Recombinant Xylella fastidiosa Elongation factor Ts (tsf)

What is Xylella fastidiosa Elongation Factor Ts (tsf) and its role in bacterial protein synthesis?

Elongation factor Ts (EF-Ts) in Xylella fastidiosa is a protein involved in the elongation phase of bacterial translation. During protein synthesis, EF-Ts functions as a guanine nucleotide exchange factor that binds to EF-Tu after GTP hydrolysis, promoting the release of GDP from EF-Tu and facilitating the regeneration of the active EF-Tu·GTP complex . This cycle is essential for delivering aminoacyl-tRNAs to the ribosome during polypeptide chain elongation.

The methodological approach to studying this function involves:

• Purification of recombinant EF-Ts and EF-Tu proteins

• In vitro GDP/GTP exchange assays using labeled nucleotides

• Measurement of the kinetics of the exchange reaction

• Analysis of the interaction between EF-Ts and EF-Tu using techniques such as surface plasmon resonance

EF-Ts is considered an attractive antimicrobial target due to its essentiality and limited homology to eukaryotic counterparts . In Xylella fastidiosa specifically, the tsf gene is likely located within the core genome rather than within the flexible gene pool that comprises up to 18% of the genome .

How is the tsf gene structured in Xylella fastidiosa compared to other bacterial species?

The tsf gene in Xylella fastidiosa, like in other bacteria, encodes the elongation factor Ts protein. Based on molecular cloning approaches used in related studies, the structure of the tsf gene can be analyzed through the following methods:

• PCR amplification using high-fidelity Taq polymerase with specific primers targeting the tsf sequence

• Cloning into vectors such as pCR2.1 using TA cloning kits

• Sequence confirmation to verify the gene structure

• Restriction digestion analysis (e.g., using NdeI and XhoI) for further characterization

When comparing the tsf gene across different subspecies of X. fastidiosa (such as multiplex, fastidiosa, and sandyi), researchers should be aware that intersubspecific homologous recombination (IHR) may have influenced gene structure in some strains .

What methods are commonly used to express recombinant Xylella fastidiosa EF-Ts?

Expression of recombinant Xylella fastidiosa EF-Ts can be accomplished using established bacterial expression systems, primarily based on methodologies employed for other bacterial EF-Ts proteins. The following protocol outlines the recommended approach:

• Gene Amplification and Cloning:

  • Amplify the tsf gene from Xylella fastidiosa genomic DNA using PCR with high-fidelity polymerase

  • Design primers with appropriate restriction sites (e.g., NdeI and XhoI)

  • Clone the amplified fragment into an intermediate vector (e.g., pCR2.1)

  • Sequence-verify the cloned fragment

  • Subclone into an expression vector (e.g., pET28a) to attach a purification tag

• Expression Conditions:

  • Transform the expression construct into E. coli BL21(DE3) or similar expression strain

  • Grow cultures at 37°C to mid-log phase (OD600 of 0.6-0.8)

  • Induce protein expression with IPTG (typically 1.0 mM for EF-Ts)

  • Continue expression for 3-4 hours

• Optimization Considerations:

  • Temperature (30-37°C)

  • IPTG concentration (0.2-1.0 mM)

  • Expression duration (3-16 hours)

  • Media composition (rich vs. minimal)

Table 1: Optimization Parameters for Recombinant Xylella fastidiosa EF-Ts Expression

How does natural competence in Xylella fastidiosa affect recombinant protein expression strategies?

Xylella fastidiosa has been demonstrated to be naturally competent, capable of taking up exogenous DNA and incorporating it into its genome through homologous recombination . This natural competence has significant implications for recombinant protein expression strategies:

• Direct Transformation Potential:

  • X. fastidiosa can incorporate exogenous DNA at rates of approximately 1 in 10^6 cells when provided with plasmid DNA and 1 in 10^7 cells in co-culture conditions

  • This allows for direct transformation of expression constructs without the need for electroporation or other artificial competence methods

• Optimization Factors:

  • Nutrient availability significantly affects transformation efficiency

  • Growth stage influences competence levels

  • DNA methylation status of transforming DNA impacts recombination efficiency

• Homologous Recombination Considerations:

  • Expression constructs designed with homologous flanking regions can be integrated into the genome

  • This approach can be used for chromosomal integration of expression cassettes

  • PCR confirmation of integration events is essential using primers that span the recombination junctions

• Experimental Design Guidelines:

  • Use modified liquid XFM medium without selection for initial transformation

  • Allow sufficient time for recombination to occur before applying selection

  • Confirm recombination events through PCR and sequencing

  • Be aware that different strains may have varying competence levels

This natural competence also highlights the potential for developing X. fastidiosa-specific expression systems that leverage the organism's own genetic machinery, potentially improving the authenticity of expressed proteins.

