Recombinant Xylella fastidiosa UPF0307 protein PD_0419 (PD_0419)

What is the genomic context of PD_0419 within Xylella fastidiosa?

PD_0419 encodes a UPF0307 family protein in Xylella fastidiosa, a bacterial plant pathogen that threatens agricultural crops worldwide. This gene is part of the core genome shared across Xylella species and subspecies, including the well-studied X. fastidiosa subsp. fastidiosa that causes Pierce's disease in grapevines. The gene appears to be conserved across different Xylella isolates, suggesting potential functional importance. UPF0307 proteins belong to a family of uncharacterized proteins that are structurally similar across different bacterial species, including those found in Pseudomonas putida . Genomic analyses of Xylella subspecies have revealed that while core functional genes remain largely conserved, there is considerable variation in virulence-associated genes across strains, highlighting the importance of studying individual proteins like PD_0419 to understand pathogenicity mechanisms .

How does PD_0419 compare to other UPF0307 family proteins in bacteria?

UPF0307 family proteins, including PD_0419 from Xylella fastidiosa, share conserved structural features across bacterial species. Computational structure models of similar proteins, like the UPF0307 protein PputW619_4274 from Pseudomonas putida, show high confidence scores (pLDDT global score of 91.77), suggesting stable tertiary structures . The structural conservation often extends across bacterial genera despite sequence variations. When comparing PD_0419 to other bacterial UPF0307 proteins, researchers should examine both sequence homology and predicted structural similarities. Key conserved domains may provide insights into potential functions, though sequence identity can vary significantly between species. The functional roles of these proteins remain largely uncharacterized, making comparative analyses particularly valuable for generating hypotheses about PD_0419's role in Xylella's biology and pathogenicity .

What bioinformatic tools are recommended for initial characterization of PD_0419?

For initial characterization of PD_0419, researchers should employ a comprehensive bioinformatic workflow that includes multiple complementary approaches. Begin with sequence analysis using BLAST against various databases to identify potential homologs, followed by multiple sequence alignment with CLUSTAL or MUSCLE to identify conserved regions. For structural prediction, AlphaFold2 provides high-confidence models similar to those available for related UPF0307 proteins . Domain prediction tools like InterProScan and SMART can identify functional domains, while subcellular localization predictors (SignalP, TMHMM) help determine the protein's cellular location. Secondary structure prediction using PSIPRED can reveal structural elements, and molecular docking simulations may suggest potential binding partners. Integrating these analyses with genome context analysis using tools like STRING and genomic island predictors provides a foundation for subsequent experimental design. Cross-reference findings with virulence factor databases to evaluate potential roles in pathogenicity comparable to other characterized Xylella virulence factors .

What expression systems are most effective for recombinant production of Xylella fastidiosa PD_0419?

The optimal expression system for recombinant PD_0419 production depends on research objectives and downstream applications. E. coli remains the most accessible and widely used system, with BL21(DE3) and its derivatives offering good expression yields for bacterial proteins. For functional studies where post-translational modifications may be important, consider Pseudomonas-based expression systems which provide a more native-like environment for proteins from related Gram-negative bacteria. Expression vector selection should prioritize inducible promoters (T7, tac) for controlled expression and fusion tags (His6, GST, MBP) to facilitate purification and potentially enhance solubility. Codon optimization is crucial when expressing Xylella genes in heterologous hosts due to potential codon bias differences. Culture conditions require careful optimization, including induction temperature (typically 16-25°C for improved solubility), IPTG concentration (0.1-1.0 mM), and media composition. For difficult-to-express proteins, specialized approaches such as cell-free expression systems or the addition of solubility enhancers may be necessary .

What purification strategy would yield the highest purity of functional PD_0419 protein?

A multi-step purification strategy is recommended to obtain high-purity, functional PD_0419 protein. Begin with affinity chromatography using a fusion tag appropriate for your expression construct—Ni-NTA for His-tagged proteins (elution with 250-300 mM imidazole) or glutathione sepharose for GST-tagged proteins. This initial step should be followed by ion-exchange chromatography to remove contaminants with different charge properties; use anion exchange (Q-sepharose) at pH >pI of PD_0419 or cation exchange (SP-sepharose) at pH <pI. Size exclusion chromatography as a final polishing step separates any remaining contaminants and aggregates while providing information about oligomeric state. Throughout purification, maintain protein stability with optimized buffer conditions (typically 20-50 mM Tris or phosphate, pH 7.4-8.0, with 150-300 mM NaCl) and consider adding reducing agents (1-5 mM DTT or β-mercaptoethanol) to prevent oxidation of cysteine residues. Quality assessment using SDS-PAGE, Western blotting, and mass spectrometry confirms purity and integrity. For functional studies, verify proper folding with circular dichroism spectroscopy and dynamic light scattering to assess homogeneity and aggregation status .

How can researchers troubleshoot low solubility issues when expressing recombinant PD_0419?

Low solubility of recombinant PD_0419 can be addressed through systematic optimization of multiple parameters. First, examine expression conditions by reducing induction temperature (16-20°C), lowering inducer concentration, and testing different media formulations. Adding solubility-enhancing fusion partners such as MBP, SUMO, or Thioredoxin often improves recovery of soluble protein. If these approaches prove insufficient, co-expression with bacterial chaperones (GroEL/ES, DnaK/J) can assist proper folding. Buffer optimization is crucial—screen various pH conditions (6.0-9.0), salt concentrations (100-500 mM), and additives including glycerol (5-10%), arginine (50-100 mM), or mild detergents (0.05-0.1% Triton X-100). For proteins that remain insoluble, controlled denaturation and refolding protocols using dialysis or dilution methods may recover functional protein from inclusion bodies. Cell-free expression systems represent an alternative approach that bypasses cellular barriers. Throughout troubleshooting, use small-scale parallel experiments with high-throughput screening methods to efficiently identify optimal conditions before scaling up. When reporting methods, document all conditions tested to provide valuable guidance for other researchers working with similar proteins from Xylella or related bacteria .

How can researchers determine the biochemical function of PD_0419?

Determining the biochemical function of PD_0419 requires a systematic, multi-faceted approach. Begin with in silico analyses through structural homology searches against proteins of known function, active site prediction, and molecular docking with potential substrates. For experimental validation, employ activity-based protein profiling using activity-based probes to identify potential enzymatic functions. Screen for enzymatic activities using substrate libraries relevant to bacterial metabolism and virulence, including various classes of hydrolases, transferases, and oxidoreductases. Protein-protein interaction studies via pull-down assays, yeast two-hybrid, or proximity labeling methods may reveal functional associations with other Xylella proteins, particularly those involved in virulence pathways described for Xylella fastidiosa . Binding assays with potential ligands using techniques such as isothermal titration calorimetry, surface plasmon resonance, or microscale thermophoresis can identify specific interactions. Complementation studies in related bacterial systems with gene knockouts of UPF0307 family proteins may demonstrate functional conservation. Throughout these investigations, consider the genomic context of PD_0419 and its potential relationship to the virulence mechanisms and pathogenicity factors characterized in Xylella fastidiosa, such as type IV pili genes or other virulence factors that are highly conserved across Xylella species .

What advanced spectroscopic methods reveal protein-ligand interactions for PD_0419?

Advanced spectroscopic methods provide crucial insights into protein-ligand interactions for PD_0419 characterization. Nuclear Magnetic Resonance (NMR) spectroscopy, particularly chemical shift perturbation experiments using 15N-HSQC, maps binding interfaces with atomic resolution by monitoring chemical shift changes upon ligand titration (protein concentrations of 0.1-0.5 mM). For larger complexes, paramagnetic relaxation enhancement (PRE) provides distance constraints by measuring effects of spin-labeled ligands. Surface Plasmon Resonance (SPR) and Bio-Layer Interferometry (BLI) determine binding kinetics (kon, koff) and equilibrium dissociation constants (KD), requiring only microgram quantities of purified protein. Isothermal Titration Calorimetry (ITC) measures thermodynamic parameters (ΔH, ΔS, ΔG) of binding events, providing mechanistic insights. Microscale Thermophoresis (MST) detects binding-induced changes in thermophoretic mobility with minimal sample consumption. Fluorescence-based techniques, including intrinsic tryptophan fluorescence and Förster resonance energy transfer (FRET), monitor conformational changes upon ligand binding. For high-throughput screening, differential scanning fluorimetry (thermal shift assays) identifies stabilizing ligands through melting temperature shifts. When applying these methods to PD_0419, consider testing molecules relevant to Xylella fastidiosa's lifestyle as a plant pathogen, including plant cell wall components, signaling molecules, or compounds produced during host-pathogen interactions .

How might PD_0419 contribute to Xylella fastidiosa virulence mechanisms?

While the specific role of PD_0419 in Xylella fastidiosa virulence remains to be fully characterized, we can hypothesize potential contributions based on known virulence mechanisms in this pathogen. PD_0419, as a conserved UPF0307 family protein, may participate in fundamental cellular processes that indirectly support virulence. Xylella fastidiosa employs several major pathogenicity strategies, including type IV pili (T4P) for twitching motility within infected plants, adhesins for attachment, and various hydrolytic enzymes for tissue degradation . The four major T4P gene clusters identified in Xylella contain 25 homologs highly conserved across representative genomes . PD_0419 could potentially interact with these systems, perhaps playing a role in regulating motility, adhesion, or biofilm formation—key processes in Xylella infection. Alternatively, it might function in metabolic pathways that support bacterial survival in the nutrient-limited xylem environment. Expression analysis during different stages of infection, co-immunoprecipitation studies with known virulence factors, and phenotypic characterization of gene knockout mutants would help elucidate its role in pathogenicity. The pattern of conservation across Xylella subspecies may provide additional clues—highly conserved proteins often serve essential functions in bacterial fitness or virulence .

What methodologies are optimal for studying PD_0419 function in planta?

Studying PD_0419 function in planta requires specialized methodologies that account for the complexities of host-pathogen interactions. Gene knockout or knockdown approaches using homologous recombination or CRISPR-Cas systems create PD_0419 mutants for comparative virulence studies. These mutants should be characterized for growth in culture before plant inoculation to distinguish direct virulence effects from general fitness defects. Complementation with wild-type PD_0419 confirms phenotype specificity. For plant inoculations, use needle puncture or insect vector transmission methods with appropriate host plants for the Xylella strain (grapevines for subsp. fastidiosa). Monitor bacterial colonization through quantitative PCR, bioluminescent/fluorescent reporters, or immunodetection methods at multiple time points (early, mid, and late infection). Disease progression assessment should include both visual symptom scoring and quantitative measurements of physiological parameters (water potential, photosynthetic efficiency). Molecular analyses of plant responses using RNA-seq or proteomics reveal host pathways affected by the presence/absence of PD_0419. Confocal microscopy with fluorescently tagged PD_0419 visualizes protein localization during infection, while in situ expression analysis using reporter fusions identifies conditions triggering PD_0419 expression. These approaches should be integrated with knowledge of previously characterized virulence mechanisms in Xylella, including type IV pili function, adhesin properties, and toxin-antitoxin systems that show variable distribution across subspecies .

How does PD_0419 expression vary across different Xylella subspecies and infection conditions?

Understanding PD_0419 expression patterns requires comparative analysis across Xylella subspecies and infection conditions. Quantitative RT-PCR analysis comparing expression levels in subspecies fastidiosa, pauca, multiplex, and newly described species like Xylella taiwanensis provides baseline data on natural expression variation . RNA-sequencing (RNA-seq) with differential expression analysis reveals subspecies-specific regulatory patterns that may correlate with host range or virulence potential. For condition-dependent expression, design experiments examining multiple environmental parameters: temperature ranges (18-32°C), pH variations (5.0-7.0), nutrient availability, and oxidative stress conditions that mimic plant defense responses. Time-course studies during infection stages capture dynamic expression changes from early attachment through biofilm formation and systemic spread. Single-cell RNA-seq can identify expression heterogeneity within bacterial populations during infection. Promoter reporter fusions (using fluorescent proteins or luciferase) enable real-time monitoring of expression dynamics in planta. Integrating expression data with genomic context analysis may reveal co-regulated gene clusters suggesting functional relationships. When analyzing differential expression, consider the genetic diversity and population structure of Xylella, as allopatric populations show divergence through both changes in gene content and nucleotide sequence variations . These approaches should be complemented with protein-level detection using specific antibodies to confirm that transcriptional changes translate to protein abundance differences .

What are the most promising approaches for targeting PD_0419 in disease management strategies?

Developing disease management strategies targeting PD_0419 requires evidence of its essential role in Xylella fastidiosa virulence or survival. If such evidence is established, several approaches warrant investigation. Structure-based drug design utilizing high-resolution structural data of PD_0419 can identify potential binding pockets for small molecule inhibitors. Virtual screening of compound libraries followed by biochemical validation identifies candidates for further development. Peptide inhibitors designed to disrupt protein-protein interactions involving PD_0419 offer alternative intervention strategies. RNA interference (RNAi) approaches using double-stranded RNA targeting PD_0419 delivered through transgenic plants or nanoparticle systems could suppress expression in vivo. CRISPR-Cas delivery systems adapted for bacterial pathogens present emerging opportunities for targeted gene disruption. For agricultural applications, consider deployment strategies that minimize selection pressure and resistance development, such as rotation of different molecular targets or combination approaches. Field testing should evaluate efficacy across different Xylella subspecies, as genetic variation affects intervention success—studies show PD-causing Xylella populations have diverged through both gene content changes and nucleotide sequence variations . Economic analysis of implementation costs versus disease reduction benefits provides practical context for stakeholders. Throughout development, consider regulatory requirements for agricultural deployment and environmental impact assessments .

What computational approaches can predict potential interacting partners for PD_0419?

Computational prediction of PD_0419 interacting partners requires integration of multiple bioinformatic approaches for robust results. Sequence-based methods include co-evolution analysis using tools like EVcouplings or GREMLIN, which identify correlated mutations between potentially interacting proteins. Gene neighborhood analysis examines genomic context conservation across bacterial species, as functionally related genes often cluster together. Protein-protein interaction (PPI) network analysis utilizing databases like STRING incorporates evidence from co-expression, text mining, and experimentally validated interactions, with confidence scores above 0.7 suggesting reliable predictions. Structure-based approaches include protein-protein docking with tools like HADDOCK, ClusPro, or ZDOCK, using available structural data or AlphaFold models similar to those for related UPF0307 proteins . Machine learning approaches trained on known bacterial PPIs can predict novel interactions based on sequence and structural features. When applying these methods to PD_0419, prioritize analysis within the context of known Xylella fastidiosa virulence mechanisms, particularly focusing on type IV pili components, adhesins, and regulatory systems that are highly conserved across Xylella species . Cross-reference predictions with transcriptomic data identifying co-expressed genes during infection. Integration of multiple prediction methods with consensus scoring improves prediction accuracy. Laboratory validation of top-ranked predictions using techniques like bacterial two-hybrid, co-immunoprecipitation, or proximity labeling is essential for confirming computational predictions .

What statistical approaches are recommended for analyzing PD_0419 functional assay data?

Statistical analysis of PD_0419 functional assay data requires tailored approaches based on experimental design and data characteristics. For enzyme kinetics experiments, nonlinear regression fitting to appropriate models (Michaelis-Menten, allosteric, inhibition) with tools like GraphPad Prism or R provides key parameters (Km, Vmax, kcat) with confidence intervals. When comparing multiple experimental conditions, use Analysis of Variance (ANOVA) with appropriate post-hoc tests (Tukey, Dunnett) for multiple comparisons, ensuring data meet assumptions of normality (Shapiro-Wilk test) and homoscedasticity (Levene's test). For non-parametric data, consider Kruskal-Wallis with Mann-Whitney U tests. Time-course experiments benefit from repeated measures ANOVA or mixed-effects models that account for within-subject correlations. Dose-response experiments should use four-parameter logistic regression to determine EC50/IC50 values. For high-throughput screening data, implement robust Z-factor analysis to assess assay quality, with Z' > 0.5 indicating excellent assay performance. When analyzing complex datasets from multiple experiments, consider multivariate approaches such as principal component analysis or partial least squares regression to identify patterns and relationships. For reproducibility, maintain detailed records of statistical methods, including software versions, parameter settings, and data transformations. Sample sizes should be determined through power analysis based on expected effect sizes, with transparency about biological and technical replicates .

How can researchers integrate structural and functional data to develop a comprehensive model of PD_0419 activity?

Integrating structural and functional data for PD_0419 requires systematic correlation of information across multiple scales. Begin by mapping functional data onto structural elements—annotate the high-confidence structural model (similar to those available for related UPF0307 proteins with pLDDT scores >90) with biochemical activity findings, identifying potential catalytic residues or binding interfaces. Use site-directed mutagenesis to validate the functional importance of specific residues, measuring activity changes quantitatively. Molecular dynamics simulations reveal conformational dynamics not captured in static structures, potentially identifying cryptic binding sites or allosteric mechanisms. For protein-ligand interactions, overlay binding data from spectroscopic studies onto structural models to visualize interaction interfaces. Network analysis of protein-protein interactions places PD_0419 in the broader context of Xylella fastidiosa cellular processes, particularly virulence mechanisms involving type IV pili and other pathogenicity factors . Implement data integration frameworks using Cytoscape or dedicated integrative modeling platforms like IMP (Integrative Modeling Platform) to visualize relationships between different data types. For conflicting data points, develop alternative hypotheses and design discriminatory experiments. The final model should be presented with clear indication of confidence levels for different aspects and explicit identification of knowledge gaps. This integration approach helps position PD_0419 within known virulence networks of Xylella fastidiosa, potentially connecting it to the highly conserved pathogenicity mechanisms observed across different subspecies .

How can high-throughput methodologies accelerate research on PD_0419 and related proteins?

High-throughput methodologies can dramatically accelerate PD_0419 research through parallel processing and automation. Implement parallel cloning strategies using Gateway or Gibson Assembly for generating comprehensive mutation libraries or truncation series, enabling systematic structure-function analyses. Automated expression screening in multiple host systems (E. coli, Pseudomonas, cell-free) with varied conditions identifies optimal production parameters. For purification, miniaturized chromatography platforms enable parallel buffer optimization across pH ranges (5.0-9.0) and salt concentrations (0-500 mM). Functional characterization benefits from microplate-based activity assays with automated liquid handling, allowing screening of hundreds of potential substrates or interaction partners simultaneously. Label-free interaction analysis using bio-layer interferometry arrays can test dozens of binding partners in parallel. For structural studies, crystallization robots testing thousands of conditions increase the likelihood of obtaining diffraction-quality crystals. Integration with laboratory information management systems (LIMS) ensures comprehensive data capture and facilitates machine learning approaches to identify patterns across large datasets. When applying these methods to PD_0419, design screens relevant to Xylella fastidiosa biology, focusing on potential roles in virulence mechanisms such as type IV pili function, adhesion, or interactions with plant substrates . Compare results across Xylella subspecies to identify conserved functions, recognizing that pathogen populations have diversified through various evolutionary mechanisms . These approaches generate comprehensive datasets that support systems-level understanding of PD_0419's role in bacterial physiology and pathogenesis .

What single-cell approaches reveal heterogeneity in PD_0419 expression during infection?

Single-cell approaches provide unprecedented insights into PD_0419 expression heterogeneity during infection, revealing subpopulation behaviors masked in bulk analyses. Single-cell RNA sequencing (scRNA-seq) adapted for bacterial cells through specialized lysis protocols and unique molecular identifiers captures transcriptional heterogeneity across thousands of individual bacteria. For visualization of expression in situ, RNA fluorescence in situ hybridization (RNA-FISH) with signal amplification detecting PD_0419 transcripts provides spatial context within infected plant tissues. Fluorescent reporter fusions (transcriptional or translational) combined with time-lapse microscopy track expression dynamics in real-time during infection processes. Flow cytometry and fluorescence-activated cell sorting (FACS) quantify expression distributions and isolate specific subpopulations for further characterization. Single-cell proteomics using mass cytometry (CyTOF) with metal-labeled antibodies against PD_0419 correlates protein levels with other bacterial markers. Microfluidic devices creating artificial xylem environments enable controlled perturbation experiments while monitoring single-cell responses. When implementing these approaches, consider potential contributions of PD_0419 to known heterogeneous behaviors in Xylella fastidiosa populations, such as differential expression of virulence factors like type IV pili components that are highly conserved across Xylella species . Analysis should employ computational methods specifically designed for single-cell data, including trajectory inference algorithms that reconstruct expression dynamics during infection progression and spatial statistical approaches that correlate expression patterns with microenvironmental features .

How might CRISPR-Cas technology advance functional studies of PD_0419 in Xylella fastidiosa?

CRISPR-Cas technology offers transformative approaches for studying PD_0419 function in Xylella fastidiosa with unprecedented precision. Implement CRISPR-Cas9 genome editing for generating clean knockouts, point mutations, or tagged versions at the native locus, avoiding polar effects on neighboring genes. Design guide RNAs with high specificity using algorithms that account for the GC content and genomic context specific to Xylella. For delivery into this Gram-negative bacterium, optimize electroporation protocols or develop specialized conjugation systems. CRISPRi (CRISPR interference) using catalytically inactive Cas9 (dCas9) enables tunable gene repression without genome modification, allowing titration of PD_0419 expression levels to identify threshold effects. Conversely, CRISPRa (CRISPR activation) can upregulate expression to assess gain-of-function phenotypes. Multiplex editing targeting PD_0419 alongside potential interaction partners identified through computational predictions allows investigation of genetic interactions and pathway relationships. For high-throughput functional genomics, CRISPR screens with pooled guide RNA libraries can identify genetic interactions across the genome. When phenotyping edited strains, comprehensively assess virulence-associated traits including biofilm formation, twitching motility, and plant colonization capacity, connecting PD_0419 function to established virulence mechanisms in Xylella fastidiosa such as type IV pili function . Consider subspecies-specific optimization of CRISPR protocols, as genetic diversity between populations may affect editing efficiency . Integration with 'omics approaches provides context for functional findings, positioning PD_0419 within the broader pathogenicity networks that have evolved in this important plant pathogen .