Recombinant Xylella fastidiosa Acyl carrier protein (acpP)

What is Xylella fastidiosa Acyl carrier protein (acpP) and what is its significance in research?

Xylella fastidiosa Acyl carrier protein (acpP) is a small protein (79 amino acids) that plays a crucial role in bacterial fatty acid biosynthesis. The protein has the sequence: "MSDIEARVRK IVAEKLNVDE EKVTNTSTFV DELGADSLDT VELVMALEDE FQCEIGDEAA EKMTSVQHAI DYIKSNAKC" .

As part of the four-helix bundle family of proteins, acpP covalently holds metabolites and secondary metabolites, such as fatty acids, polyketides, and non-ribosomal peptides . These proteins mediate the production of many pharmaceutically important compounds, including antibiotics and anticancer agents. In X. fastidiosa specifically, acpP is essential for membrane formation and cellular processes, making it a potential target for pathogen control strategies.

What are the optimal storage and handling conditions for recombinant Xylella fastidiosa acpP?

For optimal stability and functionality of recombinant X. fastidiosa acpP, researchers should adhere to the following protocol:

Before opening the vial, briefly centrifuge to bring contents to the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL and add 5-50% glycerol (final concentration) for long-term storage .

How can researchers verify the purity and integrity of recombinant Xylella fastidiosa acpP?

Methodological approach for verifying purity and integrity:

• SDS-PAGE analysis: Commercial preparations typically show >85% purity by SDS-PAGE . Run your protein alongside molecular weight markers to confirm the expected size (~9 kDa).

• Mass spectrometry: For precise molecular weight determination and to verify the absence of unexpected modifications or degradation.

• Circular dichroism: To assess secondary structure integrity, as acyl carrier proteins should display characteristic alpha-helical content.

• Functional assays: Test the ability of the protein to be modified with a phosphopantetheine group, which is essential for its biological activity in fatty acid biosynthesis.

• Western blotting: Using antibodies specific to acpP or to any tags present on the recombinant protein.

What expression systems are suitable for producing recombinant Xylella fastidiosa acpP?

• Yeast systems (e.g., Pichia pastoris, Saccharomyces cerevisiae): Provide eukaryotic processing with typically good yields for small proteins like acpP.

• Bacterial systems (E. coli): Often provide high yields but may lack certain post-translational modifications. Consider strains optimized for rare codon usage if needed.

• Insect cell systems: Useful if more complex processing is required or if the protein forms inclusion bodies in simpler systems.

When designing expression constructs, consider including affinity tags (His, GST, MBP) for purification, and determine whether these tags should be cleavable for downstream applications.

What role does Xylella fastidiosa play as a plant pathogen and how does this relate to acpP research?

Xylella fastidiosa is a high-consequence bacterial plant pathogen affecting numerous agricultural crops globally. It shows significant strain-to-strain variability in virulence and host specificity . Detection and control of this pathogen are critical for agricultural sustainability, especially given its status as a quarantine priority pest in Europe and other regions .

AcpP research relates to this pathogenicity in several ways:

• As a component of fatty acid biosynthesis, acpP is essential for bacterial survival

• Understanding acpP function may reveal vulnerabilities that could be exploited for pathogen control

• Recombinant acpP can be used to develop detection methods or as a target for antimicrobial compounds

• Structural studies of acpP may inform the design of inhibitors specific to Xylella fastidiosa

How does genetic recombination in Xylella fastidiosa affect its evolution, and what implications might this have for acpP diversity?

Xylella fastidiosa demonstrates significant genomic plasticity through natural competence and horizontal gene transfer, which influences its evolution and host adaptation . Type I restriction-modification (R-M) systems within the X. fastidiosa genome play a crucial role in this process by potentially affecting recombination rates and patterns.

Research methodology to assess acpP diversity:

• Comparative genomic analysis across the 129 X. fastidiosa genome assemblies representing all known subspecies and 32 sequence types

• Phylogenetic analysis of acpP sequences to identify evolutionary relationships and selection pressures

• Identification of recombination events affecting the acpP gene or its regulatory regions

• Correlation of acpP variants with strain-specific traits like host specificity or virulence

Preliminary data suggests that while core metabolic genes like acpP may be more conserved than virulence factors, the genomic context and regulation of acpP could vary between strains due to recombination events in adjacent genomic regions.

What experimental approaches can be used to study the role of acpP in Xylella fastidiosa virulence mechanisms?

To elucidate the role of acpP in X. fastidiosa virulence, researchers can employ the following methodological approaches:

• Gene knockout and complementation:

  • Construct acpP deletion mutants using recently developed plasmid vectors for X. fastidiosa

  • Complement with wild-type or modified acpP to verify phenotypes

  • Assess impact on biofilm formation, cell attachment, and virulence in planta

• Conditional expression systems:

  • Develop inducible promoter constructs to modulate acpP expression levels

  • Monitor physiological responses under different expression conditions

  • Correlate acpP expression with virulence-associated phenotypes

• Protein interaction studies:

  • Identify acpP interaction partners using pull-down assays, yeast two-hybrid, or co-immunoprecipitation

  • Characterize protein complexes involving acpP using structural biology approaches

  • Map interaction domains through site-directed mutagenesis

• In planta studies:

  • Track bacterial colonization and movement in plant hosts using fluorescently tagged acpP

  • Compare wild-type and acpP-modified strains in various host plants

  • Analyze plant defense responses to different bacterial strains

• Anti-virulence approach:

  • Design small molecules targeting acpP function based on structural information

  • Test compounds for inhibition of bacterial growth or biofilm formation

  • Evaluate efficacy in reducing disease symptoms in plant models

How can researchers effectively study post-translational modifications of Xylella fastidiosa acpP?