What purification techniques are most effective for recombinant Xylella fastidiosa EF-Ts?

Purification of recombinant Xylella fastidiosa EF-Ts typically follows established protocols for bacterial translation factors, with modifications to address specific properties of the protein. The following methodological approach is recommended:

• Affinity Chromatography (Primary Method):

  • Expression with a 6×His-tag facilitates purification using nickel affinity chromatography

  • Cell lysis is performed using standard methods (sonication or French press)

  • Initial capture on Ni-NTA resin with binding buffer (typically containing 20-50 mM imidazole)

  • Washing steps with increasing imidazole concentrations

  • Elution with high imidazole (250-500 mM)

• Secondary Purification Methods:

  • Ion exchange chromatography (typically Q-Sepharose)

  • Size exclusion chromatography for further purification and buffer exchange

  • Hydrophobic interaction chromatography if needed for specific contaminants

• Optimization Considerations:

  • Buffer composition affects stability (typically 50 mM Tris-HCl pH 7.5, 100 mM KCl, 10 mM MgCl₂, 5% glycerol)

  • Addition of reducing agents (1-5 mM DTT or β-mercaptoethanol)

  • Temperature management (4°C for all steps)

  • Protease inhibitors during initial extraction

• Quality Control Assessment:

  • SDS-PAGE for purity evaluation

  • Western blotting for identity confirmation

  • Mass spectrometry for accurate mass determination

  • Activity assays to confirm functional integrity

Applying these methods typically yields protein of sufficient purity (>95%) for structural and functional studies, including crystallization attempts and biochemical assays.

How can genomic island and prophage regions in Xylella fastidiosa affect recombinant tsf expression?

Xylella fastidiosa possesses one of the largest flexible gene pools characterized in bacteria, with horizontally acquired elements such as prophages, plasmids, and genomic islands (GIs) contributing up to 18% of its genome . These elements can significantly impact recombinant tsf expression through several mechanisms:

• Regulatory Influence:

  • Prophages and GIs often contain transcriptional regulators that can act in trans

  • Microarray analysis has shown that expression of genes within these elements is transcriptionally active and can be influenced by environmental stimuli in a coordinated manner

  • This regulatory cross-talk could potentially affect expression from recombinant constructs

• Codon Usage Divergence:

  • Horizontally acquired elements often display altered codon bias and GC content compared to the core genome

  • Analysis of X. fastidiosa strain 9a5c chromosome reveals regions with distinct codon usage patterns

  • When designing expression constructs, codon optimization should account for these variations to maximize expression efficiency

• Mobile Genetic Element Activity:

  • Active transposition of mobile elements can disrupt expression constructs

  • Research has shown remarkable levels of transpositional activity during the evolution of X. fastidiosa groups

  • Experimental design should include monitoring for potential insertions or rearrangements

• Strain-Specific Considerations:

  • Different X. fastidiosa strains show variable presence of genomic islands and prophages

  • GI 1 is partly or entirely duplicated in citrus strains but absent in bacteria from other hosts (except coffee)

  • When working with different strains, characterization of the specific genomic context around the tsf gene is advisable

Table 2: Major Genomic Islands in Xylella fastidiosa with Potential Impact on Recombinant Expression

What are the implications of strain-specific variations in the tsf gene for recombinant protein functionality?

Strain-specific variations in the Xylella fastidiosa tsf gene can have significant implications for recombinant protein functionality, particularly given the evidence of extensive recombination in this species:

• Subspecies Divergence:

  • X. fastidiosa comprises multiple subspecies (multiplex, fastidiosa, sandyi) with genetic variations

  • Intersubspecific homologous recombination (IHR) has been detected in multiple loci

  • These variations may affect protein structure and function in subspecies-specific ways

• Functional Adaptation:

  • Different X. fastidiosa strains infect different plant hosts, suggesting adaptive evolution

  • Variations in translation machinery proteins like EF-Ts may contribute to host-specific adaptation

  • Recombinant proteins from different strains may exhibit subtle functional differences relevant to pathogenicity

• Experimental Approaches to Address Variation:

  • Comparative sequence analysis of tsf genes from multiple strains

  • Expression and characterization of EF-Ts variants from different strains

  • Functional complementation assays to test interchangeability

  • Structural studies to identify critical regions affected by variation

• Methodological Considerations:

  • When studying X. fastidiosa EF-Ts, researchers should:

    • Clearly specify the strain/isolate source

    • Sequence-verify the tsf gene before expression

    • Consider expressing multiple strain variants for comparative studies

    • Test functionality in homologous and heterologous systems

Table 3: Analysis of Strain Variation Impact on Recombinant Protein Function

How do post-translational modifications of recombinant Xylella fastidiosa EF-Ts compare to native protein modifications?

