Recombinant Xylella fastidiosa UPF0162 protein PD_0709 (PD_0709)

What is Xylella fastidiosa UPF0162 protein PD_0709 and what is known about its function?

UPF0162 protein PD_0709 is a full-length 281 amino acid protein encoded by the PD_0709 gene in Xylella fastidiosa strain Temecula1 (ATCC 700964) . The protein belongs to the UPF0162 family, which consists of uncharacterized proteins with conserved function. While the precise function remains to be fully elucidated, sequence analysis suggests potential involvement in cellular processes related to bacterial adaptation and pathogenicity.

The UPF0162 designation indicates that this is a protein of unknown function that has been identified through genomic sequencing efforts. Based on the prevalence of natural recombination in X. fastidiosa and its significance in bacterial evolution, studying proteins like PD_0709 may provide insights into adaptive mechanisms of this plant pathogen .

What expression systems are optimal for producing recombinant PD_0709?

Multiple expression systems have been validated for the production of recombinant PD_0709, each offering distinct advantages depending on research requirements:

What purification strategies are most effective for isolating recombinant PD_0709?

When working with recombinant PD_0709, purification strategy should be determined based on the expression system and intended downstream applications. The protein can be purified using affinity chromatography approaches, with the specific method depending on the tag utilized during recombinant expression.

For biotinylated versions (CSB-EP803224XAT-B), streptavidin-based affinity purification is highly effective due to the specific Avi-tag biotinylation . This approach utilizes the E. coli biotin ligase (BirA) that covalently attaches biotin to the 15 amino acid AviTag peptide, creating a strong binding interaction with streptavidin matrices.

Standard purification protocols typically achieve >85% purity as verified by SDS-PAGE , which is suitable for most research applications. For higher purity requirements, secondary purification steps such as ion exchange or size exclusion chromatography can be implemented.

How can recombinant PD_0709 be used to study natural competence and recombination in X. fastidiosa?

Recombinant PD_0709 can serve as a valuable tool for investigating natural competence and recombination mechanisms in X. fastidiosa. Researchers can design experimental approaches utilizing tagged versions of the protein to:

• Perform pull-down assays to identify potential protein-protein interactions with known competence factors

• Develop antibodies against PD_0709 for localization studies during transformation processes

• Create PD_0709 knockout or modified strains to assess impacts on transformation efficiency

X. fastidiosa demonstrates natural competence with transformation rates of approximately one recombination event per 10^6 cells when exposed to exogenous plasmid DNA, and one event per 10^7 cells when different strains are grown together in vitro . Using recombinant PD_0709 in conjunction with these established transformation protocols can help elucidate whether this protein plays a role in these processes.

What role might PD_0709 play in X. fastidiosa pathogenicity research?

While the specific role of PD_0709 in pathogenicity has not been fully characterized, researchers can design experiments using recombinant PD_0709 to investigate its potential contributions to virulence. As X. fastidiosa is responsible for economically significant plant diseases across multiple agricultural regions , understanding protein factors involved in pathogenicity is critical.

Experimental approaches may include:

• Expression analysis of PD_0709 under different infection-relevant conditions

• Integration of the protein into advanced interaction studies with plant host factors

• Development of modified strains with altered PD_0709 expression for virulence assessment

The wide-scale genomic sequencing efforts mentioned in the literature provide context for studying individual proteins like PD_0709 within the broader perspective of X. fastidiosa evolution and host adaptation mechanisms .

How does the methylation status of PD_0709 impact its function in relation to type I restriction-modification systems?

Genome methylation is a critical factor in horizontal gene transfer and bacterial recombination in X. fastidiosa . Although specific methylation patterns of PD_0709 have not been comprehensively documented, researchers should consider potential epigenetic regulation when studying this protein.

Type I restriction-modification (R-M) systems play important roles in regulating DNA uptake and recombination in X. fastidiosa. These systems consist of restriction enzymes that cleave unmethylated DNA and methyltransferases that protect host DNA by adding methyl groups to specific sequences .

When designing experiments to study PD_0709 function:

• Consider whether the protein might interact with components of R-M systems

• Assess if PD_0709 expression is regulated by methylation-sensitive promoters

• Evaluate if the protein itself undergoes methylation that affects its functionality

What advanced structural approaches can be used to characterize PD_0709?

Advanced structural characterization of PD_0709 can provide crucial insights into its functional mechanisms. Several methodological approaches are recommended:

For optimal results with PD_0709, researchers should consider using the E. coli-expressed version (CSB-EP803224XAT) for initial crystallization trials due to its high yield and purity. If complexes with other proteins are of interest, co-expression or reconstitution approaches using the recombinant protein can be implemented.

