Recombinant Burkholderia phytofirmans Protein CrcB homolog (crcB)

What is the Recombinant Burkholderia phytofirmans Protein CrcB homolog (crcB)?

The Recombinant Burkholderia phytofirmans Protein CrcB homolog (crcB) is a full-length protein derived from Burkholderia phytofirmans strain DSM 17436/PsJN. It is encoded by the crcB gene (locus name: Bphyt_1600) and has the UniProt accession number B2T349. The protein consists of 126 amino acids with the sequence: MYWSILAVGIGGALGSLFRWFLGIRLNGVFSGLPLGTFAANVIAGYVIGVAVAGFARAPQ IAPEWRLFVITGLMGGLSTFSTFSAEVVQRLQDGRLGWAAGEIVIHVGASLLMTmLGIAT VSLLSR . This protein belongs to a family of membrane proteins that may play roles in cellular functions related to ion transport and homeostasis.

What are the optimal storage conditions for working with Recombinant B. phytofirmans CrcB protein?

For optimal stability and activity, the Recombinant B. phytofirmans CrcB protein should be stored in a Tris-based buffer with 50% glycerol at -20°C or -80°C for extended storage periods. When working with the protein, it is recommended to prepare small working aliquots and store them at 4°C for up to one week to minimize freeze-thaw cycles, which can compromise protein integrity. Repeated freezing and thawing should be avoided as this can lead to protein denaturation and loss of biological activity . For experimental reproducibility, it is advisable to document the number of freeze-thaw cycles and storage duration in research protocols.

How does B. phytofirmans CrcB protein structure compare to homologs in other Burkholderia species?

The B. phytofirmans CrcB protein shares structural similarities with homologs in other Burkholderia species, such as B. phymatum. Comparative sequence analysis reveals conservation in key domains while showing species-specific variations. The B. phymatum CrcB homolog (UniProt: B2JCE3) has the sequence: MYLSILAVGIGGALGSLFRWFLGLRLNALFPALPLGTLASNVIAGYIIGAAVAYFGRNPQ IAPEWRLFIITGLMGGLSTFSTFSAEVVQHLQQGRLNWAAGEIAIHVTASVIATVLGITT VAVVTR . Alignment analysis indicates conserved hydrophobic regions typical of membrane proteins, with both proteins having similar length (126 amino acids) and presumed membrane topology. These structural similarities suggest conserved functions across Burkholderia species, while the amino acid variations may reflect adaptations to specific environmental niches or host interactions.

What roles does CrcB homolog play in B. phytofirmans interaction with plant hosts?

B. phytofirmans PsJN is a well-documented plant growth-promoting bacterium (PGPB) that forms beneficial associations with various plant species. Though the specific role of CrcB homolog has not been fully characterized, research suggests it may function within the broader context of plant-microbe interactions. B. phytofirmans colonization induces significant changes in plant cell anatomy and influences iron accumulation in host tissues . The bacterium possesses multiple genes related to iron acquisition and storage, including ferritin genes (Bphyt_3313, Bphyt_0219, Bphyt_1412) and mini-ferritin Dps genes (Bphyt_0714, Bphyt_5727) . Given that CrcB family proteins in other organisms are often associated with ion transport, the B. phytofirmans CrcB homolog may potentially contribute to mineral homeostasis during plant colonization or stress response mechanisms in this beneficial plant-microbe interaction.

How do transcriptomic profiles change in plants colonized by B. phytofirmans, and what does this suggest about CrcB function?

Transcriptomic analyses of plants colonized by B. phytofirmans reveal distinct expression patterns depending on the plant species and tissue. In Arabidopsis, B. phytofirmans colonization leads to detectable expression of several bacterial genes, including those related to iron acquisition and storage . Interestingly, comparative transcriptomic studies between rice responses to native and non-native Burkholderia strains reveal that B. phytofirmans PsJN (a non-native strain) elicits markedly different responses compared to rice-native Burkholderia strains . The transcriptional regulation of genes related to secondary metabolism, immunity, and phytohormones appears to be strain-specific . While the specific transcriptomic signature of CrcB expression requires further investigation, these differential responses suggest that proteins like CrcB may participate in strain-specific adaptation mechanisms that influence bacterial colonization and plant response pathways.

