Recombinant Xylella fastidiosa Phenylalanine--tRNA ligase beta subunit (pheT), partial

What is the role of Phenylalanine--tRNA ligase beta subunit (pheT) in Xylella fastidiosa?

The Phenylalanine--tRNA ligase beta subunit (pheT) plays a crucial role in protein synthesis in Xylella fastidiosa by catalyzing the attachment of phenylalanine to its cognate tRNA molecule. This aminoacylation process is essential for accurate translation of genetic information. In X. fastidiosa, which exhibits high rates of horizontal gene transfer and recombination, the pheT gene may contribute to the organism's adaptability across different plant hosts. The protein functions within the complex protein synthesis machinery that allows X. fastidiosa to proliferate in plant xylem vessels, contributing to its pathogenicity in numerous economically important crops .

How do the genetics of pheT differ among Xylella fastidiosa subspecies?

The genetic diversity of the pheT gene varies across the five recognized X. fastidiosa subspecies (fastidiosa, multiplex, pauca, sandyi, and morus). Comparative genomic analyses reveal sequence variations in pheT that often align with subspecies classifications. These genetic differences may contribute to the varying host ranges observed among subspecies. For instance, X. fastidiosa subsp. fastidiosa and subsp. multiplex carry distinct allelic variants of many genes, including those involved in protein synthesis . Similar to other genetic elements in X. fastidiosa, pheT sequences can serve as molecular markers for strain identification and may contribute to the pathogen's adaptation to different environmental niches or host specialization.

What sequencing approaches are recommended for identifying pheT variants in clinical samples?

For identifying pheT variants in field samples of X. fastidiosa, a multi-tiered approach is recommended:

• Initial detection using real-time PCR targeting conserved regions of the X. fastidiosa genome

• Follow-up with nested PCR protocols for increased sensitivity, particularly for insect vector samples where bacterial loads may be lower

• Amplification of the pheT gene region using specific primers

• Sequencing using either:

  • Traditional Sanger sequencing for individual gene analysis

  • Whole-genome sequencing for comprehensive genetic characterization

This approach has proven effective in detecting X. fastidiosa in both plant tissues and insect vectors such as Philaenus spumarius, where bacterial loads typically range from 10³ to 10⁴ cells per insect . Custom nested PCR protocols have demonstrated superior sensitivity compared to standard qPCR methods when working with vector specimens, revealing that X. fastidiosa is often more widely distributed than previously detected using less sensitive methods .

How does horizontal gene transfer affect the genetic diversity of pheT in X. fastidiosa populations?

Horizontal gene transfer (HGT) significantly impacts the genetic diversity of genes like pheT in X. fastidiosa populations through several mechanisms:

• Natural transformation: X. fastidiosa demonstrates high rates of natural competence, allowing for the uptake of extracellular DNA from the environment .

• Conjugative transfer: Some strains possess tra and trb operons that encode type IV secretion systems facilitating plasmid transfer between cells .

• Recombination events: Following DNA acquisition, recombination can incorporate foreign genetic material into the recipient genome.

This genetic exchange can occur between different subspecies and strains of X. fastidiosa that occupy the same ecological niche, particularly within insect vectors that might harbor multiple strains simultaneously. Research has shown that approximately 6% of P. spumarius specimens carry two different subspecies of X. fastidiosa, creating opportunities for genetic exchange . These processes contribute to the mosaic nature of the X. fastidiosa genome and may accelerate adaptation to new hosts or environmental conditions, potentially affecting genes involved in essential functions like protein synthesis, including pheT.

What role do restriction-modification systems play in regulating recombination of pheT genes?

