Recombinant Burkholderia phytofirmans UPF0060 membrane protein Bphyt_4776 (Bphyt_4776)

What expression systems are recommended for recombinant production of Bphyt_4776?

For the recombinant expression of Bphyt_4776, several expression systems can be employed, each with distinct advantages:

For Bphyt_4776 specifically, insect cell expression systems often provide the best balance between yield and proper folding of membrane proteins . When using prokaryotic systems like E. coli, codon optimization may be necessary to address potential rare codon issues that could impede efficient translation. Additionally, fusion tags are typically attached during the production process to facilitate purification and may be determined during specific production protocols .

How should Bphyt_4776 be handled and stored for optimal stability?

Recombinant Bphyt_4776 requires specific storage conditions to maintain structural integrity and function:

• Primary storage: Store at -20°C for regular use or -80°C for extended storage

• Working solution: Prepare working aliquots and store at 4°C for up to one week

• Buffer composition: Tris-based buffer with 50% glycerol, specifically optimized for this protein

• Avoid degradation: Repeated freezing and thawing is not recommended

• Aliquoting strategy: Divide into single-use aliquots immediately after initial thawing

For experimental work, it is advisable to maintain the protein in a buffer that mimics its native environment, potentially incorporating mild detergents to maintain solubility of this membrane protein. Stability assays should be performed prior to functional studies to ensure the protein remains in its native conformation.

What are the optimal conditions for performing structural studies on Bphyt_4776?

Structural characterization of Bphyt_4776 requires specialized approaches due to its membrane-embedded nature. The following methodological pipeline is recommended:

• Detergent screening: Test a panel of detergents (e.g., DDM, LMNG, CHAPS) at varying concentrations to identify optimal solubilization conditions

• Purification optimization: Implement two-step purification using affinity chromatography followed by size exclusion chromatography

• Structural techniques:

  • X-ray crystallography: Utilize lipidic cubic phase (LCP) crystallization

  • Cryo-EM: Consider amphipol-stabilized or nanodisc-reconstituted preparations

  • NMR spectroscopy: Isotope labeling with 15N and 13C for solution NMR of detergent-solubilized protein

Researchers should be aware that the UPF0060 family proteins often present challenges in crystallization due to their flexibility and hydrophobic nature. Recent advances in AlphaFold2 prediction may provide preliminary structural insights that can guide experimental design .

How can researchers investigate potential functional interactions between Bphyt_4776 and other proteins in Burkholderia phytofirmans?

To explore the protein-protein interaction network of Bphyt_4776, employ the following methodological approaches:

• Co-immunoprecipitation (Co-IP): Using antibodies specific to Bphyt_4776 to pull down interaction partners, similar to techniques used in other membrane protein studies . This approach can be modeled after successful Co-IP studies of membrane proteins as demonstrated in case studies with other proteins like TSHR and CD40 .

• Proximity labeling approaches:

  • BioID: Fuse Bphyt_4776 with a promiscuous biotin ligase

  • APEX2: Utilize ascorbate peroxidase fusion for proximity labeling

  • These methods allow identification of proteins in close proximity to Bphyt_4776 in vivo

• Membrane-based yeast two-hybrid (MYTH): This specialized Y2H system is designed for membrane proteins and can identify direct interactors

• Functional assays: Design experiments to test whether Bphyt_4776 influences known pathways in Burkholderia phytofirmans, particularly those related to plant growth promotion and stress tolerance

Analyzing interaction data through pathway enrichment can reveal the functional network in which Bphyt_4776 operates, potentially connecting to the bacterium's role in plant growth promotion.

What methodologies are recommended for investigating the role of Bphyt_4776 in plant-microbe interactions?