Acyl carrier proteins require post-translational modification with a phosphopantetheine group to function in fatty acid biosynthesis. Studying these modifications in X. fastidiosa acpP requires specialized approaches:

• In vitro modification assay:

  • Express and purify recombinant phosphopantetheinyl transferase (PPTase) from X. fastidiosa

  • Incubate unmodified acpP with PPTase and coenzyme A

  • Monitor conversion using mass spectrometry or mobility shift assays

• Mass spectrometry approaches:

  • Intact protein MS to determine exact mass shifts corresponding to modifications

  • Tandem MS/MS after proteolytic digestion to map modification sites

  • Top-down proteomics for comprehensive characterization of all forms

• NMR spectroscopy:

  • 15N/13C-labeled acpP for structural characterization

  • Compare chemical shifts between unmodified and modified forms

  • Analyze conformational changes induced by phosphopantetheinylation

• Activity-based probes:

  • Synthesize fluorescent or affinity-tagged CoA analogs

  • Monitor incorporation into acpP by PPTases

  • Use for visualization or enrichment of modified protein

• Antibody development:

  • Generate antibodies specific to modified vs. unmodified acpP

  • Apply in Western blotting and immunoprecipitation

  • Use for quantification of modification states in different conditions

What approaches can be used to investigate acpP's role in Xylella fastidiosa's response to environmental stresses?

Research by the USDA indicates interest in X. fastidiosa's physiological responses to environmental factors such as cold exposure . To investigate acpP's potential role in these responses:

• Transcriptomic analysis:

  • Perform RNA-seq or qRT-PCR under various stress conditions (temperature, pH, osmotic pressure)

  • Quantify acpP expression changes relative to housekeeping genes

  • Identify co-regulated genes that may function with acpP in stress responses

• Proteomics approaches:

  • Quantitative proteomics to measure acpP protein levels under stress conditions

  • Phosphoproteomics to detect potential regulatory modifications

  • Protein-protein interaction studies under normal vs. stress conditions

• Functional assays:

  • Measure fatty acid biosynthesis rates under stress conditions

  • Compare membrane composition in response to environmental changes

  • Assess acpP-dependent metabolic adaptations during stress

• Mutant phenotyping:

  • Compare stress tolerance of wild-type, acpP mutant, and complemented strains

  • Analyze growth curves, survival rates, and recovery after stress exposure

  • Test plant colonization efficiency following stress treatment

• Structural biology:

  • Determine if acpP structure changes under different environmental conditions

  • Assess thermal stability using differential scanning fluorimetry

  • Measure binding affinities to partner proteins at different temperatures

How can recombinant Xylella fastidiosa acpP be used in the development of novel detection methods for the pathogen?

Early detection of X. fastidiosa is crucial to reduce crop losses and prevent bacterial spread . Recombinant acpP could contribute to detection method development:

• Antibody-based detection:

  • Use purified recombinant acpP as an immunogen to produce polyclonal or monoclonal antibodies

  • Develop ELISA, lateral flow assays, or immunofluorescence methods

  • Validate specificity against other bacterial species common in plant environments

• Aptamer development:

  • Screen DNA/RNA aptamer libraries against recombinant acpP

  • Optimize selected aptamers for binding affinity and specificity

  • Incorporate into biosensor platforms for field detection

• MS-based detection:

  • Identify acpP-specific peptide markers through proteomics

  • Develop targeted MS assays (MRM/PRM) for these markers

  • Apply to plant extract samples with minimal processing

• Comparison with established methods:

  • Benchmark new acpP-based detection against current standards like Harper's qPCR assay

  • Evaluate sensitivity, specificity, cost, and field applicability

  • Consider complementarity with recombinase polymerase amplification methods like AmplifyRP

• Multiplexed detection systems:

  • Combine acpP detection with other X. fastidiosa biomarkers

  • Develop assays capable of distinguishing different subspecies/strains

  • Create field-deployable kits requiring minimal technical expertise

What structural biology techniques are most appropriate for studying Xylella fastidiosa acpP interactions with partner proteins?

Understanding the structural basis of acpP interactions is critical for elucidating its function in X. fastidiosa metabolism and potentially developing targeted interventions:

How might comparative analysis of acpP across Xylella fastidiosa strains contribute to understanding host specificity?

