Recombinant Xylella fastidiosa tRNA (guanine-N (7)-)-methyltransferase (trmB)

Basic Research Questions

• What is Xylella fastidiosa and why is it significant for research?

Xylella fastidiosa is a fastidious, gram-negative bacterium belonging to the family Xanthomonadaceae. It is a xylem-limited plant pathogen with extraordinary biological properties that make it a significant research subject. X. fastidiosa possesses a remarkably broad host range, infecting over 600 plant species across 63 diverse plant families . This bacterium exhibits a unique dual lifestyle, acting either as a pathogen causing severe diseases or as a benign commensal depending on the host plant .

The economic impact of X. fastidiosa has been severe in the Americas, Europe, and parts of Asia, where it causes devastating diseases in agricultural crops including Pierce's disease in grapevines, citrus variegated chlorosis, and diseases affecting almond, coffee, and ornamental plants . Its xylem-limited nature makes it an excellent model for studying vascular pathogens , while its genetic recombination capabilities and restriction-modification systems provide insights into bacterial evolution and adaptation .

Understanding X. fastidiosa is crucial for developing disease management strategies, particularly since it is transmitted by xylem sap-feeding insects like sharpshooter leafhoppers , adding complexity to its epidemiology and control.

• What is tRNA (guanine-N(7)-)-methyltransferase (trmB) and what is its function in X. fastidiosa?

tRNA (guanine-N(7)-)-methyltransferase (trmB) is an enzyme in X. fastidiosa that catalyzes the methylation of the N7 position of guanine in tRNA molecules. The enzyme is classified under EC number 2.1.1.33 . In the X. fastidiosa M12 strain, the trmB protein consists of 244 amino acids , and its structure has been computationally modeled through AlphaFold (AF-B0U509-F1) .

The primary function of trmB is to modify specific tRNA molecules, which is essential for:

• Maintaining proper translational fidelity and efficiency

• Ensuring correct tRNA folding and stability

• Contributing to cellular stress responses

• Potentially regulating gene expression through tRNA modifications

While not explicitly stated in the search results, trmB may contribute to X. fastidiosa's adaptation to different environmental conditions or hosts by modulating translation through tRNA modifications. The methylation patterns established by enzymes like trmB could also interact with the broader epigenetic landscape of the bacterium, which includes the restriction-modification systems extensively studied in X. fastidiosa .

Table 1: Key characteristics of X. fastidiosa tRNA (guanine-N(7)-)-methyltransferase

• How does X. fastidiosa interact with host plants?

X. fastidiosa interacts with host plants through complex mechanisms that determine whether it establishes a pathogenic or commensal relationship. The bacterium specifically colonizes the xylem tissue of plants, where it forms biofilms that can obstruct water transport . Interestingly, biofilm formation appears to attenuate virulence in some hosts. Mutant strains impaired in biofilm formation and locked in a planktonic phase exhibit hypervirulent phenotypes in grapevines, suggesting that X. fastidiosa enters the surface adhesive biofilm state as a means to attenuate its own virulence .

Several key factors influence plant-pathogen interactions:

• The compatibility between xylem pit membrane carbohydrate composition and X. fastidiosa-secreted cell wall-degrading enzymes mediates disease onset and progression .

• The O antigen component of X. fastidiosa is critical for evading initial immune recognition in susceptible plants like grapevines .

• The self-limiting behavior during parasitism in symptomatic hosts may be a remnant from its lifestyle as a commensal in nonsymptomatic hosts .

• Recent research has focused on how X. fastidiosa interacts with the plant immune system and influences the host's microbiome .

The mechanism by which X. fastidiosa causes disease in certain hosts but acts as a commensal in others is not fully elucidated, making this an active area of research .

• What are restriction-modification systems in X. fastidiosa?