Post-translational modifications (PTMs) of bacterial elongation factors can affect their function and interactions. Comparing PTMs between recombinant and native Xylella fastidiosa EF-Ts requires systematic analysis:

• Identification of Native PTMs:

  • Mass spectrometry-based proteomics of X. fastidiosa cell extracts

  • Enrichment methods for specific modifications (phosphorylation, methylation, etc.)

  • Western blot analysis with modification-specific antibodies

  • 2D gel electrophoresis to separate protein variants

• Expression System Considerations:

  • E. coli expression systems may not reproduce all native X. fastidiosa PTMs

  • Alternative expression hosts (Pseudomonas, other Gram-negative bacteria) might better reproduce relevant modifications

  • Cell-free expression systems allow controlled addition of modification enzymes

• Analytical Methods for Comparison:

  • High-resolution mass spectrometry (LC-MS/MS)

  • Peptide mapping with modification-specific detection

  • Functional assays comparing native and recombinant protein activities

  • Structural studies (X-ray crystallography, cryo-EM) to visualize modification sites

• Strategies to Preserve or Mimic Native Modifications:

  • Co-expression with relevant modification enzymes

  • In vitro enzymatic modification after purification

  • Site-directed mutagenesis to mimic constitutive modifications (e.g., Glu for phospho-Ser)

  • Chemical modification approaches

The environmental context is particularly important, as transcriptome analysis shows that expression in X. fastidiosa can be influenced by environmental stimuli in a coordinated manner . This suggests that growth conditions may influence the PTM profile of native EF-Ts, which should be considered when comparing to recombinant versions.

What experimental approaches can differentiate between EF-Ts functions in different Xylella fastidiosa subspecies?

Differentiating between EF-Ts functions across Xylella fastidiosa subspecies requires multifaceted experimental approaches that address both structural and functional aspects:

• Comparative Genomic Analysis:

  • Sequence alignment of tsf genes from different subspecies (fastidiosa, multiplex, sandyi)

  • Identification of subspecies-specific variations and potential recombination events

  • Phylogenetic analysis to understand evolutionary relationships

• Recombinant Protein Expression and Characterization:

  • Expression of EF-Ts variants from multiple subspecies

  • Comparative biochemical characterization:

    • GDP/GTP exchange kinetics with cognate and non-cognate EF-Tu proteins

    • Thermal stability studies (differential scanning fluorimetry)

    • Protein-protein interaction analysis (surface plasmon resonance, isothermal titration calorimetry)

• Cross-complementation Studies:

  • Construction of tsf knockout or conditional mutants in different subspecies

  • Complementation with tsf genes from other subspecies

  • Assessment of growth rates, translation efficiency, and stress responses

  • Analysis of subspecies-specific phenotypes (biofilm formation, virulence)

• In vitro Translation Assays:

  • Reconstituted translation systems using components from different subspecies

  • Measurement of translation rates and fidelity using reporter constructs

  • Competition assays between subspecies variants

• Structural Studies:

  • Crystal structures or homology models of EF-Ts variants

  • Molecular dynamics simulations to identify functional differences

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

Table 4: Methods for Functional Comparison of EF-Ts Across X. fastidiosa Subspecies

How can structural studies of recombinant Xylella fastidiosa EF-Ts inform antimicrobial drug development?

Structural studies of recombinant Xylella fastidiosa EF-Ts can provide valuable insights for antimicrobial drug development, leveraging the fact that bacterial elongation factors are essential proteins with limited homology to eukaryotic counterparts :

• Structure Determination Methods:

  • X-ray crystallography of purified recombinant EF-Ts

  • Cryo-electron microscopy of EF-Ts/EF-Tu complexes

  • NMR spectroscopy for dynamic structural information

  • Computational modeling based on homologous structures

• Target Site Identification:

  • The interface between EF-Ts and EF-Tu represents a primary target

  • Nucleotide binding pockets involved in GDP/GTP exchange

  • Unique structural features of X. fastidiosa EF-Ts compared to human elongation factors