Given that Burkholderia phytofirmans PsJN is known for its plant growth-promoting properties and ability to enhance abiotic stress tolerance in plants , investigating Bphyt_4776's role in these processes requires multi-faceted approaches:

• Gene knockout/knockdown studies:

  • Generate Bphyt_4776 deletion mutants in B. phytofirmans

  • Create conditional expression strains

  • Assess mutant phenotypes in plant colonization assays

• Plant inoculation experiments:

  • Compare wildtype and Bphyt_4776-mutant strains for:

    • Root colonization efficiency

    • Plant growth promotion

    • Abiotic stress tolerance induction (particularly salinity tolerance)

  • Quantify bacterial populations in rhizosphere and endosphere

• Transcriptomic and metabolomic analyses:

  • Perform RNA-seq on plants inoculated with wildtype vs. mutant bacteria

  • Analyze metabolite profiles to identify differential responses

  • Focus on salinity stress responses and sodium accumulation in tissues

• Localization studies:

  • Generate fluorescently tagged Bphyt_4776 to track protein localization during plant colonization

  • Use confocal microscopy to visualize bacterial-plant interfaces

This methodological framework can provide insights into whether Bphyt_4776 contributes to the documented ability of B. phytofirmans to enhance plant tolerance to salinity and other stresses.

What strategies can address the challenges in expressing hydrophobic regions of Bphyt_4776?

The hydrophobic nature of Bphyt_4776, particularly its transmembrane domains, presents significant expression challenges. The following strategies can improve expression outcomes:

• Fusion partners optimization:

  • N-terminal fusions: MBP, thioredoxin, or SUMO to enhance solubility

  • C-terminal stability tags: Consider GFP fusion to monitor folding

  • Cleavable tags: Incorporate precision protease sites for tag removal

• Expression optimization matrix:

• Membrane mimetics:

  • Detergent screening: Test DDM, LMNG, CHAPS at varying concentrations

  • Nanodiscs: Consider MSP1D1 or SMA copolymer systems

  • Liposome reconstitution: POPC/POPE mixtures may stabilize function

• Codon optimization: Analyze the sequence for rare codons, particularly if multiple rare codons are linked together, as this can cause expression difficulties in heterologous systems .

These methodologies collectively address the expression challenges documented for membrane proteins similar to Bphyt_4776.

How can researchers troubleshoot purification issues with Bphyt_4776?

Purification of membrane proteins like Bphyt_4776 often encounters specific challenges. The following troubleshooting guidance addresses common issues:

• Poor solubilization:

  • Implement systematic detergent screening

  • Optimize detergent:protein ratios

  • Consider mixed micelle approaches with secondary detergents

• Low binding to affinity resins:

  • Ensure tag accessibility (N vs. C-terminal positioning)

  • Screen different affinity tag systems (His, FLAG, Strep)

  • Modify binding conditions (salt, pH, imidazole concentration)

• Protein aggregation:

  • Identify aggregation onset using dynamic light scattering

  • Incorporate stabilizing agents (glycerol, specific lipids)

  • Consider buffer optimization (pH range 6.5-8.0, salt concentration 100-500 mM)

• Contaminant co-purification:

  • Implement more stringent washing (increased imidazole for His-tagged proteins)

  • Add secondary purification steps (ion exchange, size exclusion)

  • For truncated products, use dual-tagging strategies to isolate only full-length protein

• Activity loss during purification:

  • Incorporate activity assays at each purification step

  • Minimize time at room temperature

  • Consider rapid purification protocols

Each batch of purified Bphyt_4776 should undergo quality control via SDS-PAGE, Western blotting, and if possible, mass spectrometry to confirm identity and integrity.

What methodologies can elucidate the role of Bphyt_4776 in bacterial stress responses?

Investigating Bphyt_4776's potential role in bacterial stress responses requires multidisciplinary approaches:

• Comparative expression analysis:

  • qRT-PCR to quantify Bphyt_4776 expression under various stresses (osmotic, pH, temperature)

  • Western blotting to confirm protein-level changes

  • Promoter-reporter fusions to visualize expression patterns

• Phenotypic characterization of mutants:

• Molecular interaction studies:

  • Identify proteins that interact with Bphyt_4776 under stress conditions

  • Map stress-response signaling pathways

  • Determine whether Bphyt_4776 participates in sensing or responding to environmental changes

• Comparative genomics:

  • Analyze conservation of UPF0060 family proteins across bacterial species

  • Correlate presence/absence with ecological niches and stress resilience

Given that Burkholderia phytofirmans PsJN enhances plant tolerance to salinity , understanding whether Bphyt_4776 contributes to the bacterium's own stress responses may provide insights into the mechanisms of plant-microbe interactions under stress.

How can researchers investigate potential signaling functions of Bphyt_4776 in plant-microbe interactions?

To explore whether Bphyt_4776 participates in signaling processes during plant-microbe interactions, researchers should consider:

• Bacterial-plant co-culture systems:

  • Design split co-culture systems to separate bacterial and plant components

  • Analyze whether wildtype vs. Bphyt_4776-mutant bacteria differ in volatile organic compound (VOC) production

  • Determine if Bphyt_4776 influences plant hormone levels (auxin, ethylene, etc.)

• Receptor activation assays:

  • Generate purified Bphyt_4776 for direct application to plant tissues

  • Monitor early signaling responses (Ca²⁺ flux, MAPK activation)

  • Screen for plant genes responsive to Bphyt_4776 treatment

• In planta visualization:

  • Create fluorescently tagged Bphyt_4776 to track localization during plant colonization

  • Use FRET/BRET approaches to monitor potential interaction with plant proteins

  • Implement live-cell imaging to capture dynamic interactions

• Biochemical characterization:

  • Assess whether Bphyt_4776 binds specific ligands

  • Test for enzymatic activities that could generate signaling molecules

  • Determine if post-translational modifications occur during plant interaction

This research direction connects to findings that B. phytofirmans influences plant responses to abiotic stress, potentially through volatile organic compound signaling pathways .

What mass spectrometry approaches are most effective for characterizing post-translational modifications of Bphyt_4776?

Membrane proteins like Bphyt_4776 may undergo post-translational modifications (PTMs) that affect their function. The following mass spectrometry (MS) approaches are recommended:

• Sample preparation optimization:

  • In-gel digestion: Enables visualization of protein integrity

  • Filter-aided sample preparation (FASP): Effective for detergent removal

  • Specialized proteases: Use combinations of trypsin, chymotrypsin, and Glu-C for improved coverage

• MS techniques for specific PTM types:

• Data analysis pipelines:

  • Implement specialized search algorithms (e.g., ModifiComb, PTMiner)

  • Utilize spectral counting or MS1 intensity for PTM quantification

  • Validate with site-directed mutagenesis of modified residues

• Spatial proteomics:

  • Combine subcellular fractionation with MS analysis

  • Track PTM differences across cellular compartments

  • Correlate modifications with protein localization

These approaches can identify whether Bphyt_4776 undergoes modifications that might regulate its function in different cellular contexts or during plant-microbe interactions.

What bioinformatic approaches can predict functional domains and evolutionary conservation of Bphyt_4776?

Computational analyses can provide valuable insights into Bphyt_4776's potential functions:

• Domain and motif prediction:

  • Apply TMHMM, HMMTOP for transmembrane topology

  • Use Pfam, SMART, and InterPro for domain identification

  • Implement MEME and GLAM2 to discover novel motifs

• Evolutionary analysis workflow:

  • Collect UPF0060 family homologs across bacterial species

  • Perform multiple sequence alignment using MAFFT or Clustal Omega

  • Generate phylogenetic trees using maximum likelihood methods

  • Calculate selection pressures (dN/dS) across the sequence

• Structural bioinformatics:

  • Utilize AlphaFold2 for 3D structure prediction

  • Perform molecular dynamics simulations in membrane environments

  • Identify potential ligand binding sites using CASTp or FTMap

• Functional association networks:

  • Employ STRING and GeneMANIA to predict functional partners

  • Perform gene neighborhood analysis across bacterial genomes

  • Correlate with known pathways in plant-associated bacteria

A comprehensive bioinformatic analysis can generate testable hypotheses about Bphyt_4776's function, particularly in the context of Burkholderia phytofirmans' role in promoting plant growth and stress tolerance.

What are the major knowledge gaps regarding Bphyt_4776's function in plant-growth promoting activities?

Despite understanding that Burkholderia phytofirmans PsJN promotes plant growth and stress tolerance , several knowledge gaps remain regarding Bphyt_4776's specific role:

• Functional characterization:

  • The precise molecular function of UPF0060 family proteins remains largely unknown

  • Whether Bphyt_4776 participates in bacterial adaptation to plant environments is unclear

  • Its potential role in mediating volatile organic compound effects on plants requires investigation

• Regulatory networks:

  • How Bphyt_4776 expression is regulated during plant colonization

  • Whether it responds to plant signals or environmental stresses

  • If it participates in quorum sensing or other bacterial communication systems

• Structure-function relationships:

  • The three-dimensional structure remains unsolved

  • Critical residues for function have not been identified

  • Potential ligands or interacting molecules are unknown

• Plant response pathways:

  • Whether plants recognize Bphyt_4776 directly or indirectly

  • How it might contribute to plant stress response signaling

  • Its potential role in modulating sodium accumulation in plant tissues during salt stress

Addressing these gaps requires interdisciplinary approaches combining structural biology, molecular genetics, plant physiology, and systems biology.

How can CRISPR-Cas9 technology be applied to study Bphyt_4776 function in Burkholderia phytofirmans?

CRISPR-Cas9 technology offers precise genetic manipulation capabilities for studying Bphyt_4776:

• Gene editing strategies:

  • Complete gene knockout: Disrupt the Bphyt_4776 coding sequence

  • Domain-specific mutations: Introduce targeted modifications to specific protein regions

  • Promoter modifications: Alter expression patterns without affecting protein sequence

  • Fluorescent protein tagging: Create fusion proteins for localization studies

• Delivery methods for Burkholderia:

  • Conjugation-based plasmid delivery

  • Electroporation of ribonucleoprotein complexes

  • Transposon-based systems for higher efficiency

• Phenotypic analysis pipeline:

• Off-target assessment:

  • Whole genome sequencing to confirm specificity

  • Complementation studies to validate phenotypes

  • Transcriptome analysis to identify compensatory mechanisms

CRISPR-based approaches can overcome traditional challenges in manipulating Burkholderia species, providing unprecedented precision in dissecting Bphyt_4776's functions in plant-microbe interactions and stress tolerance mechanisms.

What interdisciplinary approaches could reveal novel functions of Bphyt_4776 in environmental adaptation?

Understanding Bphyt_4776's potential roles in environmental adaptation requires combining multiple disciplinary perspectives:

• Systems biology approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Network analysis to position Bphyt_4776 in stress response pathways

  • Flux balance analysis to identify metabolic roles

• Environmental microbiology methods:

  • Mesocosm studies with wildtype vs. mutant strains

  • Competition assays under various environmental stresses

  • In situ expression analysis using reporter systems

• Synthetic biology applications:

  • Heterologous expression in non-native hosts

  • Construction of minimal systems to test sufficiency for function

  • Engineering chimeric proteins to dissect domain functions

• Advanced imaging techniques:

  • Super-resolution microscopy for subcellular localization

  • Correlative light and electron microscopy

  • Label-free chemical imaging (FTIR, Raman) for associated metabolites

• Computational modeling:

  • Molecular dynamics simulations in membrane environments

  • Protein-protein docking to predict interaction partners

  • Machine learning to identify patterns in functional datasets

These interdisciplinary approaches can contextualize Bphyt_4776's function within the broader ecological role of Burkholderia phytofirmans as a plant growth-promoting rhizobacterium that enhances tolerance to chilling, drought, and salinity .