Restriction-modification (R-M) systems in X. fastidiosa are genetic elements that protect the bacterium from foreign DNA and influence horizontal gene transfer. These systems are particularly important in understanding X. fastidiosa's evolution and genetic diversity. The search results provide detailed information specifically about Type I R-M systems in X. fastidiosa:

Type I R-M systems consist of three subunits: restriction endonuclease (HsdR), methyltransferase (HsdM), and specificity (HsdS) components . Genomic analyses have identified four main Type I R-M systems in X. fastidiosa:

• Three systems (XfaI, XfaII, XfaIII) are conserved across all 129 X. fastidiosa genome assemblies examined .

• One system (XfaIV) is found only in subspecies multiplex and pauca .

• One of these systems shares homology with a Type I R-M system present in genome assemblies of 31 Xylella taiwanensis strains .

The HsdS subunits contain target recognition domains (TRDs) that determine DNA sequence specificity. These TRDs can recombine between HsdS subunits to generate novel alleles with new target specificities . Researchers identified 44 unique TRDs among 50 HsdS alleles across X. fastidiosa strains, arranged in 31 distinct allele profiles .

Importantly, transformation efficiency of some X. fastidiosa strains is increased by inhibition or deletion of Type I R-M systems , highlighting their role in mediating horizontal gene transfer. Inactivating mutations were identified in Type I R-M systems of specific strains, showing heterogeneity in the complement of functional systems across X. fastidiosa lineages .

Table 2: Type I Restriction-Modification Systems in X. fastidiosa

• How is the structure of X. fastidiosa tRNA (guanine-N(7)-)-methyltransferase characterized?

The structure of X. fastidiosa tRNA (guanine-N(7)-)-methyltransferase has been computationally modeled rather than experimentally determined. According to search result , the structure model is available in the RCSB Protein Data Bank with the identifier AF_AFB0U509F1 . This model was generated using AlphaFold and represents the trmB protein from X. fastidiosa M12 strain (UniProtKB: B0U509) .

The computational model provides several insights into the potential structure:

It's important to note that this is a computed structure model with no experimental data to verify its accuracy . The actual experimental determination of the structure would require techniques such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, or cryo-electron microscopy.

The model can provide hypotheses about the enzyme's catalytic site, substrate binding regions, and potential structural changes during catalysis, but these would need experimental validation.

Advanced Research Questions

• What methodologies are used to study recombination in X. fastidiosa restriction-modification systems?

Research into recombination in X. fastidiosa restriction-modification systems employs several sophisticated methodologies that provide insights into genetic diversity and epigenetic regulation:

Genomic comparative analysis forms the foundation of these studies, with researchers comparing Type I R-M systems across 129 X. fastidiosa genome assemblies representing all known subspecies and 32 sequence types . This comprehensive approach allowed for the identification of conserved R-M systems and detailed analysis of sequence variation across the species.

Target Recognition Domain (TRD) classification methodologies involve categorizing TRDs within HsdS genes into homology groups (e.g., TRD-1 "A", TRD-1 "B", etc.) to track recombination events and allele diversity . This classification system revealed that 44 unique TRDs combine to form at least 50 unique HsdS alleles across the four Type I R-M systems .

Researchers employed positional analysis of TRDs to detect domain shuffling. For instance, in the XfaII system, two TRDs ("Y" and "Z") were found to have switched positions, suggesting domain position recombination in addition to sequence variation .

Methylome analysis provided functional validation by characterizing DNA methylation patterns in 20 X. fastidiosa strains and associating these patterns with Type I R-M system allele profiles . This approach helps link genetic variation in R-M systems to functional differences in DNA methylation.

Additional methodologies included identification of inactivating mutations (frameshift mutations, SSR length variations) and phylogenetic analysis that examined HsdS allele profiles in relation to monophyletic strain clusters of X. fastidiosa .

These complementary approaches have revealed that HsdS genes recombine among Xylella strains and/or unknown donors, and the resulting TRD reassortment establishes differential epigenetic modifications across Xylella lineages .