  • Allosteric sites that could affect protein function

• Drug Discovery Approaches:

  • Structure-based virtual screening against identified binding sites

  • Fragment-based drug discovery using NMR or X-ray crystallography

  • High-throughput screening using assays that measure:

    • Inhibition of EF-Ts/EF-Tu interaction

    • Interference with GDP/GTP exchange function

    • Impact on bacterial translation in vitro

• Optimization Strategies:

  • Lead compounds can be optimized based on structural information

  • Medicinal chemistry to improve:

    • Binding affinity to the target site

    • Selectivity over human translation factors

    • Cell penetration into bacterial cells

    • Resistance to efflux mechanisms

Previous research has identified several compound classes with inhibitory properties against bacterial elongation factors, including indole dipeptides, benzimidazole amidines, 2-arylbenzimidazoles, N-substituted imidazoles, and N-substituted guanidines . These provide starting points for X. fastidiosa-specific inhibitors.

Table 5: Potential Binding Sites on X. fastidiosa EF-Ts for Inhibitor Development

What are the comparative interaction kinetics between recombinant Xylella fastidiosa EF-Ts and EF-Tu versus those from other bacterial species?

Understanding the comparative interaction kinetics between Xylella fastidiosa EF-Ts/EF-Tu and those from other bacterial species provides insights into potential species-specific translation mechanisms:

• Methodological Approaches for Kinetic Analysis:

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Fluorescence-based assays for real-time interaction monitoring

  • Scintillation proximity assay for high-throughput screening

• Key Parameters for Comparison:

  • Association rate constants (kon)

  • Dissociation rate constants (koff)

  • Equilibrium dissociation constants (KD)

  • Thermodynamic parameters (ΔH, ΔS, ΔG)

  • Nucleotide exchange rates

• Experimental Design:

  • Expression and purification of recombinant EF-Ts and EF-Tu from:

    • Xylella fastidiosa (multiple strains/subspecies)

    • E. coli (as reference)

    • Other plant pathogens (comparative analysis)

  • Preparation of protein variants with appropriate tags for immobilization

  • Development of standardized assay conditions

• Data Analysis and Interpretation:

  • Comparison of kinetic parameters across species

  • Correlation with structural differences

  • Assessment of the impact of temperature, pH, and ionic conditions

  • Evaluation of species-specific inhibitors

Table 6: Comparative Binding Kinetics Framework for EF-Ts/EF-Tu Interactions

A novel scintillation proximity assay has been developed for the detection of inhibitors of EF-Tu and EF-Ts interaction , which could be adapted for comparative studies of X. fastidiosa and other species to identify both conserved and species-specific aspects of this interaction.

How can site-directed mutagenesis of recombinant Xylella fastidiosa EF-Ts help elucidate pathogenicity mechanisms?

Site-directed mutagenesis of recombinant Xylella fastidiosa EF-Ts can provide valuable insights into bacterial pathogenicity mechanisms through systematic analysis of protein function:

• Rational Design of Mutations:

  • Target conserved residues in active sites based on structural data

  • Focus on residues unique to X. fastidiosa compared to non-pathogenic bacteria

  • Investigate subspecies-specific residues that correlate with host range

  • Examine positions implicated in intersubspecific recombination events

• Mutagenesis and Expression Strategy:

  • Use overlap extension PCR or commercial site-directed mutagenesis kits

  • Express in systems allowing controlled induction (e.g., pET28a with IPTG)

  • Purify mutant proteins using established protocols for wild-type EF-Ts

  • Verify structural integrity through circular dichroism or thermal shift assays

• Functional Characterization:

  • In vitro GDP/GTP exchange assays to measure catalytic activity

  • Protein-protein interaction studies with EF-Tu

  • Thermal stability analysis to assess structural impact

  • Translation efficiency assays using cell-free systems

• In vivo Pathogenicity Studies:

  • Complementation of EF-Ts knockout strains with mutant variants

  • Assessment of growth rates in different media conditions

  • Evaluation of biofilm formation capabilities

  • Plant infection assays to measure virulence directly

  • Analysis of bacterial survival under environmental stresses relevant to plant infection

Table 7: Site-Directed Mutagenesis Strategy for X. fastidiosa EF-Ts

This approach leverages the understanding that translation machinery plays a critical role in bacterial adaptation to different environments, including plant hosts. The extensive genomic flexibility of Xylella fastidiosa, with its large flexible gene pool and ability to undergo homologous recombination , suggests that even subtle changes in essential proteins like EF-Ts could contribute to host specificity and virulence.