Type I restriction-modification (R-M) systems function as bacterial immune systems and significantly influence horizontal gene transfer and recombination of genes including pheT in X. fastidiosa:

• R-M systems recognize and cleave foreign DNA lacking appropriate methylation patterns

• The specificity subunit (hsdS) determines target sequence recognition

• Through recombination, target recognition domains (TRDs) can be exchanged between hsdS genes

• This process generates novel specificities, affecting which DNA sequences can be successfully transferred

Analysis of 129 X. fastidiosa genome assemblies identified 44 unique TRDs among 50 hsdS alleles, arranged in 31 different allele profiles that generally correspond to monophyletic strain clusters . Some strains exhibit inactivating mutations in their R-M systems, creating heterogeneity in functional R-M system complements across X. fastidiosa populations . This variability likely influences the potential for successful horizontal transfer of genes like pheT between strains, potentially affecting evolutionary trajectories and functional adaptation.

How can conjugative plasmid systems be utilized for experimental manipulation of pheT in X. fastidiosa?

Conjugative plasmid systems offer valuable tools for experimental manipulation of genes like pheT in X. fastidiosa strains that may be difficult to transform using standard methods:

• Donor strain selection: Utilize strains like M23 (X. fastidiosa subsp. fastidiosa) or Dixon (X. fastidiosa subsp. multiplex) that carry functional tra and trb operons .

• Vector construction: Employ mobilizable broad-host-range vectors like pBBR5pemIK, which has demonstrated successful transfer between X. fastidiosa strains .

• Recombinant construction: Clone the pheT gene (wild-type or modified) into the vector under appropriate regulatory elements.

• Conjugation protocol: Co-culture donor and recipient strains under conditions that promote cell contact and plasmid transfer.

• Selection: Apply appropriate antibiotic selection to identify successful transconjugants.

This approach has been validated for plasmid transfer between different subspecies of X. fastidiosa, with transfer efficiency dependent on both tra and trb operon functions . The method enables genetic modification of strains recalcitrant to natural transformation and allows for the introduction of larger genetic constructs than might be possible through transformation.

What expression systems are most effective for producing recombinant X. fastidiosa pheT for structural studies?

For structural studies of recombinant X. fastidiosa pheT, several expression systems have demonstrated effectiveness:

• E. coli-based systems:

  • BL21(DE3) with pET expression vectors under T7 promoter control

  • Arctic Express strains for expression at lower temperatures to enhance proper folding

  • Fusion tags: His6, MBP (maltose-binding protein), or SUMO tags to enhance solubility

• Cell-free expression systems:

  • Particularly valuable for potentially toxic proteins

  • Allows rapid screening of various conditions

• Optimized expression parameters:

The choice of expression system should be guided by the specific experimental goals. For crystallography or cryo-EM studies requiring large amounts of highly pure protein, E. coli systems with affinity tags offer the best yield-to-effort ratio. For functional studies where proper folding is critical, slower expression at reduced temperatures with chaperone co-expression may be preferable.

What are the key considerations for designing site-directed mutagenesis experiments on pheT to study functional domains?

When designing site-directed mutagenesis experiments for X. fastidiosa pheT functional domain analysis, researchers should consider:

• Domain identification:

  • Class II aaRS enzymes like PheRS typically contain catalytic, anticodon-binding, and editing domains

  • Sequence alignment with structurally characterized homologs (e.g., from E. coli) to identify conserved residues

• Target selection strategy:

  • Conserved catalytic residues (HIGH and KMSKS motifs)

  • Residues specific to X. fastidiosa compared to other organisms

  • Interface residues between α and β subunits

  • Residues potentially involved in tRNA recognition

• Mutation types:

  • Conservative substitutions (maintain chemical properties)

  • Non-conservative substitutions (alter chemical properties)

  • Alanine-scanning mutagenesis (systematic replacement with alanine)

• Functional assays:

  • Aminoacylation activity measurements (ATP-PPi exchange, tRNA charging)

  • Binding assays (fluorescence anisotropy, ITC)

  • Thermal stability determination (thermal shift assays)

• Controls:

  • Wild-type pheT as positive control

  • Known inactive mutants from homologous systems

  • Multiple independent mutant clones to ensure reproducibility

When interpreting results, researchers should consider potential long-range structural effects of mutations and not simply attribute phenotypes to direct functional roles of the mutated residues. Combining mutagenesis with structural studies provides the most comprehensive understanding of structure-function relationships.