• How do epigenetic modifications affect X. fastidiosa virulence and host range?

The relationship between epigenetic modifications and X. fastidiosa virulence/host range represents an emerging frontier in understanding this pathogen. DNA methylation patterns vary across X. fastidiosa strains and are associated with Type I R-M system allele profiles . These differential epigenetic modifications likely influence gene expression patterns that could affect various aspects of bacterial behavior.

While direct experimental evidence linking specific methylation patterns to virulence is limited in the search results, several mechanisms can be proposed based on current knowledge:

• Epigenetic regulation may control the expression of genes involved in biofilm formation, which is known to attenuate virulence in some hosts . Differential methylation could potentially shift the balance between planktonic and biofilm lifestyles.

• Type I R-M systems influence horizontal gene transfer , potentially facilitating or restricting the acquisition of virulence factors or genes involved in host adaptation. This could indirectly impact the evolution of virulence and host range.

• Methylation patterns could affect the expression of cell wall-degrading enzymes, which interact with xylem pit membrane carbohydrates and are implicated in determining disease outcomes .

• Epigenetic modifications might influence how X. fastidiosa interacts with the plant immune system, potentially affecting the O antigen composition that is critical for evading immune recognition .

The search results indicate that X. fastidiosa strains show significant variability in virulence on specific host plant species , but the direct connection between epigenetic modifications and this variability requires further research. The study referenced in suggests that understanding methylation associated with Type I R-M systems across X. fastidiosa strains could provide insight into the bacterium's biology and evolution through epigenetics, potentially explaining aspects of host specificity and virulence.

Table 3: Potential Relationships Between Epigenetic Modifications and X. fastidiosa Virulence Factors

• What approaches can be used to express and purify recombinant X. fastidiosa trmB for structural studies?

Successful expression and purification of recombinant X. fastidiosa trmB for structural studies requires a systematic approach addressing the unique challenges of this enzyme. While the search results don't specifically address expression and purification of recombinant X. fastidiosa trmB, methodological approaches can be proposed based on standard techniques for bacterial methyltransferases.

The expression system selection is a critical first step. Several options should be considered:

• Escherichia coli-based expression systems using vectors such as pET series for high-level expression

• Codon optimization for E. coli, considering the potential codon bias between X. fastidiosa and E. coli

• Testing different fusion tags (His-tag, GST-tag, MBP-tag) to improve solubility and facilitate purification

• Evaluation of various expression conditions (temperature, IPTG concentration, expression duration) to maximize yield of soluble protein

Purification strategies should follow a multi-step approach:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged proteins)

• Intermediate purification using ion exchange chromatography

• Final polishing using size exclusion chromatography

• On-column or post-purification tag removal using appropriate proteases (TEV, thrombin)

Protein solubility and stability optimization is essential for structural studies:

• Systematic buffer screening (pH, salt concentration, additives)

• Addition of stabilizing agents or cofactors (potentially S-adenosylmethionine as a methyl donor)

• Testing different storage conditions for long-term stability

• Using thermal shift assays to identify stabilizing conditions

Quality assessment should be rigorous:

• SDS-PAGE and Western blot analysis for purity and identity

• Mass spectrometry for accurate molecular weight determination and confirmation of intact protein

• Activity assays to confirm functional integrity of the recombinant enzyme

• Dynamic light scattering to assess homogeneity and detect aggregation

The AlphaFold-predicted structure mentioned in search result could guide experimental design by identifying potentially disordered regions that might interfere with crystallization or stable protein expression. For structural studies specifically, crystallization screening should proceed using commercial sparse matrix kits, followed by optimization of promising conditions and potential co-crystallization with substrate analogs or cofactors.

• How can genomic data be used to analyze the evolution of trmB genes across X. fastidiosa subspecies?