What transformation protocols are most effective when working with X. fastidiosa and recombinant PD_0709?

When designing transformation experiments involving X. fastidiosa and recombinant PD_0709, researchers should follow established protocols that maximize efficiency. Based on published methodologies, the following approach is recommended:

• Harvest X. fastidiosa cells from solid PWG medium after approximately 7 days of growth

• Dilute cells in modified XFM to a final OD600 of 0.0025-0.05 (approximately 10^6 to 2×10^7 CFU/ml)

• Allow growth for 2 days at 28°C with constant shaking at 180 rpm

• Add DNA (such as plasmids expressing or targeting PD_0709) to a final concentration of 5 μg/ml

• Continue growth for an additional 24 hours before plating on selective media

• Count antibiotic-resistant colonies after approximately 14 days of growth

• Confirm recombination events through PCR analysis

This protocol has been validated for gene integration studies in X. fastidiosa and can be adapted for specific experiments involving PD_0709, whether introducing modified versions of the gene or creating knockout strains.

How can protein-protein interaction studies be designed to identify PD_0709 binding partners?

To elucidate the functional network of PD_0709, comprehensive protein-protein interaction studies can be conducted using various complementary approaches:

• Yeast Two-Hybrid (Y2H) Screening:

  • Clone PD_0709 into bait vectors

  • Screen against X. fastidiosa genomic prey libraries

  • Validate interactions with targeted approaches

• Pull-Down Assays using Biotinylated PD_0709:

  • Utilize the Avi-tag biotinylated version (CSB-EP803224XAT-B)

  • Immobilize on streptavidin beads

  • Incubate with X. fastidiosa lysates

  • Identify binding partners via mass spectrometry

• Co-Immunoprecipitation (Co-IP):

  • Generate antibodies against PD_0709

  • Perform Co-IP from X. fastidiosa extracts

  • Identify co-precipitated proteins

• Cross-linking Mass Spectrometry (XL-MS):

  • Use chemical cross-linkers to stabilize transient interactions

  • Perform tryptic digestion and MS analysis

  • Identify cross-linked peptides indicating proximity

These methodologies can reveal whether PD_0709 interacts with proteins involved in natural competence, recombination, or other cellular processes relevant to X. fastidiosa biology and pathogenicity.

What are the current limitations in studying PD_0709 and how might they be addressed?

Current research on PD_0709 faces several significant challenges:

• Limited Functional Annotation: As a UPF0162 family protein, PD_0709's precise function remains unclear. This can be addressed through comprehensive comparative genomics approaches and systematic phenotypic studies of mutant strains.

• Technical Challenges in X. fastidiosa Manipulation: X. fastidiosa is relatively slow-growing (colonies visible after approximately 14 days) , making genetic manipulation time-consuming. Developing optimized transformation protocols specifically for PD_0709 studies could improve efficiency.

• Integration with Broader X. fastidiosa Biology: Connecting PD_0709 function to known virulence mechanisms requires integrative approaches. Researchers should consider experimental designs that place PD_0709 in the context of established pathogenicity pathways.

• Structural Information Gap: The three-dimensional structure of PD_0709 is not well-characterized. Prioritizing structural studies would significantly advance functional understanding.

Addressing these limitations will require collaborative approaches drawing on expertise in molecular biology, structural biology, plant pathology, and bioinformatics.

How might PD_0709 research contribute to understanding X. fastidiosa evolution and host adaptation?

Research on PD_0709 has significant potential to advance our understanding of X. fastidiosa evolution and host adaptation through several avenues:

• Comparative Genomics: Analyzing PD_0709 sequence conservation and variation across X. fastidiosa strains with different host preferences could reveal evolutionary patterns relevant to host adaptation.

• Recombination Mechanisms: If PD_0709 is involved in natural competence or genomic recombination, its study could illuminate mechanisms underlying X. fastidiosa's genetic diversity. Research has shown that recombination occurs at relatively high rates and plays a significant role in shaping X. fastidiosa genetic diversity .

• Type I R-M System Interactions: Investigating potential interactions between PD_0709 and the type I restriction-modification systems in X. fastidiosa could provide insights into how these systems influence bacterial evolution through regulation of DNA uptake and recombination .

• Host-Pathogen Interface: Determining whether PD_0709 has any role at the host-pathogen interface could contribute to understanding the mechanisms of X. fastidiosa's broad host range and the emergence of new plant diseases.

As economic impacts from X. fastidiosa continue to affect agricultural production globally , research into proteins like PD_0709 becomes increasingly valuable for developing comprehensive models of pathogen adaptation and evolution.