How can researchers effectively analyze the evolutionary relationships between pheT variants using bioinformatic approaches?

Effective bioinformatic analysis of evolutionary relationships between X. fastidiosa pheT variants requires a multi-faceted approach:

• Sequence acquisition and alignment:

  • Collect pheT sequences from diverse X. fastidiosa strains representing all subspecies

  • Include pheT sequences from related Xanthomonadaceae for outgroup comparison

  • Use progressive alignment algorithms (MUSCLE, MAFFT) with refinement steps

• Phylogenetic analysis:

  • Maximum likelihood methods (RAxML, IQ-TREE) with appropriate substitution models

  • Bayesian inference (MrBayes) for posterior probability assessment

  • Test multiple models and select based on likelihood ratio tests or AIC/BIC criteria

• Recombination detection:

  • Employ multiple detection methods (RDP, GENECONV, MaxChi)

  • Sliding window analyses to identify potential breakpoints

  • Phylogenetic network approaches (SplitsTree) to visualize reticulate evolution

• Selection analysis:

  • Calculate dN/dS ratios to identify selection pressures

  • Codon-based tests (PAML, HyPhy) to detect site-specific selection

  • Assess functional constraints across different domains

• Structural mapping:

  • Map variable sites onto predicted 3D structures

  • Assess clustering of variable positions in functional domains

This comprehensive approach can reveal patterns of evolution, including evidence of horizontal gene transfer events, recombination breakpoints, and sequence type associations. Such analyses may identify pheT regions that contribute to host specificity or environmental adaptation, particularly when correlated with strain metadata such as host range, geographic origin, and pathogenicity profiles.

How can recombinant pheT be utilized to develop novel diagnostic tools for X. fastidiosa detection?

Recombinant pheT-based diagnostic tools offer promising approaches for X. fastidiosa detection with several advantages:

• Antibody-based detection systems:

  • Generate anti-pheT antibodies using purified recombinant protein

  • Develop ELISA-based detection systems for field diagnostics

  • Create lateral flow immunoassays for rapid testing

• Aptamer-based biosensors:

  • Select DNA/RNA aptamers with high affinity for pheT

  • Develop electrochemical or optical biosensors using these aptamers

  • Enable real-time detection in plant tissues or insect vectors

• Molecular beacon probes:

  • Design fluorescent probes targeting pheT sequence variations

  • Develop multiplex assays detecting different subspecies simultaneously

  • Integrate with portable nucleic acid amplification platforms

These approaches can complement existing PCR-based methods, which sometimes underestimate X. fastidiosa prevalence, particularly in insect vectors . When developing such diagnostic tools, researchers should validate with diverse strain collections representing all known sequence types and subspecies. Field testing should include both plant tissues and insect vectors, with particular attention to P. spumarius populations, where bacterial loads typically range from 10³ to 10⁴ cells per insect but can reach 10⁵ or 10⁶ cells in approximately 13% of individuals .

What approaches can resolve contradictory phylogenetic signals between pheT and other housekeeping genes in X. fastidiosa?

Resolving contradictory phylogenetic signals between pheT and other housekeeping genes requires sophisticated analytical approaches:

• Concatenated gene analysis vs. gene-by-gene approach:

  • Construct phylogenies using both methods

  • Compare topologies to identify incongruences

  • Quantify statistical support for conflicting nodes

• Recombination detection and filtering:

  • Apply recombination detection algorithms to identify potential recombination events

  • Remove recombining regions or model recombination explicitly

  • Reconstruct phylogenies with and without putative recombinant regions

• Coalescent-based species tree estimation:

  • Implement methods that account for incomplete lineage sorting (ASTRAL, *BEAST)

