Recombinant Xylella fastidiosa Thiol:disulfide interchange protein DsbE (dsbE), partial

What is the biological significance of DsbE in Xylella fastidiosa pathogenicity?

The thiol:disulfide interchange protein DsbE in Xylella fastidiosa likely plays a crucial role in the pathogen's virulence mechanisms. Disulfide bonds provide structural stability to proteins and regulate enzyme activity across all domains of life . In bacterial pathogens, including X. fastidiosa, these bonds are particularly important for the proper functioning of secreted proteins and virulence factors.

The function of many Gram-positive and Gram-negative bacteria virulence factors depends on the formation of correct disulfide bonds, including those important in adhesion to host cells, bacterial spread and growth, and host cell manipulation . Given that X. fastidiosa is a xylem-limited bacterium causing several economically important plant diseases including grapevine Pierce's disease, citrus variegated chlorosis, and olive scorch , the DsbE protein likely contributes to the pathogen's ability to colonize plant hosts and cause disease symptoms by ensuring proper protein folding and function in the challenging xylem environment.

Research methodologies to investigate this question should include gene knockout studies, complementation assays, and virulence comparisons between wild-type and DsbE-deficient strains across multiple host plant species.

How does X. fastidiosa DsbE differ structurally from homologous proteins in other bacterial species?

Understanding the structural differences between X. fastidiosa DsbE and homologous proteins in other bacterial species requires comparative structural biology approaches. Begin with sequence alignment of DsbE across diverse bacterial species using tools like MUSCLE or CLUSTALW. Next, perform structural predictions using homology modeling software such as SWISS-MODEL or Phyre2, followed by validation through molecular dynamics simulations.

Key structural features to analyze include the active site CXXC motif, which is likely conserved across species but may have subtle differences in the intervening amino acids that affect redox potential . Additionally, examine substrate binding regions that may reflect adaptation to X. fastidiosa's specific environmental niche as a xylem-limited plant pathogen .

The distinct evolutionary pressures faced by X. fastidiosa subspecies (fastidiosa, pauca, multiplex, sandyi, tashke, and morus) may have resulted in subspecies-specific adaptations in DsbE structure. These differences could be visualized through structural superimposition and electrostatic surface potential analysis to identify unique binding interfaces or catalytic properties.

What experimental approaches are most effective for purifying active recombinant X. fastidiosa DsbE?

Purification of active recombinant X. fastidiosa DsbE requires careful consideration of expression systems and purification strategies to maintain the protein's native conformation and enzymatic activity.

Recommended methodology:

• Cloning and expression optimization:

  • Clone the partial or complete dsbE gene from X. fastidiosa into pET expression vectors with either N- or C-terminal His-tags

  • Test multiple E. coli expression strains (BL21(DE3), Origami, SHuffle) - the latter two are recommended as they enhance disulfide bond formation

  • Optimize expression conditions using a temperature range (16-30°C) and IPTG concentrations (0.1-1.0 mM)

• Purification protocol:

  • Lyse cells in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

  • Include reducing agents (2-5 mM DTT) during initial purification steps

  • Employ immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography

  • Consider on-column refolding protocols if the protein forms inclusion bodies

• Activity verification:

  • Assess thiol:disulfide interchange activity using standard insulin reduction assays

  • Verify correct folding through circular dichroism spectroscopy

  • Confirm redox potential using redox equilibrium studies with glutathione

This methodological approach addresses the challenges specific to disulfide bond-containing proteins while maximizing yield and activity of the recombinant DsbE.

How does recombination influence DsbE functional diversity across X. fastidiosa subspecies?

Recombination is a significant driver of X. fastidiosa evolution, with genomic analyses revealing that individual nucleotides are three times more likely to change due to recombination than due to point mutation . This genomic plasticity may directly impact the diversity and function of proteins involved in pathogenicity, including DsbE.

