter oxidative stress within the xylem environment to successfully colonize and persist .

• Metabolic adaptation: The enzyme may facilitate metabolic adaptation to nutrient-limited xylem environments by participating in alternative metabolic pathways enabling carbon source utilization specific to plant xylem composition.

• Biofilm formation contribution: Reductase activity might influence redox-dependent signaling pathways involved in biofilm formation - a critical virulence factor for X. fastidiosa colonization of xylem vessels .

Methodological approaches to investigate these roles include:

• Creating PD_1032 knockout mutants through homologous recombination techniques

• Complementation studies with wild-type and catalytically inactive variants

• Competitive index assays comparing mutant versus wild-type strains in planta

• Transcriptomic comparisons of wild-type and mutant strains under oxidative stress conditions

• Microscopic evaluation of biofilm formation in vitro and in planta

Research on similar metabolic enzymes in X. fastidiosa suggests that even seemingly auxiliary metabolic functions can significantly impact virulence through effects on bacterial fitness within the specialized xylem environment.

What structural features and catalytic residues are essential for PD_1032 enzymatic function?

The essential structural features and catalytic residues of PD_1032 can be identified through a combination of bioinformatic analysis, structural studies, and experimental validation:

• Conserved domains: Bioinformatic analysis likely reveals conserved reductase domains, potentially including a Rossmann fold for NAD(P)H binding characteristic of oxidoreductases.

• Catalytic motifs: Critical functional motifs would include:

  • Cofactor binding site (typically G-X-G-X-X-G or similar motif)

  • Substrate binding pocket residues

  • Catalytic residues for proton transfer

  • Potential regulatory sites

• Essential residues: Site-directed mutagenesis studies targeting predicted catalytic residues (likely including conserved cysteines, histidines, and acidic residues) would demonstrate their importance for catalytic function.

Methodological approaches to determine these features include:

• Homology modeling based on structurally characterized reductases

• Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

• Site-directed mutagenesis of predicted key residues followed by activity assays

• Crystallization and X-ray diffraction analysis for definitive structural determination

• Molecular dynamics simulations to understand conformational changes during catalysis

Comparative analysis with restriction-modification systems in X. fastidiosa, which undergo recombination exchanging target recognition domains (TRDs) , may provide insight into the evolutionary forces shaping enzyme specificity in this pathogen.

How can PD_1032 be targeted for potential disease management strategies?

PD_1032 putative reductase can be targeted for disease management through several strategic approaches:

• Small molecule inhibitors: Develop specific inhibitors targeting PD_1032's active site, potentially disrupting X. fastidiosa metabolism within plants. The methodological approach involves:

  • Structure-based virtual screening against PD_1032 homology models

  • High-throughput enzymatic assays to identify inhibitory compounds

  • Validation in bacterial cultures and plant infection models

  • Optimization of lead compounds for efficacy and specificity

• Transgenic approaches: Similar to the rpfF-expressing transgenic grape plants that produced diffusible signal factor (DSF) causing "pathogen confusion" , transgenic plants expressing inhibitors of PD_1032 or antibodies targeting the enzyme could disrupt bacterial metabolism.

• Peptide-based inhibitors: Design peptides that mimic substrate binding but prevent catalysis, potentially delivered through engineered endophytes colonizing the same xylem space.

The efficacy of these approaches requires evaluation in appropriate model systems, including:

• In vitro enzyme inhibition assays

• Growth inhibition assays with X. fastidiosa cultures

• Greenhouse trials with model plants (tobacco, periwinkle)

• Field trials with economically important host plants (grape, olive, citrus)

Success would be measured by reduced bacterial multiplication, decreased symptom development, and limited systemic movement in plants, similar to metrics used in other X. fastidiosa intervention studies .

What techniques can detect and quantify PD_1032 expression during X. fastidiosa infection?