  • Compare gene trees to species trees to identify discordance patterns

  • Quantify genealogical discordance using appropriate metrics

• Network-based approaches:

  • Use phylogenetic networks to visualize conflicting signals

  • Apply consensus network methods to summarize contradictory patterns

  • Identify potential hybrid origins or introgression events

• Time-calibrated analyses:

  • Develop dated phylogenies to estimate timing of divergence events

  • Compare chronology across different genes

  • Identify temporal patterns consistent with HGT events

Contradictory signals may result from horizontal gene transfer events facilitated by the high rates of natural transformation in X. fastidiosa or plasmid-mediated conjugative transfer . Additionally, restriction-modification systems with variable target specificities across strains may create uneven barriers to gene flow, resulting in mosaic evolutionary patterns . Researchers should consider that approximately 6% of insect vectors may simultaneously harbor multiple X. fastidiosa subspecies, creating opportunities for genetic exchange .

How might the study of pheT contribute to understanding X. fastidiosa host adaptation mechanisms?

The study of pheT can provide unique insights into X. fastidiosa host adaptation mechanisms through several research approaches:

• Comparative genomics across host-specific strains:

  • Analyze pheT sequence variations between strains with different host preferences

  • Identify amino acid substitutions that correlate with host range

  • Compare evolutionary rates in strains infecting different hosts

• Experimental evolution studies:

  • Track pheT sequence changes during adaptation to new hosts

  • Monitor translation efficiency during host switching

  • Assess selection pressures on pheT in different plant environments

• Proteome-wide translation efficiency analysis:

  • Examine codon usage patterns in relation to tRNA abundance

  • Analyze potential host-specific optimization of translation

  • Assess phenylalanine usage in proteins critical for host adaptation

• Functional characterization of variant pheT alleles:

  • Express and purify pheT variants from different host-adapted strains

  • Compare aminoacylation efficiency and accuracy

  • Assess thermal stability and pH optima in relation to host xylem conditions

Understanding how translation machinery components like pheT adapt to different host environments may reveal subtle mechanisms of host specialization. Since X. fastidiosa strains carrying conjugative transfer genes can belong to different subspecies and frequently differ in host ranges , analyzing the correlation between pheT variants and host specificity could provide insights into the genetic basis of host adaptation. This knowledge could inform strategies for disease management in agricultural settings where X. fastidiosa causes significant economic losses.

What strategies can overcome difficulties in genetic manipulation of X. fastidiosa strains with active restriction-modification systems?

Genetic manipulation of X. fastidiosa strains with active restriction-modification systems presents significant challenges. These strategies can improve success rates:

• Host-mimicking methylation approaches:

  • Express X. fastidiosa-specific methyltransferases in E. coli

  • Prepare plasmid DNA with strain-specific methylation patterns

  • Design vectors that avoid recognition sequences of active R-M systems

• Restriction inhibitor supplementation:

  • Include TypeOne Restriction Inhibitor during transformation

  • Supplement with GTP as a competitive inhibitor for type I R-M systems

• Conjugation-based approaches:

  • Utilize the tra and trb operons for conjugative transfer

  • Employ broad-host-range vectors like pBBR5pemIK that have demonstrated successful transfer

  • Optimize conjugation conditions for specific donor-recipient pairs

• Natural transformation optimization:

  • Induce competence via growth on specific media or starvation

  • Provide high concentrations of DNA to overwhelm restriction systems

  • Use DNA fragments with homologous flanking regions for recombination

• CRISPR-Cas-based genome editing:

  • Deliver CRISPR components via conjugative plasmids

  • Target and inactivate restriction-modification genes

  • Create restriction-deficient strains for subsequent manipulations

These approaches should be tailored to the specific X. fastidiosa strain, as R-M system complements vary significantly across strains. Analysis of 129 X. fastidiosa genome assemblies revealed substantial heterogeneity in functional R-M systems, with some strains carrying inactivating mutations . Understanding the specific methylation patterns of the target strain can guide the selection of appropriate genetic manipulation strategies.