To investigate the influence of recombination on DsbE functional diversity, researchers should:

• Perform comparative genomic analysis of dsbE sequences from all known X. fastidiosa subspecies (fastidiosa, pauca, multiplex, sandyi, tashke, and morus)

• Apply recombination detection methods such as:

  • RDP4 suite containing multiple algorithms for recombination detection

  • GARD (Genetic Algorithm for Recombination Detection)

  • ClonalFrameML for phylogenetic analysis accounting for recombination

• Identify potential recombination breakpoints within and flanking the dsbE gene by analyzing:

  • Sequence diversity patterns

  • Linkage disequilibrium

  • Phylogenetic incongruence between gene segments

• Experimentally validate functional differences between DsbE variants using:

  • Site-directed mutagenesis to create chimeric proteins

  • Redox activity assays measuring disulfide interchange kinetics

  • Cross-complementation studies in different X. fastidiosa subspecies

Research has demonstrated that each of the main X. fastidiosa subspecies is under different selective pressures , which may drive functional diversification of proteins like DsbE. Understanding how recombination shapes this diversity could provide insights into subspecies-specific host adaptation and pathogenicity mechanisms.

What is the role of DsbE in X. fastidiosa biofilm formation and how does this impact host colonization?

The formation of biofilms is a crucial aspect of X. fastidiosa pathogenicity, enabling colonization of plant xylem vessels and contributing to disease symptoms through vascular occlusion. Investigating the role of DsbE in biofilm formation requires a multifaceted approach:

• Generate dsbE knockout and complemented strains through homologous recombination or CRISPR-Cas9 genome editing

• Quantitatively assess biofilm formation using:

  • Crystal violet staining of in vitro biofilms

  • Confocal laser scanning microscopy with fluorescently labeled strains

  • Scanning electron microscopy to examine biofilm architecture

• Analyze extracellular matrix composition differences between wild-type and dsbE mutants:

  • Quantify exopolysaccharide production

  • Profile biofilm protein content through proteomics

  • Examine eDNA contributions to matrix stability

• Evaluate the oxidation state of key biofilm-associated proteins in wild-type versus dsbE mutant strains

• Perform in planta colonization studies using:

  • Gfp-tagged bacterial strains for visualization

  • qPCR quantification of bacterial populations in xylem

  • Transcriptomic analysis of dsbE expression during different stages of host colonization

Note: This table represents hypothetical results based on expected DsbE function; actual experimental data may vary.

Understanding DsbE's role in biofilm formation could reveal new targets for controlling X. fastidiosa infections in economically important crops.

How do environmental factors influence the expression and activity of DsbE in X. fastidiosa?

X. fastidiosa encounters various environmental stresses as it transitions between insect vectors and plant hosts. The expression and activity of DsbE may be modulated in response to these changing conditions. To investigate this relationship:

• Design transcriptomic experiments analyzing dsbE expression under various conditions:

  • Temperature shifts (18-32°C) simulating seasonal changes

  • pH variations (5.0-7.0) mimicking different xylem environments

  • Nutrient limitation reflecting xylem composition differences between host species

  • Oxidative stress conditions simulating plant defense responses

• Develop a reporter system to monitor dsbE promoter activity in real-time:

  • Construct dsbE promoter-GFP or -luciferase fusions

  • Monitor expression changes during transition from planktonic to biofilm growth

  • Track expression during different stages of infection in multiple host plants

• Perform in vitro enzymatic assays to assess DsbE activity under varying conditions:

  • Measure thiol:disulfide exchange rates at different temperatures

  • Determine pH optima and stability profiles

  • Assess the impact of oxidative stress on enzyme kinetics

• Analyze post-translational modifications of DsbE using mass spectrometry:

  • Identify potential regulatory modifications (phosphorylation, S-glutathionylation)

  • Determine how these modifications affect enzyme activity

  • Map the regulatory network controlling DsbE function

Understanding how environmental factors influence DsbE expression and activity could help explain the differential virulence of X. fastidiosa on various host plants and under different climatic conditions .

What is the relationship between X. fastidiosa DsbE and bacterial type I restriction-modification systems?