Multiple complementary techniques can detect and quantify PD_1032 expression during X. fastidiosa infection:

• Transcript quantification:

  • RT-qPCR targeting PD_1032 mRNA, similar to methods used for other X. fastidiosa genes like cvaC-1

  • RNA-Seq for global transcriptome analysis, allowing normalization against housekeeping genes

  • In situ hybridization to localize expression in infected plant tissues

• Protein detection:

  • Western blotting using antibodies raised against recombinant PD_1032

  • Immunohistochemistry to visualize protein localization in infected tissues

  • Selected Reaction Monitoring (SRM) mass spectrometry for sensitive protein quantification

• Activity measurement:

  • Enzyme assays from plant tissue extracts

  • Activity-based protein profiling using tagged substrate analogs

Methodological considerations include:

• Sampling strategy: differentiate between inoculation points (PI) and systemic infection sites (UP)

• Timing: assess expression changes throughout infection progression (early colonization vs. established infection)

• Controls: compare with housekeeping genes and other metabolic enzymes

Results might be presented as fold-changes in expression across infection stages:

This approach allows correlation of PD_1032 expression with infection stages and symptom development.

How do environmental factors affect PD_1032 expression and activity in planta?

Environmental factors significantly influence PD_1032 expression and activity during plant infection, with implications for disease development and management:

• Temperature effects:

  • Temperature fluctuations alter X. fastidiosa gene expression patterns

  • PD_1032 activity may exhibit temperature optima relevant to seasonal disease progression

  • Methodological approach: Compare expression and enzyme activity across temperature ranges (15-35°C) in controlled environment chambers

• Water stress influence:

  • Water stress affects xylem fluid composition and plant defense responses

  • PD_1032 expression may respond to osmotic changes in xylem environment

  • Methodology: Monitor expression in plants under different irrigation regimes using RT-qPCR

• Host plant species effects:

  • Different host plants create distinct microenvironments for X. fastidiosa

  • Comparison of PD_1032 expression across hosts like olive, periwinkle, and citrus may reveal host-specific regulation

  • Approach: Parallel infections in multiple hosts with standardized inoculum and sampling protocols

• Microbial interactions:

  • Endophytic microbiome may influence X. fastidiosa gene expression

  • Co-infection studies with beneficial endophytes could reveal interaction effects on PD_1032 expression

  • Methodology: Amplicon sequencing of microbiome alongside X. fastidiosa-specific gene expression analysis

Research findings might be presented as expression heat maps across environmental conditions, highlighting factors that significantly upregulate or downregulate PD_1032 expression, potentially identifying environmental conditions that could suppress virulence-related functions of this enzyme.

How might CRISPR-Cas9 gene editing be applied to study PD_1032 function in X. fastidiosa?

CRISPR-Cas9 gene editing offers powerful approaches to study PD_1032 function in X. fastidiosa, despite the challenges of genetic manipulation in this fastidious bacterium:

• Precise gene knockout methodology:

  • Design sgRNAs targeting conserved regions of PD_1032

  • Clone sgRNA into a Cas9-expressing vector compatible with X. fastidiosa

  • Include homology arms with selectable markers for recombination

  • Transform X. fastidiosa using electroporation protocols optimized for this species

  • Screen transformants using PCR and sequencing to confirm edits

• Domain-specific mutations:

  • Create precise point mutations in catalytic residues to generate enzymatically inactive variants

  • Design edits that maintain protein structure but eliminate activity

  • Compare phenotypes of null mutants versus catalytically inactive variants

• Promoter replacements:

  • Substitute native promoter with inducible or constitutive promoters to control expression

  • Study effects of altered expression timing on virulence and colonization

• Tagged protein variants:

  • Introduce sequences encoding epitope tags or fluorescent proteins

  • Enable visualization and purification of the native protein from infected plants

This approach must consider the type I restriction-modification systems in X. fastidiosa that may influence transformation efficiency . Molecular tools must be designed to avoid restriction sites recognized by the bacterium's 31 different allele profiles of restriction enzymes. Complementation studies should be performed to confirm phenotypes result from the specific mutation rather than polar effects or off-target modifications.