X. fastidiosa exhibits significant strain variability in host plant specificity and virulence . While acpP as a metabolic protein might not be the primary determinant of host specificity, comparative analysis could reveal important insights:

• Sequence analysis methodology:

  • Compare acpP sequences across all known X. fastidiosa subspecies

  • Identify amino acid substitutions correlated with host preference

  • Apply selection pressure analysis to detect adaptive evolution

• Functional validation:

  • Express acpP variants from different strains in a common genetic background

  • Assess impact on growth in media mimicking different host environments

  • Test complementation efficiency across strains

• Protein interaction network comparison:

  • Identify strain-specific differences in acpP interaction partners

  • Map interaction networks in strains with different host preferences

  • Correlate network differences with metabolic adaptations

• Regulatory analysis:

  • Compare acpP promoter regions across strains

  • Identify potential differences in expression regulation

  • Correlate with transcriptomic data from different host infection scenarios

Note: This table represents a hypothetical pattern based on general X. fastidiosa biology as specific acpP expression data across strains was not provided in the search results.

What are the challenges and solutions in studying acpP function in Xylella fastidiosa biofilm formation?

Biofilm formation is a critical virulence mechanism for X. fastidiosa in plant xylem vessels. Studying acpP's potential role in this process presents specific challenges:

• Challenges in experimental design:

  • Difficulty in maintaining consistent biofilm growth conditions in vitro

  • Limitations in visualizing proteins within mature biofilms

  • Potential lethality of complete acpP deletion

• Methodological solutions:

  • Develop conditional expression systems to modulate acpP levels

  • Use fluorescently tagged acpP variants that maintain functionality

  • Employ microfluidic systems to mimic xylem vessel conditions

  • Apply confocal microscopy with 3D reconstruction for spatial organization

• Analytical approaches:

  • Quantitative biofilm assays comparing wild-type and modified strains

  • Lipidomic analysis of biofilm matrix composition

  • Transcriptomic profiling during different stages of biofilm development

  • Correlative microscopy combining fluorescence and electron microscopy

• In planta validation:

  • Microscopic examination of infected plant tissues

  • Quantification of bacterial populations in xylem vessels

  • Comparison of biofilm structure between plant hosts

• Data interpretation considerations:

  • Distinguish direct vs. indirect effects of acpP modification

  • Account for growth rate differences when comparing biofilm formation

  • Consider metabolic adaptations that may compensate for acpP alterations

How can researchers design experiments to elucidate the potential of acpP as a target for controlling Xylella fastidiosa infections?

Given acpP's essential role in bacterial metabolism, it represents a potential target for controlling X. fastidiosa. An experimental roadmap for exploring this potential includes:

• Target validation:

  • Demonstrate essentiality through conditional knockout studies

  • Identify critical residues through site-directed mutagenesis

  • Assess growth inhibition when acpP function is compromised

• Inhibitor discovery pipeline:

  • Structure-based virtual screening for potential acpP inhibitors

  • High-throughput screening of compound libraries

  • Fragment-based drug discovery approaches

  • Repurposing of known inhibitors of bacterial fatty acid synthesis

• Inhibitor characterization:

  • Biochemical assays measuring acpP activity inhibition

  • Thermal shift assays to confirm direct binding

  • Co-crystallization with inhibitors to determine binding modes

  • Cellular assays to verify uptake and target engagement

• In planta efficacy testing:

  • Greenhouse trials with potential inhibitors on infected plants

  • Assessment of bacterial population reduction

  • Monitoring of symptom development and plant health

  • Evaluation of phytotoxicity and environmental impact

• Resistance development assessment:

  • In vitro evolution experiments under inhibitor pressure

  • Sequencing of resistant mutants to identify resistance mechanisms

  • Design of inhibitor combinations to reduce resistance development

What methodological approaches are most effective for studying acpP expression regulation in Xylella fastidiosa under different environmental conditions?

Understanding how acpP expression is regulated in response to environmental factors is critical for elucidating its role in X. fastidiosa adaptation and pathogenicity:

• Transcriptional analysis:

  • Promoter mapping using 5' RACE or primer extension

  • Reporter gene fusions to monitor promoter activity

  • ChIP-seq to identify transcription factors binding to the acpP promoter

  • Single-cell transcriptomics to detect population heterogeneity

• Environmental response profiling:

  • qRT-PCR arrays under various conditions (temperature, pH, osmolarity)

  • RNA-seq time course during environmental transitions

  • Correlation of acpP expression with global transcriptional networks

  • Comparison between in vitro and in planta expression patterns

• Regulatory network analysis:

  • Identification of small RNAs potentially regulating acpP

  • Construction of transcription factor overexpression libraries

  • CRISPR interference targeting putative regulators

  • Network modeling to predict regulatory interactions

• Post-transcriptional regulation:

  • mRNA stability assays under different conditions

  • Ribosome profiling to assess translation efficiency

  • Proteomics to correlate transcript and protein levels

  • Analysis of potential post-translational modifications affecting stability

• Methodology for cold response studies:

  • Controlled temperature shift experiments as mentioned in USDA research

  • Comparison between laboratory and field temperature fluctuations

  • Analysis of membrane composition changes at different temperatures

  • Correlation between cold survival and acpP expression/activity