Recombinant Xylella fastidiosa Translation initiation factor IF-2 (infB), partial

What is Xylella fastidiosa and why is it significant in research?

Xylella fastidiosa is a xylem-limited, Gram-negative bacterial pathogen that causes economically important diseases in numerous plant species throughout the Americas. The bacterium is divided into four main subspecies: fastidiosa, sandyi, multiplex, and pauca, which have diverged genetically by 1-3% due to geographical isolation over approximately 20,000 to 50,000 years . The significance of X. fastidiosa in research stems from its status as a major phytosanitary threat to global agricultural production, causing severe diseases including Pierce's disease in grapevines, citrus variegated chlorosis (CVC), and olive quick decline syndrome (OQDS) . The bacterium's ability to persist with either a commensal or pathogenic lifestyle in more than 500 plant species makes it an important model organism for studying host-pathogen interactions and bacterial adaptation mechanisms .

How do researchers differentiate between Xylella fastidiosa subspecies?

Researchers primarily differentiate between X. fastidiosa subspecies using molecular typing methods. The most widely employed approach is Multilocus Sequence Typing (MLST), which categorizes isolates into sequence types (STs) based on unique sets of alleles across multiple loci . The standard MLST protocol for X. fastidiosa utilizes 7 housekeeping loci plus additional genes such as pilU . This method provides valuable insight into the evolutionary history and genetic diversity of the taxa.

For subspecies that have undergone intersubspecific homologous recombination (IHR), more sensitive detection methods are required. The "introgression test" can identify recombination events that might be missed by standard recombination detection programs such as RDP4 and PHI . Researchers typically compare allele sequences to known non-IHR X. fastidiosa subsp. multiplex alleles and to known X. fastidiosa subsp. fastidiosa and sandyi alleles to identify evidence of intersubspecific recombination .

What is the Translation initiation factor IF-2 (infB) and what is its role in bacterial protein synthesis?

Translation initiation factor IF-2 (infB) is a crucial protein involved in the initiation phase of bacterial protein synthesis. In bacteria like X. fastidiosa, IF-2 facilitates the binding of the initiator tRNA (fMet-tRNA) to the 30S ribosomal subunit during translation initiation. This process is essential for proper start codon recognition and ensuring accurate protein synthesis.

The infB gene is highly conserved across bacterial species, making it useful for phylogenetic analysis and strain typing. In X. fastidiosa research, partial sequences of the infB gene have been utilized alongside other conserved genes in multilocus sequence typing (MLST) schemes to investigate genetic relationships between different strains and subspecies.

How does Xylella fastidiosa colonize and interact with host plants?

X. fastidiosa enters plant xylem at insect feeding sites and employs both passive movement and active migration strategies to colonize the host. The bacterium utilizes type-4 pili-mediated twitching motility to migrate to distal tissues, allowing systemic infection throughout the plant . The bacteria particularly thrive in dead xylem vessels, forming large cell aggregates that obstruct water and mineral transport, resulting in characteristic symptoms like leaf scorch .

The interactions between X. fastidiosa and host plants are complex and can result in either commensal or pathogenic relationships depending on the specific host-pathogen combination. Recent transcriptome and microbiome analyses have advanced our understanding of factors important for X. fastidiosa plant infection, including how the bacterium interacts with the plant immune system and influences the host's microbiome .

What methods are used to detect and quantify Xylella fastidiosa in laboratory settings?

Several complementary methods are employed for detection and quantification of X. fastidiosa in laboratory settings:

• Culture-based methods: X. fastidiosa is typically cultured on specialized media such as BCYE (Buffered Charcoal Yeast Extract) agar, though it is considered fastidious and slow-growing .

• Fluorescence microscopy: The LIVE/DEAD® BacLight™ viability kit can accurately assess bacterial populations and distinguish between intact and permeable cells, providing valuable information about cell viability at different time intervals .

• Spot assays: These are used to evaluate bacteriolytic effects on X. fastidiosa, with clear zones indicating susceptibility .

• Molecular methods: PCR-based techniques and DNA microarray analysis allow for both detection and expression analysis of X. fastidiosa genes .

• Primer extension analysis: This technique is used for identifying transcription start sites and mapping promoters in X. fastidiosa genes, providing information about gene regulation mechanisms .

How does intersubspecific homologous recombination impact the genetic diversity and host range of Xylella fastidiosa?

Intersubspecific homologous recombination (IHR) significantly impacts X. fastidiosa's genetic diversity and potentially facilitates host shifts. Research has shown that previously allopatric subspecies now co-occur due to human activity, resulting in genetic exchange through IHR . This recombination is detectable due to the pre-existing genetic divergence between subspecies.

Studies focusing on X. fastidiosa subsp. multiplex have identified specific sequence types (STs) as recombinant forms, carrying alleles derived from IHR with X. fastidiosa subsp. fastidiosa . Of 10 alleles unique to recombinant X. fastidiosa subsp. multiplex strains, 4 were derived entirely from X. fastidiosa subsp. fastidiosa, and 3 were chimeric for X. fastidiosa subsp. multiplex and fastidiosa . This genetic exchange may contribute to adaptive evolution and host range expansion.