What protein-protein interactions involve PD_1032 in the X. fastidiosa proteome network?

Characterizing protein-protein interactions involving PD_1032 provides insight into its broader functional context within X. fastidiosa:

• Unbiased interaction screens:

  • Bacterial two-hybrid assays using PD_1032 as bait against X. fastidiosa genomic library

  • Affinity purification-mass spectrometry (AP-MS) with tagged PD_1032

  • Cross-linking mass spectrometry to capture transient interactions

• Focused interaction studies:

  • Co-immunoprecipitation with antibodies against predicted interaction partners

  • Biolayer interferometry with purified PD_1032 and candidate partners

  • Microscale thermophoresis to determine binding affinities

• In vivo validation:

  • Bimolecular fluorescence complementation (BiFC) in model bacteria

  • Förster resonance energy transfer (FRET) analysis with fluorescently tagged proteins

  • Co-localization studies in bacterial cells

• Computational prediction:

  • Interactome predictions based on homologous proteins in related bacteria

  • Structural docking simulations to evaluate physical compatibility of interactions

Potential interaction partners may include:

• Other metabolic enzymes in related pathways

• Regulatory proteins sensing redox state

• Components of stress response systems

• Membrane proteins involved in substrate transport

The resulting interaction network could be visualized as a protein interaction map highlighting primary, secondary, and tertiary interaction partners, with edge weights representing interaction strengths determined experimentally.

How does genomic variation in PD_1032 across X. fastidiosa strains correlate with host range and virulence?

Genomic variation analysis of PD_1032 across X. fastidiosa strains provides valuable insights into evolutionary adaptation and host-pathogen interactions:

• Comparative sequence analysis:

  • Analyze PD_1032 sequences across the 129 X. fastidiosa genome assemblies representing all known subspecies and 32 sequence types

  • Identify conserved regions versus variable domains

  • Calculate selection pressures (dN/dS ratios) across the gene sequence

  • Map variations to functional domains and catalytic sites

• Structure-function correlations:

  • Model effects of amino acid substitutions on protein structure

  • Express and purify variant forms to compare enzymatic properties

  • Correlate specific substitutions with substrate preference changes

• Host range associations:

  • Group PD_1032 variants by host specificity of source strains

  • Identify amino acid signatures associated with particular host preferences

  • Test hypotheses through heterologous expression of variants in model strains

• Recombination analysis:

  • Examine evidence for horizontal gene transfer or recombination events affecting PD_1032

  • Determine if PD_1032 shows recombination patterns similar to the hsdS genes which undergo target recognition domain exchanges

Results could be presented as phylogenetic trees of PD_1032 variants alongside host range data and virulence measurements, potentially revealing correlations between specific variants and pathogenicity traits. This information could guide targeted intervention strategies for particular crop-pathogen combinations.

What integrated research strategies combine molecular, structural, and field approaches to understand PD_1032 function?

An integrated research strategy to fully characterize PD_1032 function would combine:

• Molecular approaches:

  • Gene expression studies across infection stages and hosts

  • Protein production and characterization

  • Interaction mapping within proteome networks

  • Genetic manipulation through CRISPR-Cas9 or traditional methods

• Structural biology:

  • X-ray crystallography or cryo-EM structure determination

  • Computational modeling of enzyme-substrate interactions

  • Dynamic studies through hydrogen-deuterium exchange

• Field and greenhouse studies:

  • Infection trials with mutant strains

  • Correlation of PD_1032 variation with disease parameters

  • Evaluation of potential inhibitors in planta

This integration would create a comprehensive understanding from molecular mechanisms to ecological significance. The approach should include standardized protocols for bacterial culture, plant inoculation methods similar to those used in olive, citrus and periwinkle trials , and consistent measurement of disease parameters to enable cross-comparison between studies.

How might systems biology approaches enhance our understanding of PD_1032's role in X. fastidiosa pathogenicity?

Systems biology approaches offer powerful frameworks to position PD_1032 within the broader context of X. fastidiosa pathogenicity: