Recombinant Xylella fastidiosa Elongation factor G (fusA), partial

What is Elongation factor G (fusA) and what role does it play in Xylella fastidiosa?

Elongation factor G (EF-G), encoded by the fusA gene, is a critical protein involved in bacterial protein synthesis. In Xylella fastidiosa, as in other bacteria, EF-G functions in ribosomal peptide elongation and ribosome recycling processes . The protein facilitates the translocation step of protein synthesis by catalyzing the movement of tRNAs and mRNA through the ribosome. This process is essential for bacterial survival and pathogenicity, as it enables the efficient production of proteins required for cellular functions and host interaction. X. fastidiosa, as a xylem-limited bacterial pathogen affecting numerous plant species across the Americas, relies on proper protein synthesis mechanisms to maintain its infectious capabilities .

How does fusA sequence variation differ among Xylella fastidiosa subspecies?

The fusA gene shows varying degrees of sequence conservation across the four main Xylella fastidiosa subspecies: fastidiosa, sandyi, multiplex, and pauca. These subspecies have diverged genetically by approximately 1-3%, primarily due to geographical isolation over the past 20,000 to 50,000 years . Sequence analysis of multiple loci, including fusA, has revealed that while some regions of the gene remain highly conserved due to functional constraints on the EF-G protein, other regions display subspecies-specific variations. These variations serve as useful markers for taxonomic classification and for tracking intersubspecific recombination events that may occur when previously geographically isolated subspecies come into contact .

What are the standard methods for amplifying and sequencing the fusA gene from Xylella fastidiosa isolates?

Researchers typically employ PCR-based approaches to amplify the fusA gene from Xylella fastidiosa isolates. The process begins with bacterial DNA extraction from pure cultures or infected plant tissue, followed by PCR amplification using primers targeting conserved regions flanking the fusA gene. For multi-locus sequence typing (MLST) studies that include fusA, established primer sets are available that generate amplicons suitable for sequencing and comparative analysis. Sequencing is generally performed using Sanger methodology for individual isolates or next-generation sequencing for population-level studies. When analyzing recombinant strains or novel isolates, it may be necessary to design new primers based on conserved regions identified through alignment of known fusA sequences from different X. fastidiosa subspecies .

What expression systems are most effective for producing recombinant Xylella fastidiosa EF-G protein?

Escherichia coli expression systems have proven most effective for producing recombinant Xylella fastidiosa EF-G protein. According to available product information, E. coli is the preferred host for expression of this recombinant protein . The effectiveness of E. coli stems from its well-established genetic manipulation protocols, rapid growth, and high protein yield capabilities. For optimal expression, researchers typically use T7 promoter-based expression vectors with fusion tags to facilitate purification. IPTG-inducible systems allow controlled expression, minimizing potential toxicity issues. While eukaryotic expression systems like yeast or insect cells might offer advantages for certain proteins requiring post-translational modifications, the prokaryotic nature of X. fastidiosa EF-G makes bacterial expression systems most suitable for producing functionally relevant recombinant protein .

What purification strategies yield the highest purity for recombinant fusA protein?

High-purity recombinant fusA protein (>85% as determined by SDS-PAGE) is typically achieved through multi-step purification protocols . The purification strategy begins with affinity chromatography, leveraging fusion tags engineered into the recombinant construct. Common tag options include His-tag, GST, or MBP, with the specific tag determined during the manufacturing process. Following initial capture by affinity chromatography, secondary purification steps may include ion exchange chromatography to separate proteins based on charge differences and size exclusion chromatography to remove aggregates and achieve final polishing. To maintain protein integrity throughout the purification process, appropriate buffer conditions must be maintained, typically including protease inhibitors and stabilizing agents. The final product is often supplied in a storage buffer containing glycerol (typically 5-50% final concentration) to prevent protein degradation during freeze-thaw cycles .

How should recombinant fusA protein be stored to maintain activity?

For optimal stability and activity maintenance, recombinant Xylella fastidiosa EF-G protein should be stored according to specific guidelines. The recommended storage conditions include -20°C for standard storage, with -80°C preferred for extended storage periods . Prior to opening, vials should be briefly centrifuged to bring contents to the bottom. Reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL. To prevent activity loss during storage, addition of glycerol to a final concentration of 5-50% (with 50% being typical) is recommended, followed by aliquoting to avoid repeated freeze-thaw cycles . Working aliquots can be stored at 4°C for up to one week, but long-term storage requires -20°C/-80°C. The shelf life of liquid preparations is approximately 6 months at -20°C/-80°C, while lyophilized forms maintain stability for about 12 months under the same conditions .