What are the most reliable protocols for isolating high-quality recombinant pheT for structural studies?

Isolating high-quality recombinant X. fastidiosa pheT for structural studies requires optimized protocols addressing several challenges:

• Expression optimization:

  • Test multiple expression constructs with various fusion tags

  • Optimize induction conditions (temperature, inducer concentration, time)

  • Consider co-expression with the alpha subunit (pheS) for proper folding

• Multi-step purification strategy:

• Quality control assessments:

  • Dynamic light scattering to confirm homogeneity

  • Thermal shift assays to optimize buffer conditions

  • Activity assays to confirm functional integrity

  • Mass spectrometry to verify protein identity and modifications

• Stability enhancements:

  • Screen additives using thermal shift assays

  • Identify optimal buffer conditions for long-term stability

  • Consider limited proteolysis to identify stable core domains

• Co-purification approaches:

  • Purify with pheS to maintain physiological heterodimeric complex

  • Include appropriate tRNA substrates for stabilization

  • Consider purification with substrate analogs for crystallization

Researchers should be particularly attentive to potential contamination with bacterial endotoxins, which can interfere with downstream applications. Additionally, proper folding of pheT may depend on specific chaperones or interaction with the alpha subunit, so co-expression strategies often yield better results than expressing the beta subunit alone.

How can researchers effectively analyze the impact of pheT variants on X. fastidiosa virulence in different host plants?

Analyzing the impact of pheT variants on X. fastidiosa virulence requires comprehensive experimental approaches that connect molecular mechanisms to plant pathology:

• Genetic manipulation strategies:

  • Create isogenic strains differing only in pheT alleles

  • Use conjugative plasmid transfer for strains resistant to transformation

  • Complement pheT mutants with variants from different subspecies

• In vitro characterization:

  • Assess growth rates in standard and host-mimicking media

  • Measure biofilm formation capabilities

  • Quantify protein synthesis rates and fidelity

• Plant inoculation studies:

  • Test multiple host species representing different susceptibilities

  • Quantify bacterial populations at various time points post-inoculation

  • Assess symptom development using standardized disease scales

• Transcriptomic and proteomic analyses:

  • Compare gene expression profiles between strains with different pheT variants

  • Identify differentially expressed virulence factors

  • Analyze translation efficiency of key virulence genes

• Vector transmission experiments:

  • Assess acquisition efficiency by Philaenus spumarius

  • Measure vector transmission rates between plants

  • Determine bacterial population dynamics in insect vectors

• Mixed infection experiments:

  • Co-inoculate plants with strains carrying different pheT variants

  • Track population dynamics over time

  • Assess competitive fitness in planta

This multi-faceted approach can determine whether pheT variants contribute to host adaptation, virulence, or fitness in specific plant hosts. Since approximately 6% of insect vectors can simultaneously carry multiple X. fastidiosa subspecies , understanding how different pheT variants affect bacterial fitness in mixed populations may provide insights into strain competition and succession in natural settings.

What are the most promising research directions for understanding the role of pheT in X. fastidiosa evolution and adaptation?

Several promising research directions could enhance our understanding of pheT's role in X. fastidiosa evolution and adaptation:

• Evolutionary rate analysis across ecological contexts:

  • Compare evolutionary rates of pheT across different plant hosts

  • Analyze selection pressures in different geographic regions

  • Identify potential signatures of adaptive evolution

• Experimental evolution under translation stress:

  • Subject X. fastidiosa populations to aminoglycoside pressure

  • Track mutations in translation machinery genes including pheT

  • Assess adaptation to translation errors and their consequences

• Proteome-wide mistranslation assessment:

  • Develop methods to quantify phenylalanine misincorporation rates

  • Compare mistranslation frequencies across strains with different pheT variants

  • Assess impacts on protein stability and function

• Cross-species comparative studies:

  • Compare pheT evolution in X. fastidiosa to other bacterial pathogens

  • Identify convergent adaptation patterns in translation machinery

  • Assess horizontal gene transfer of translation components across species

• Systems biology approaches:

  • Develop computational models of translation dynamics

  • Integrate transcriptomic, proteomic, and metabolomic data

  • Predict impacts of pheT variants on global cellular physiology

These approaches could reveal whether pheT variants contribute to the remarkably broad host range of X. fastidiosa as a species, despite individual strains often showing host specialization. Understanding the role of translation machinery in bacterial adaptation may provide new insights into pathogen evolution and potentially identify novel targets for disease management strategies.

How might advanced genomic techniques further our understanding of recombination events involving pheT?

Advanced genomic techniques offer powerful approaches to understanding recombination events involving pheT in X. fastidiosa:

• Long-read sequencing technologies:

  • PacBio and Oxford Nanopore for complete genome assemblies

  • Identification of structural variations and genomic rearrangements

  • Accurate placement of pheT in genomic context

• Metagenomics of mixed infections:

  • Sequence directly from infected plants or insects without isolation

  • Identify strain mixtures and potential recombinants

  • Detect cryptic genetic diversity not captured by culturing

• Single-cell genomics:

  • Sequence individual bacterial cells from infected tissues

  • Identify rare recombinant genotypes

  • Track heterogeneity within bacterial populations

• Chromosome conformation capture:

  • Map three-dimensional genome organization

  • Identify potential hotspots for recombination

  • Assess chromatin accessibility patterns

• Methylome analysis:

  • Map DNA methylation patterns genome-wide

  • Correlate with restriction-modification system profiles

  • Identify regions protected from or susceptible to restriction

These techniques could reveal the frequency and mechanisms of recombination events involving pheT and other genes. Given that approximately 6% of insect vectors carry multiple X. fastidiosa subspecies simultaneously and that restriction-modification systems show substantial variation across strains , advanced genomic approaches could identify factors that facilitate or inhibit genetic exchange. Understanding these dynamics is crucial for predicting the emergence of new pathogen variants with altered host ranges or virulence characteristics.

What interdisciplinary approaches could advance our understanding of the structure-function relationship in X. fastidiosa pheT?

Interdisciplinary approaches combining multiple scientific disciplines offer promising avenues for advancing our understanding of structure-function relationships in X. fastidiosa pheT:

• Integrated structural biology:

  • X-ray crystallography for high-resolution static structures

  • Cryo-electron microscopy for conformational ensembles

  • NMR spectroscopy for dynamics and ligand interactions

  • Molecular dynamics simulations to model conformational changes

• Systems genetics approaches:

  • GWAS across diverse X. fastidiosa strains

  • Correlation of sequence variations with phenotypic differences

  • Epistatic interaction mapping with other translation components

• Synthetic biology strategies:

  • Design chimeric pheT proteins with domains from different subspecies

  • Create minimal synthetic aminoacyl-tRNA synthetases

  • Explore non-canonical amino acid incorporation capabilities

• Chemical biology techniques:

  • Develop pheT-specific inhibitors as molecular probes

  • Use photo-crosslinking to map interaction interfaces

  • Apply activity-based protein profiling to assess functional states

• Evolutionary biochemistry:

  • Resurrect ancestral pheT sequences through phylogenetic inference

  • Characterize biochemical properties of ancestral and contemporary enzymes

  • Track functional changes along evolutionary trajectories

These interdisciplinary approaches could provide unprecedented insights into how pheT structure influences X. fastidiosa adaptation to different plant hosts. For example, comparing pheT properties between strains with different host preferences could reveal subtle adaptations in translation machinery that contribute to host specificity. Furthermore, understanding the structural basis of pheT function could guide the development of targeted antimicrobials that exploit unique features of the X. fastidiosa enzyme.