Recent research has highlighted the importance of type I restriction-modification (R-M) systems in X. fastidiosa horizontal gene transfer and recombination . These systems regulate DNA methylation patterns, which can influence gene expression and genome plasticity. Investigating the relationship between DsbE and type I R-M systems requires specialized methodologies:

• Analyze the genomic context of dsbE in relation to R-M system components across X. fastidiosa strains:

  • Examine co-occurrence patterns of specific dsbE alleles with R-M system variants

  • Identify potential co-regulation through shared regulatory elements

  • Map physical proximity on the chromosome that might indicate functional linkage

• Investigate the methylation status of the dsbE gene and its promoter region:

  • Perform bisulfite sequencing to map methylation patterns

  • Compare methylation profiles across strains with different R-M system alleles

  • Correlate methylation patterns with dsbE expression levels

• Examine the disulfide bond status of R-M system proteins:

  • Identify conserved cysteine residues in HsdS, HsdM, and HsdR subunits

  • Perform thiol-trapping experiments to determine in vivo redox state

  • Test whether DsbE can catalyze disulfide bond formation in R-M proteins

• Create double mutants lacking both dsbE and components of type I R-M systems:

  • Assess changes in transformation efficiency and natural competence

  • Measure alteration in recombination frequencies

  • Quantify impacts on genome methylation patterns

This investigation could reveal previously unrecognized connections between protein disulfide bond formation and epigenetic regulation in X. fastidiosa, potentially explaining mechanisms of strain-specific host adaptation and virulence .

What are the best molecular techniques for studying DsbE genetic variation across X. fastidiosa isolates?

Analyzing DsbE genetic variation across X. fastidiosa isolates requires a combination of established and cutting-edge molecular techniques. Based on the evolution of molecular methods used for X. fastidiosa characterization , researchers should consider:

• PCR-based approaches:

  • Design primers targeting conserved regions flanking dsbE

  • Optimize PCR conditions for high-fidelity amplification

  • Consider touchdown PCR to improve specificity when working with field isolates

• Sequencing strategies:

  • Sanger sequencing for individual isolate characterization

  • Illumina sequencing for high-throughput analysis of multiple isolates

  • Oxford Nanopore or PacBio long-read sequencing to capture genomic context

  • Targeted amplicon sequencing focusing on dsbE and flanking regions

• Comparative genomic approaches:

  • Whole genome alignment to identify structural variations affecting dsbE

  • SNP and indel analysis to characterize allelic variation

  • Synteny analysis to detect genomic rearrangements affecting dsbE expression

• Population genetics analysis:

  • Calculate nucleotide diversity (π) and Tajima's D to detect selection

  • Perform McDonald-Kreitman tests to identify adaptive evolution

  • Apply phylogenetic analyses accounting for recombination

When interpreting results, researchers should consider that recombination rates in X. fastidiosa are high, with nucleotides three times more likely to change due to recombination than mutation . This necessitates appropriate analytical methods that account for horizontal gene transfer when reconstructing evolutionary relationships of dsbE variants.

How can researchers effectively distinguish between DsbE and other Dsb family proteins in functional studies?

Distinguishing between DsbE and other Dsb family proteins (DsbA, DsbB, DsbC, DsbD, etc.) in functional studies is critical for accurate characterization. These proteins share similar catalytic mechanisms but have distinct roles in disulfide bond formation and isomerization pathways. Recommended approaches include:

• Biochemical differentiation:

  • Determine redox potential values using glutathione equilibrium methods

  • Measure enzyme kinetics with specific substrates

  • Assess protein-protein interactions with putative partners

  • Analyze pH dependence of catalytic activity

• Structural characterization:

  • Compare active site CXXC motifs (amino acid composition affects function)

  • Analyze substrate binding pockets using molecular modeling

  • Perform circular dichroism to assess secondary structure differences

  • Use thermal shift assays to compare protein stability

• Substrate specificity profiling:

  • Develop proteomic approaches to identify native substrates

  • Use peptide arrays to determine sequence preferences

  • Perform in vitro folding assays with model substrates

• Cellular localization:

  • Create fluorescent protein fusions to track subcellular localization

  • Perform fractionation studies to determine membrane association

  • Use immunogold electron microscopy for precise localization

This methodological framework ensures that functional studies accurately attribute observed phenotypes to the correct Dsb protein, avoiding misinterpretation of results due to functional overlap between family members.

What are the optimal experimental conditions for assessing DsbE activity in vitro versus in planta?