How does the fusA gene contribute to Xylella fastidiosa host adaptation?

The fusA gene's role in X. fastidiosa host adaptation is primarily indirect but nonetheless significant. While not directly involved in host-pathogen interactions like adhesins or virulence factors, the EF-G protein encoded by fusA is essential for bacterial protein synthesis. Research has demonstrated that intersubspecific homologous recombination (IHR) involving multiple loci, including potentially fusA, has facilitated host shifts in X. fastidiosa . This recombination introduces novel genetic variation that may alter protein expression patterns critical for adaptation to new plant hosts. For example, analysis of X. fastidiosa subsp. multiplex isolates revealed that recombinant types carrying genetic material from X. fastidiosa subsp. fastidiosa gained the ability to infect previously non-susceptible hosts like blueberry and blackberry . While the specific contribution of fusA variants to this host shift remains to be fully characterized, it is likely that changes in translation efficiency and fidelity resulting from altered EF-G function influence the expression of direct virulence factors, thereby contributing to the adaptation process .

What evidence exists for fusA involvement in intersubspecific recombination events?

Multiple lines of evidence support fusA involvement in intersubspecific recombination events in Xylella fastidiosa. Studies analyzing X. fastidiosa subsp. multiplex isolates have identified significant introgression from X. fastidiosa subsp. fastidiosa in multiple loci, potentially including fusA . Research has demonstrated that recombinant forms of X. fastidiosa originated via genome-wide recombination between X. fastidiosa subsp. multiplex ancestors and X. fastidiosa subsp. fastidiosa donors . Parsimony analysis using alleles as characters supports a common origin for mulberry-type and recombinant-group sequence types (STs), suggesting coordinated recombination events across multiple loci . While not all studies specifically identify fusA as a recombination hotspot, the pattern of recombination observed across the X. fastidiosa genome indicates that essential genes involved in core cellular functions, including potentially fusA, have been subject to intersubspecific exchange that has facilitated host adaptation and expanded the pathogen's host range .

How do fusA sequence variants correlate with host specificity in different Xylella fastidiosa strains?

The correlation between fusA sequence variants and host specificity in Xylella fastidiosa demonstrates a complex relationship shaped by intersubspecific recombination. Research has identified that recombinant X. fastidiosa subsp. multiplex isolates carrying genetic material from X. fastidiosa subsp. fastidiosa gained the ability to infect novel hosts, including blueberry (7 isolates from Georgia, 3 from Florida) and blackberry (1 each from Florida and North Carolina) . While non-recombinant X. fastidiosa subsp. multiplex strains can infect some plant species, the recombinant types show unique host associations not observed in their non-recombinant counterparts. This strongly supports the hypothesis that intersubspecific recombination facilitated these host shifts . Although the specific contribution of fusA variants to this host specificity hasn't been fully characterized, the protein's fundamental role in translation suggests it may influence the expression of virulence factors and other host-interaction proteins, thereby indirectly affecting host range .

How can recombinant fusA be used to study antibiotic resistance mechanisms in bacteria?

Recombinant fusA provides an excellent model for studying antibiotic resistance mechanisms, particularly against drugs that target protein synthesis. Fusidic acid (FA) specifically targets Elongation Factor G, inhibiting ribosomal peptide elongation and ribosome recycling . Using recombinant fusA in biochemical assays allows researchers to investigate the kinetics of FA inhibition at multiple stages of translation. Quench flow and stopped flow experiments have revealed that FA targets EF-G at three distinct stages during translocation: an early stage (I), a later stalling stage (II), and just before EF-G release from the post-translocation ribosome (stage III) . By introducing specific mutations into recombinant fusA constructs and assessing their effects on FA binding and inhibition, researchers can identify resistance mechanisms and characterize the structural basis for drug resistance. This approach enables the rational design of new antibiotics that can overcome resistance mechanisms and provides insights into bacterial adaptation strategies .

What are the methodological approaches for studying fusA protein-protein interactions?

Studying fusA protein-protein interactions requires a multi-faceted approach combining biochemical, biophysical, and structural techniques. For in vitro interaction studies, researchers can employ pull-down assays using recombinant fusA with affinity tags to identify binding partners, followed by mass spectrometry for identification. Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) provide quantitative measurements of binding kinetics and thermodynamics. For structural characterization of these interactions, X-ray crystallography and cryo-electron microscopy have proven particularly valuable, allowing researchers to place fusA-ribosome complexes in their functional context . In vivo interactions can be assessed using bacterial two-hybrid systems or fluorescence resonance energy transfer (FRET). To specifically study how recombinant fusA interacts with ribosomes, reconstituted translation systems containing purified components provide controlled environments for mechanistic investigations. These approaches collectively enable researchers to understand how fusA variants from different X. fastidiosa subspecies might differ in their interaction networks, potentially contributing to host adaptation .

How can comparative analysis of fusA be used to track evolutionary relationships between Xylella fastidiosa subspecies?

Comparative analysis of fusA sequences provides valuable insights into the evolutionary relationships between Xylella fastidiosa subspecies and the impact of recombination on their divergence. Researchers employ multilocus sequence typing (MLST) approaches that include fusA along with other genetic markers (such as leuA, cysG, malF, petC, holC, nuoL, gltT, and pilU) to construct phylogenetic trees and track introgression events . Specific analytical methods include nucleotide diversity calculations (π within and π total), introgression tests, and parsimony analysis using alleles as characters . These approaches have revealed that X. fastidiosa subspecies diverged genetically by 1-3% over approximately 20,000-50,000 years of geographical isolation, with subsequent human-mediated contact facilitating intersubspecific recombination . By analyzing sequence mismatches between putative recombinant regions and potential donor sequences, researchers can trace the geographic origin of introgressed DNA fragments. For example, research has demonstrated that some IHR regions in X. fastidiosa subsp. multiplex were inconsistent with X. fastidiosa subsp. fastidiosa alleles found in the United States but matched alleles from Central America .

How does fusA mutation or recombination affect Xylella fastidiosa's response to environmental stressors?

The impact of fusA mutation or recombination on X. fastidiosa's response to environmental stressors remains an active area of research. As an essential component of the translation machinery, alterations in EF-G structure or function could significantly affect protein synthesis under stress conditions. Recombinant strains carrying fusA alleles from different subspecies may exhibit altered translation efficiency or accuracy, potentially affecting the expression of stress-response proteins. This could manifest as changes in thermal tolerance, desiccation resistance, or survival during nutrient limitation - all critical factors for a xylem-limited pathogen facing variable environmental conditions within plant hosts. Research has demonstrated that intersubspecific recombination has facilitated host shifts in X. fastidiosa , suggesting that recombinant strains may possess enhanced stress tolerance in certain host environments. Studying the specific contribution of fusA recombination to stress adaptation requires comparative proteomics approaches to identify differentially expressed proteins between recombinant and non-recombinant strains under controlled stress conditions, followed by functional characterization of candidate stress-response pathways.

How does the biochemical activity of recombinant fusA differ between Xylella fastidiosa subspecies?

The biochemical activity differences of fusA between Xylella fastidiosa subspecies have yet to be fully characterized, presenting an important research opportunity. Potential differences might include variations in GTP hydrolysis rates, ribosome binding affinity, or translocation efficiency. These differences could arise from amino acid substitutions in key functional domains resulting from subspecies divergence or intersubspecific recombination . To investigate these differences, researchers would need to express and purify recombinant fusA proteins from different X. fastidiosa subspecies and recombinant strains, then conduct comparative biochemical assays. These could include GTPase activity measurements using malachite green phosphate detection assays, ribosome binding studies using fluorescence anisotropy or surface plasmon resonance, and in vitro translation assays measuring translocation rates and accuracy. Of particular interest would be comparing fusA activity from non-recombinant X. fastidiosa subsp. multiplex strains with recombinant strains carrying X. fastidiosa subsp. fastidiosa-derived fusA alleles to determine if recombination confers altered translational properties that might contribute to the observed host shifts in recombinant strains .

What statistical approaches are recommended for designing surveys to detect Xylella fastidiosa in field populations?

Statistically sound and risk-based surveys for detecting Xylella fastidiosa require careful methodological consideration. The European Food Safety Authority (EFSA) guidelines recommend a three-step approach . First, clearly define survey aims (detection, delimitation, or buffer zone monitoring) and characterize the host plant population and detection methods. Second, calculate required sample sizes using statistical tools like RiBESS+ with inputs including design prevalence, test sensitivity, and desired confidence level. Finally, strategically allocate samples across the survey area based on population distribution and risk factors . For detection surveys substantiating pest freedom, assume a low design prevalence (often 1%). Delimiting surveys to determine infestation boundaries require higher sampling intensity near known infected areas. Buffer zone surveys monitor areas at low prevalence levels, requiring intermediate sampling intensity . The statistical rigor of these approaches enables comparison across time and space, contributing to harmonization of X. fastidiosa surveys across regions while maintaining flexibility to account for specific host plants, vectors, and climate conditions .

How can researchers effectively design experiments to study recombination between fusA alleles from different Xylella fastidiosa subspecies?