The ability of X. fastidiosa to acquire novel genetic material through IHR is supported by experimental evidence confirming the bacterium's transformational competence and the presence of conjugative plasmids in some isolates . This genetic plasticity likely plays a critical role in the bacterium's ability to adapt to new environmental niches and host plants.

What experimental approaches are most effective for studying recombinant Xylella fastidiosa proteins like Translation initiation factor IF-2?

Effective experimental approaches for studying recombinant X. fastidiosa proteins include:

• Gene cloning and expression systems: Optimized expression systems in E. coli or other suitable hosts can produce recombinant X. fastidiosa proteins including Translation initiation factor IF-2 (infB).

• Protein purification techniques: Affinity chromatography, particularly using His-tagged recombinant proteins, allows for isolation of pure protein samples for functional studies.

• Structural biology methods: X-ray crystallography and cryo-electron microscopy provide insights into protein structure-function relationships.

• Functional assays: In vitro translation systems can be used to assess the activity of recombinant IF-2 in initiating protein synthesis.

• Mutation studies: Site-directed mutagenesis of key residues can reveal functional domains within the protein.

• Gene expression analysis: DNA microarray analysis and primer extension techniques can be used to study gene expression patterns under various conditions, as demonstrated in studies of the extracytoplasmic-function sigma factor in X. fastidiosa .

How can bacteriophages be utilized in controlling Xylella fastidiosa infections?

Bacteriophages represent a promising approach for controlling X. fastidiosa infections, as demonstrated by recent research on Xylella phage MATE 2. This novel lytic bacteriophage, isolated from sewage water in southern Italy, shows significant potential as an antibacterial agent against X. fastidiosa subsp. pauca .

Key characteristics that make MATE 2 an effective control agent include:

• Broad-spectrum activity: MATE 2 exhibits antibacterial activity against various phytobacteria within the family Xanthomonadaceae .

• Rapid adsorption time: The phage requires only 10 minutes for adsorption to bacterial cells .

• Environmental resilience: MATE 2 demonstrates high resistance to a broad range of pH (4–10) and temperatures (4–60°C), making it suitable for field applications .

• Sustained efficacy: The phage successfully suppressed the growth of X. fastidiosa subsp. pauca cells in liquid culture for 7 days .

The application methodology for bacteriophage control typically involves direct application of phage suspensions. In laboratory settings, spot assays are used to evaluate bacteriolytic effects, with clear zones indicating susceptibility . For field applications, optimization of delivery methods would be necessary to ensure effective distribution throughout plant vascular systems.

What stress response mechanisms does Xylella fastidiosa employ, and how are they regulated?

X. fastidiosa employs several stress response mechanisms regulated by specialized sigma factors. The extracytoplasmic-function (ECF) sigma factor, encoded by the rpoE gene, plays a crucial role in stress response . An rpoE null mutant shows increased sensitivity to heat shock and ethanol exposure, indicating the importance of this sigma factor in stress adaptation .

The X. fastidiosa σᴱ regulon includes 21 genes involved in various stress response functions:

• Protein homeostasis: Enzymes for protein folding and degradation

• Signal transduction: Components for cellular signaling pathways

• DNA restriction modification: Systems to protect against foreign DNA

• Various hypothetical proteins: Likely involved in specialized stress responses

Unlike many other ECF sigma factors, X. fastidiosa rpoE is not autoregulated. Instead, it positively regulates the gene encoding its putative anti-sigma factor, rseA . Upon heat shock, rpoE expression remains constant while rseA and XF2241 (the third gene in the operon) are highly induced at a σᴱ-dependent promoter internal to the operon .

The consensus sequence for the σᴱ-binding motif has been determined through primer extension analysis of σᴱ-dependent genes, revealing a characteristic sequence pattern: AAC-16/17 nucleotides-TnnA . This sequence is critical for the recognition and binding of the sigma factor to its target promoters.

How do genomic features of Xylella fastidiosa contribute to its pathogenicity in different host plants?

The pathogenicity of X. fastidiosa across different host plants is influenced by several genomic features:

• Subspecies-specific genes: The four main subspecies (fastidiosa, sandyi, multiplex, and pauca) have diverged genetically and possess unique genetic elements that contribute to host specificity and virulence .

• Recombination events: Intersubspecific homologous recombination (IHR) introduces novel genetic variation that may facilitate host shifts . Evidence suggests that recombinant strains may acquire the ability to infect new host plants through genetic exchange between subspecies.

• Virulence factors: X. fastidiosa produces various cell wall-degrading enzymes, adhesins, and other virulence factors that facilitate colonization and damage to host tissues .

• Regulatory systems: Stress response regulatory systems, such as the ECF sigma factor encoded by rpoE, play important roles in adaptation to host environments . These systems allow the bacterium to respond to stresses encountered within the host.

• Type-4 pili genes: These structures mediate twitching motility, allowing the bacterium to migrate through xylem vessels and establish systemic infections .

The complex interplay between these genomic features determines whether X. fastidiosa establishes a commensal or pathogenic relationship with a particular host plant. The bacterium's ability to colonize more than 500 plant species with varying disease outcomes highlights the importance of these genomic adaptations in host-pathogen interactions .