Evaluating DsbE activity requires different experimental approaches depending on whether investigations are conducted in vitro or in planta. Each environment presents unique challenges and considerations:

In vitro activity assessment:

• Enzyme kinetic assays:

  • Use synthetic peptides containing appropriate disulfide bonds as substrates

  • Monitor thiol:disulfide exchange using fluorescence-based assays

  • Optimize buffer conditions (pH 6.0-7.5, 50-150 mM NaCl)

  • Include physiologically relevant concentrations of glutathione to mimic in vivo redox environment

• Thermal stability profiles:

  • Perform differential scanning fluorimetry across pH range 5.0-8.0

  • Test stability in the presence of plant xylem extract components

  • Assess the impact of divalent cations (Ca²⁺, Mg²⁺) on activity and stability

• Interaction studies:

  • Identify binding partners using pull-down assays coupled with mass spectrometry

  • Quantify binding affinities through isothermal titration calorimetry

  • Visualize protein-protein interactions via biolayer interferometry

In planta activity assessment:

• Expression systems:

  • Develop X. fastidiosa strains expressing tagged DsbE variants

  • Create reporter systems linking DsbE activity to fluorescent output

  • Establish methods to extract active enzyme from infected plant tissue

• Plant infection protocols:

  • Standardize inoculation procedures across different host plants

  • Develop sampling techniques for xylem sap at various infection stages

  • Create micro-environmental sensors to monitor conditions in xylem vessels

• Activity measurements:

  • Assess redox state of known DsbE substrates in planta

  • Monitor changes in disulfide bond patterns during infection progression

  • Correlate DsbE activity with virulence phenotypes across host range

• Considerations for environmental variables:

  • Account for diurnal temperature fluctuations

  • Consider seasonal changes in plant physiology

  • Standardize plant growth conditions to minimize variability

These methodological considerations ensure that DsbE activity assessments accurately reflect the protein's function in both controlled laboratory settings and the complex environment of infected plant tissues.

How should researchers interpret conflicting data between genomic predictions and experimental results for DsbE function?

Discrepancies between genomic predictions and experimental results for DsbE function can arise from various sources. A systematic approach to resolving these conflicts includes:

• Re-evaluate bioinformatic predictions:

  • Assess the quality of genome assembly and annotation

  • Consider alternative start codons or splice variants

  • Examine the possibility of sequencing errors in key functional domains

  • Compare predictions across multiple algorithms and databases

• Review experimental methodology:

  • Verify protein expression and correct folding

  • Ensure assay conditions reflect physiological environment

  • Check for interfering factors in experimental systems

  • Validate results using complementary techniques

• Consider biological explanations:

  • Post-translational modifications not predicted from sequence

  • Protein moonlighting (multiple functions of the same protein)

  • Context-dependent functionality based on interaction partners

  • Genetic background effects in different X. fastidiosa strains

• Integration strategies:

  • Develop a hypothesis reconciliation framework

  • Design targeted experiments to address specific discrepancies

  • Use structural modeling to predict the impact of sequence variations

  • Apply systems biology approaches to place DsbE in broader cellular context

When working with X. fastidiosa, remember that high rates of recombination can lead to mosaic genes with complex evolutionary histories . This genomic plasticity may explain functional variations not easily predicted from sequence data alone and necessitates careful interpretation of experimental results across different strains and subspecies.

What statistical approaches are most appropriate for analyzing DsbE sequence variation in the context of X. fastidiosa evolution?

The high rates of recombination in X. fastidiosa necessitate specialized statistical approaches when analyzing DsbE sequence variation:

• Recombination-aware phylogenetic methods:

  • ClonalFrameML or Gubbins to detect and account for recombination events

  • STRUCTURE or fastGEAR for population structure analysis

  • PhiPack for detecting recombination breakpoints

  • BAPS for Bayesian clustering of sequence types

• Selection analysis approaches:

  • PAML for detecting positive selection on specific codons

  • FUBAR for fast unconstrained Bayesian approximation

  • MEME for detecting episodic diversifying selection

  • BUSTED for gene-wide tests of selection

• Appropriate measures of genetic diversity:

  • π (nucleotide diversity) calculated separately for recombining/non-recombining regions

  • dN/dS ratios with sliding window analysis

  • Linkage disequilibrium decay rates as indicators of recombination frequency

  • FST values for population differentiation based on DsbE sequences

• Comparative statistical frameworks:

  • Bayesian skyline plots to infer historical population dynamics

  • Mantel tests to correlate genetic and geographic/host distances

  • AMOVA to partition genetic variance among hierarchical groups

  • Network-based approaches (SplitsTree) to visualize complex evolutionary relationships

When interpreting results, researchers should account for the known subspecies structure within X. fastidiosa (fastidiosa, pauca, multiplex, sandyi, tashke, and morus) and consider how this structure might influence DsbE evolution through differential selective pressures and opportunities for horizontal gene transfer.