Designing experiments to study recombination between fusA alleles from different Xylella fastidiosa subspecies requires a multi-faceted approach. Researchers should first establish a comprehensive collection of isolates representing diverse subspecies, geographical regions, and host plants to capture the full range of fusA genetic diversity. Sequencing of the fusA gene and flanking regions from these isolates provides the foundation for identifying potential recombination events using specialized software packages that include multiple detection methods, such as those found in RDP4 program and the PHI program . To study recombination experimentally, researchers can develop in vitro transformation systems utilizing natural competence of X. fastidiosa or explore the role of conjugative plasmids in facilitating genetic exchange . Experimental evolution studies exposing mixed populations of different subspecies to selective pressures can reveal whether recombination occurs preferentially in specific regions of fusA and what selective advantages recombinant alleles might confer. Combining these experimental approaches with comparative genomic analyses of natural recombinants provides complementary perspectives on the mechanisms and consequences of fusA recombination .

What methodological considerations are important when comparing fusA sequences for population genetics studies?

When comparing fusA sequences for population genetics studies of Xylella fastidiosa, several methodological considerations are critical for robust analysis. First, sampling design must ensure adequate representation across geographic regions, host plants, and temporal periods to capture the full genetic diversity. For sequence analysis, consistent protocols for DNA extraction, PCR amplification, and sequencing are essential to minimize technical artifacts . Researchers should employ multiple sequence alignment tools with parameters optimized for the expected level of sequence divergence (1-3% between subspecies) . Population genetic analyses should include calculation of genetic diversity indices (such as nucleotide diversity π) both within and between populations, and formal tests for recombination using methods like the introgression test . When analyzing potential recombinant sequences, researchers should compare them against comprehensive databases of known alleles from different subspecies and geographic regions to accurately identify the source of introgressed fragments . Finally, interpretation of results must consider the biological context, including the known history of subspecies introduction and contact, to distinguish between recent recombination events and shared ancestral polymorphisms .

What are the key differences in fusA allele distributions among Xylella fastidiosa isolates from different host plants?

The distribution of fusA alleles among Xylella fastidiosa isolates demonstrates clear patterns related to host plant associations, particularly in recombinant strains. Research has identified specific plant hosts for recombinant X. fastidiosa types carrying genetic material potentially including fusA variants from different subspecies. Below is a comparative analysis of host associations:

This table demonstrates that while some plant hosts (oak, elm, sycamore) support both non-recombinant and recombinant X. fastidiosa types, blueberry and blackberry appear uniquely associated with recombinant types . This pattern strongly supports the hypothesis that intersubspecific recombination, potentially involving fusA, facilitated host shifts to these berry crops .

How do different analytical methods compare in their ability to detect recombination events in the fusA gene?

Different analytical methods vary considerably in their sensitivity and specificity for detecting recombination events in the fusA gene and other loci in Xylella fastidiosa. The table below compares the effectiveness of various recombination detection methods based on research findings:

Research has demonstrated that the introgression test detected significant evidence of intersubspecific recombination in X. fastidiosa when other methods (RDP4 and PHI) failed to identify recombination events . This highlights the importance of employing multiple complementary analytical approaches when investigating recombination in bacterial genomes .

What molecular and functional characteristics distinguish fusA protein variants found in different Xylella fastidiosa subspecies?

The molecular and functional characteristics of fusA protein variants across Xylella fastidiosa subspecies reveal important differences that may influence bacterial physiology and host interactions. While comprehensive biochemical characterization of fusA variants remains limited, available data allow for the following comparative analysis:

This comparative analysis highlights that while core functional domains remain conserved across subspecies due to selective constraints, recombination events have introduced subspecies-specific variations that may contribute to the observed differences in host range, particularly in recombinant X. fastidiosa subsp. multiplex strains that have acquired sequences from X. fastidiosa subsp. fastidiosa .

What are the most promising approaches for studying the structural biology of fusA and its interactions with the ribosome?

The most promising approaches for studying fusA structural biology combine advanced imaging technologies with functional assays. Cryo-electron microscopy (cryo-EM) represents the gold standard for visualizing fusA-ribosome complexes at near-atomic resolution, allowing researchers to capture different conformational states during the translation cycle . This technique has already provided valuable insights into the mechanism of fusidic acid inhibition of EF-G . X-ray crystallography remains valuable for high-resolution structures of isolated fusA domains. Emerging approaches include hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map conformational changes and interaction surfaces, and integrative structural biology combining multiple experimental data types with computational modeling. For X. fastidiosa specifically, comparative structural analysis of fusA variants from different subspecies using these techniques could reveal how sequence variations affect protein function and drug interactions. Single-molecule FRET and optical tweezers approaches offer opportunities to study the dynamics of fusA-ribosome interactions in real-time. These structural insights would significantly advance understanding of how fusA variants contribute to X. fastidiosa subspecies diversification and host adaptation .