Genomic data analysis for studying trmB evolution across X. fastidiosa subspecies requires a comprehensive approach similar to that employed for R-M systems in the search results. The following methodological framework would be appropriate:

Comparative genomic analysis forms the foundation:

• Identification and extraction of trmB gene sequences from multiple X. fastidiosa genomes representing different subspecies

• Multiple sequence alignment using tools like MUSCLE, MAFFT, or ClustalW to identify conserved and variable regions

• Calculation of nucleotide and amino acid sequence identity across strains

• Identification of single nucleotide polymorphisms (SNPs) and insertion/deletion events

Phylogenetic analysis provides evolutionary context:

• Construction of phylogenetic trees using different algorithms (Maximum Likelihood, Bayesian inference)

• Comparison of trmB gene phylogeny with whole-genome phylogeny to detect potential horizontal gene transfer events

• Analysis of selection pressures using dN/dS ratios to identify regions under purifying or diversifying selection

• Ancestral sequence reconstruction to infer the evolutionary trajectory of trmB

Functional domain analysis connects sequence to function:

• Identification of conserved catalytic and substrate-binding domains

• Comparison with trmB genes from other bacterial species

• Prediction of functional consequences of observed sequence variations

• Structural modeling of variant proteins to assess potential functional changes

Association with bacterial phenotypes provides biological relevance:

• Correlation analysis between trmB sequence variants and known phenotypic differences between X. fastidiosa strains

• Investigation of potential links between trmB variations and host specificity or virulence

• Integration with transcriptomic data to assess if trmB variants correlate with expression differences

The approach demonstrated in the search results for R-M systems, where researchers identified system components across 129 genome assemblies , provides a robust template for how such an analysis might be structured for trmB genes. This comprehensive analysis could reveal whether trmB has undergone adaptive evolution in different subspecies of X. fastidiosa in response to different host environments.

• What experimental designs are optimal for studying the role of trmB in X. fastidiosa pathogenicity?

To rigorously investigate the role of trmB in X. fastidiosa pathogenicity, a multi-faceted experimental approach is necessary. The following experimental designs would provide comprehensive insights:

Gene knockout studies provide the foundation:

• Creation of trmB deletion mutants using homologous recombination or CRISPR-Cas systems

• Construction of conditional mutants if trmB is essential for viability

• Complementation studies to confirm phenotypes are directly related to trmB deletion

• Development of point mutants in catalytic sites to distinguish enzymatic from structural roles

Phenotypic characterization reveals functional consequences:

• Assessment of growth rates in different media and conditions

• Biofilm formation assays, which are particularly relevant as biofilm formation is linked to virulence attenuation in X. fastidiosa

• Cell adhesion and motility assays to evaluate changes in bacterial behavior

• Stress response experiments (temperature, oxidative stress) to assess bacterial resilience

Plant infection experiments connect to disease processes:

• Inoculation of wild-type and trmB mutant strains into susceptible host plants

• Quantification of bacterial population in planta over time using qPCR

• Assessment of disease symptom development and progression

• Comparison across multiple host plants to determine if trmB affects host range

Molecular mechanism studies provide mechanistic understanding:

• RNA-seq to compare gene expression profiles between wild-type and trmB mutant strains

• Ribosome profiling to assess translational impacts of altered tRNA modification

• tRNA modification analysis using mass spectrometry to quantify changes in tRNA methylation patterns

• Proteomics approaches to identify differences in protein expression and post-translational modifications

Vector transmission experiments assess ecological relevance:

• Assessment of acquisition and transmission efficiency by insect vectors for wild-type and trmB mutant strains

• Analysis of bacterial persistence in the vector

• Evaluation of gene expression changes during vector colonization

Table 4: Experimental Approaches for Studying trmB in X. fastidiosa Pathogenicity

Research should consider the unique aspects of X. fastidiosa biology, including its fastidious nature, xylem colonization, and relationship with insect vectors . Furthermore, the dual lifestyle of X. fastidiosa as either pathogen or commensal depending on the host suggests that experiments should be conducted in multiple plant species to fully understand the role of trmB in different host interactions.