How can researchers effectively integrate DsbE functional data with broader X. fastidiosa pathogenomics research?

Integrating DsbE functional data with broader X. fastidiosa pathogenomics requires multidimensional approaches that connect molecular mechanisms to phenotypic outcomes:

• Multi-omics integration strategies:

  • Correlate DsbE expression (transcriptomics) with protein abundance (proteomics)

  • Map post-translational modifications using phosphoproteomics and redox proteomics

  • Link metabolomic profiles to DsbE activity under various conditions

  • Integrate methylome data to examine epigenetic regulation of dsbE expression

• Network analysis approaches:

  • Construct protein-protein interaction networks centered on DsbE

  • Develop gene co-expression networks to identify functionally related genes

  • Create metabolic models incorporating DsbE-dependent pathways

  • Apply Bayesian networks to infer causal relationships

• Functional genomics validation:

  • Use transposon mutagenesis libraries to identify genetic interactions

  • Apply CRISPR interference for conditional knockdowns

  • Develop high-throughput phenotyping assays for DsbE-related functions

  • Create reporter strains for in planta visualization of DsbE activity

• Data visualization and sharing:

  • Develop interactive visualizations connecting genotype to phenotype

  • Contribute standardized datasets to community databases

  • Adopt FAIR data principles (Findable, Accessible, Interoperable, Reusable)

  • Establish common ontologies for X. fastidiosa functional annotations

The high genomic diversity and recombination rates in X. fastidiosa make integration particularly challenging but also potentially revealing. Researchers should explicitly account for strain-specific differences when aggregating data and consider how DsbE function might vary across the different subspecies and their associated host ranges .

What emerging technologies hold the most promise for advancing understanding of DsbE function in X. fastidiosa?

Several cutting-edge technologies show particular promise for elucidating DsbE function in X. fastidiosa:

• CRISPR-Cas systems for precise genome editing:

  • Development of CRISPR-Cas9 or base editing systems optimized for X. fastidiosa

  • Creation of conditional knockdowns using CRISPRi

  • Domain-specific mutagenesis to map functional regions of DsbE

  • High-throughput variant screening through CRISPR libraries

• Advanced imaging technologies:

  • Super-resolution microscopy to visualize DsbE localization at nanoscale

  • Single-molecule tracking to follow DsbE dynamics in living cells

  • Correlative light and electron microscopy for structural context

  • In planta imaging using minimally invasive techniques to track infection processes

• Structural biology advances:

  • Cryo-electron microscopy for high-resolution structure determination

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic regions

  • AlphaFold2 and other AI-based structure prediction tools

  • Time-resolved structural studies to capture conformational changes during catalysis

• Single-cell technologies:

  • Single-cell RNA-seq to capture heterogeneity in bacterial populations

  • Spatial transcriptomics to map gene expression in biofilms

  • CyTOF mass cytometry for high-dimensional phenotypic profiling

  • Microfluidic systems for tracking individual bacterial cells during host interaction

• Synthetic biology approaches:

  • Designer disulfide bond systems with tunable redox properties

  • Orthogonal translation systems for site-specific incorporation of non-canonical amino acids

  • Cell-free expression systems for rapid protein engineering

  • Biosensors that report on DsbE activity in real-time

These emerging technologies can help overcome the technical challenges associated with studying fastidious pathogens like X. fastidiosa and provide unprecedented insights into the molecular mechanisms of DsbE function in bacterial pathogenesis and host adaptation.

What are the most significant knowledge gaps in understanding the relationship between DsbE and X. fastidiosa host specificity?