How might CRISPR-Cas9 gene editing be applied to study the function of fusA variants in Xylella fastidiosa?

CRISPR-Cas9 gene editing represents a powerful approach for investigating fusA function in Xylella fastidiosa through precise genetic manipulation. This technology could enable researchers to generate isogenic strains differing only in their fusA alleles, allowing direct assessment of how variants from different subspecies affect bacterial phenotypes. Specific applications include: (1) Allelic replacement experiments swapping fusA alleles between subspecies to determine their contribution to host specificity observed in recombinant strains ; (2) Introduction of targeted mutations in conserved functional domains to probe structure-function relationships; (3) Creation of fluorescently-tagged fusA variants to study localization and dynamics in living cells; and (4) Development of inducible expression systems to control fusA levels and study dosage effects. Technical considerations include optimizing transformation protocols for X. fastidiosa, which is naturally competent but may require specialized approaches for different subspecies . Screening methods must account for the essential nature of fusA, potentially requiring conditional approaches. The resulting engineered strains would enable unprecedented insights into how fusA variants contribute to X. fastidiosa biology, pathogenicity, and host adaptation .

What new insights might be gained from comparative genomics studies of fusA across the entire Xylellaceae family?

Expanding comparative genomics studies of fusA beyond Xylella fastidiosa to encompass the entire Xylellaceae family would provide valuable evolutionary context and functional insights. Such broad-scale analysis would reveal the degree of fusA conservation across related genera and identify lineage-specific adaptations. By constructing phylogenetic trees based on fusA sequences, researchers could determine whether this gene follows the expected species tree or shows evidence of horizontal gene transfer beyond the documented intersubspecific recombination in X. fastidiosa . Selective pressure analysis (dN/dS ratios) across the family would identify regions under purifying, neutral, or positive selection, potentially correlating with functional domains or bacterial lifestyle adaptations. Synteny analysis of the genomic regions surrounding fusA might reveal conservation patterns or rearrangements affecting gene regulation. Comparative analysis of recombination frequencies across the family could determine whether the intersubspecific recombination observed in X. fastidiosa is exceptional or common within Xylellaceae. These insights would contextualize current understanding of fusA variation in X. fastidiosa and potentially identify novel functional elements contributing to host adaptation and bacterial evolution that could inform disease management strategies .

What are the key considerations for researchers working with recombinant fusA in Xylella fastidiosa studies?

Researchers working with recombinant Xylella fastidiosa Elongation Factor G (fusA) should consider several critical factors to ensure successful and meaningful experiments. First, proper storage and handling of the recombinant protein is essential, including storage at -20°C/-80°C, addition of glycerol (5-50%), and minimizing freeze-thaw cycles to maintain activity . Second, experimental design should account for the biological context of fusA in X. fastidiosa, particularly its role in protein synthesis and potential involvement in intersubspecific recombination events that facilitate host shifts . Third, when comparing fusA variants from different subspecies, researchers should employ multiple complementary analytical approaches, as different methods vary in their sensitivity for detecting recombination events . Fourth, interpretation of results should consider the evolutionary history of X. fastidiosa subspecies, including their geographical isolation and subsequent human-mediated contact that facilitated recombination . Finally, researchers should explore the functional consequences of fusA variation through structural and biochemical analyses, potentially revealing how sequence differences affect protein function and contribute to bacterial adaptation to different plant hosts .

How does current research on fusA contribute to our understanding of Xylella fastidiosa evolution and host adaptation?

Research on fusA provides significant insights into the evolution and host adaptation mechanisms of Xylella fastidiosa, particularly through the lens of intersubspecific recombination. Studies have demonstrated that recombination between previously allopatric subspecies has introduced novel genetic variation that facilitates host shifts . The identification of recombinant X. fastidiosa types with unique host associations, such as blueberry and blackberry, strongly supports the hypothesis that intersubspecific recombination enables adaptation to new plant hosts . While fusA's primary role in protein synthesis may seem disconnected from host interaction, variations in this essential gene could affect translation efficiency and accuracy, influencing the expression of virulence factors and other host-interaction proteins. The finding that recombinant regions in some loci match alleles from Central America rather than the United States suggests complex evolutionary histories involving multiple introduction events . By studying fusA alongside other loci involved in recombination events, researchers can reconstruct the evolutionary trajectories of X. fastidiosa lineages and identify the genetic changes that enable this pathogen to expand its host range, with significant implications for disease management in agricultural settings .