Despite advances in X. fastidiosa research, several critical knowledge gaps remain regarding DsbE's role in host specificity:

• Substrate specificity across host environments:

  • Limited understanding of DsbE's natural substrates in different plant hosts

  • Unknown adaptation mechanisms of DsbE to various xylem chemical compositions

  • Unclear relationship between DsbE activity and host-specific virulence factors

  • Poor characterization of redox environments across plant host species

• Evolutionary dynamics across X. fastidiosa subspecies:

  • Incomplete picture of how DsbE variants correlate with host range differences

  • Limited understanding of recombination's role in DsbE functional diversification

  • Unknown selective pressures driving DsbE evolution in different hosts

  • Unclear contribution of DsbE to subspecies-specific ecological niches

• Regulatory networks:

  • Minimal data on host-specific regulation of dsbE expression

  • Poor understanding of how plant defense responses affect DsbE function

  • Limited knowledge of signaling pathways connecting environmental sensing to DsbE activity

  • Unknown epigenetic mechanisms potentially regulating dsbE through DNA methylation

• Methodological limitations:

  • Challenges in directly studying protein folding in planta

  • Difficulty isolating sufficient quantities of native DsbE from infected plants

  • Technical barriers to real-time monitoring of disulfide bond formation during infection

  • Lack of standardized assays for comparing DsbE function across strains

Addressing these knowledge gaps requires developing novel experimental approaches that can bridge molecular mechanisms with ecological outcomes, potentially revealing new strategies for controlling X. fastidiosa diseases across its extensive host range.

How might understanding DsbE function contribute to developing novel control strategies for X. fastidiosa-related diseases?

Understanding DsbE function could lead to innovative control strategies for X. fastidiosa diseases through several promising avenues:

• Targeted inhibitor development:

  • Design of small-molecule inhibitors specifically targeting DsbE active site

  • Development of peptidomimetics that compete with natural substrates

  • Creation of transition state analogs that block catalytic activity

  • Identification of allosteric modulators affecting DsbE function

• Host-induced gene silencing approaches:

  • Design of RNA interference constructs targeting dsbE expression

  • Development of transgenic plants expressing anti-dsbE constructs

  • Creation of spray-applied RNA molecules for temporary suppression

  • Exploitation of natural plant small RNAs that may regulate bacterial genes

• Immune modulation strategies:

  • Engineering plant immune responses to recognize DsbE-dependent virulence factors

  • Development of decoy substrates that sequester DsbE activity

  • Priming of plant defense responses targeting bacterial protein folding

  • Selection of plant varieties with xylem chemistry unfavorable to DsbE function

• Biocontrol approaches:

  • Engineering competitor microbes expressing DsbE inhibitors

  • Development of phage therapy targeting X. fastidiosa surface proteins dependent on DsbE

  • Creation of synthetic communities that modulate xylem redox environment

  • Design of protective endophytes competing for X. fastidiosa colonization sites

• Diagnostic applications:

  • Development of DsbE-based markers for strain identification

  • Creation of biosensors detecting X. fastidiosa-specific disulfide bond patterns

  • Design of rapid field tests based on DsbE antibodies or aptamers

  • Implementation of early detection systems for pre-symptomatic infections

These approaches leverage fundamental knowledge of DsbE function to develop targeted, environmentally sustainable control strategies that could protect economically important crops from the devastating effects of X. fastidiosa diseases .

What are the key considerations for researchers designing comprehensive studies of X. fastidiosa DsbE?

Researchers designing comprehensive studies of X. fastidiosa DsbE should consider a multifaceted approach that integrates evolutionary, structural, functional, and applied aspects. Key considerations include:

• Evolutionary context:

  • Account for the high genomic diversity and recombination rates in X. fastidiosa

  • Include representative strains from all subspecies and sequence types

  • Consider both vertical inheritance and horizontal gene transfer patterns

  • Analyze selection pressures across different ecological niches

• Methodological robustness:

  • Employ multiple complementary approaches for each research question

  • Validate recombinant protein studies with in planta observations

  • Include appropriate controls for strain background effects

  • Standardize experimental conditions to allow cross-laboratory comparisons

• Biological relevance:

  • Focus on environmentally realistic conditions mimicking xylem environments

  • Consider temperature, pH, and nutrient variations across host plants

  • Account for interactions with other microbial community members

  • Validate laboratory findings with field observations when possible

• Translational potential:

  • Design experiments with potential applications in disease management

  • Consider how findings relate to agricultural practices

  • Assess potential for development of resistant plant varieties

  • Explore opportunities for diagnostic tool development