What is the significance of B. phytofirmans iron-related genes in relation to CrcB function and plant interaction studies?

B. phytofirmans possesses a complex array of iron-related genes that have been detected in colonized plant tissues, including classic ferritin (Bphyt_3313), mini-ferritin Dps (Bphyt_0714, Bphyt_5727), TonB-dependent siderophore receptor (Bphyt_4626), and L-ornithine 5-monooxygenase (PvdA; Bphyt_4074) . The expression of these genes in planta suggests active bacterial iron acquisition and storage during colonization. While direct functional relationships between CrcB and these iron-related systems remain to be established, CrcB homologs in other organisms have been implicated in various transport processes. Research investigating potential interactions between CrcB and iron homeostasis systems would provide valuable insights into B. phytofirmans adaptation during plant colonization and the molecular mechanisms underlying beneficial plant-microbe interactions.

What are the recommended protocols for analyzing B. phytofirmans gene expression in plant tissues?

Analysis of B. phytofirmans gene expression in plant tissues requires specialized protocols to distinguish bacterial transcripts from plant host RNA. A recommended workflow includes:

• RNA Extraction and Purification: Total RNA extraction from colonized plant tissues followed by DNase treatment to remove genomic DNA contamination.

• Plant rRNA Depletion: Depletion of plant rRNAs using a Ribo-Zero rRNA removal kit to enrich for bacterial RNA, as described by Sheibani-Tezerji et al.

• cDNA Synthesis: Reverse transcription of the resulting RNA to cDNA using random hexamers and a high-capacity cDNA reverse transcription kit.

• Primer Design: Design of specific primers for B. phytofirmans genes of interest, specifying optimal Tm values between 58-62°C and amplicon sizes between 100-250 bp.

• RT-PCR Analysis: Quantification of gene expression using real-time PCR with appropriate reference genes (e.g., 16S rRNA) for normalization.

This methodology enables detection of bacterial transcripts in planta and can be applied to analyze CrcB homolog expression during plant colonization, providing insights into its potential roles in host interaction .

What considerations should be made when designing experiments to study CrcB function in plant-microbe interactions?

When designing experiments to study CrcB function in plant-microbe interactions, researchers should consider:

• Selection of Plant Models: Different plant species show varying responses to B. phytofirmans colonization. For comprehensive understanding, multiple plant models should be tested, considering that responses to rice-native vs. non-native Burkholderia strains differ significantly .

• Tissue-Specific Analysis: Responses to B. phytofirmans vary between plant tissues. Studies indicate more conserved responses to endophytes in leaves compared to roots , suggesting the importance of analyzing multiple tissue types.

• Time-Course Experiments: Colonization and plant responses evolve over time, necessitating sampling at multiple time points to capture dynamic changes.

• Mutant Construction: Development of CrcB deletion or overexpression mutants to directly assess its function through comparative phenotypic and transcriptomic analyses.

• Control Strains: Inclusion of closely related Burkholderia species (e.g., B. phymatum) as controls to distinguish species-specific from general Burkholderia-induced responses.

• Environmental Variables: Consideration of growth conditions that might influence CrcB expression or function, particularly parameters affecting ion homeostasis.

These experimental design considerations will help establish the specific contribution of CrcB to plant-microbe interactions and distinguish its functions from other bacterial factors.

What techniques are recommended for purification and functional characterization of recombinant CrcB protein?

For purification and functional characterization of recombinant CrcB protein, the following techniques are recommended:

• Expression System Selection: Choose an appropriate heterologous expression system considering the membrane protein nature of CrcB. E. coli-based systems with specialized strains for membrane protein expression or eukaryotic systems may be considered depending on research objectives.

• Purification Strategy:

  • Extraction with mild detergents optimized for membrane proteins

  • Affinity chromatography using appropriate tags (determined during production process )

  • Size exclusion chromatography for final purification

• Structural Analysis:

  • Circular dichroism spectroscopy to assess secondary structure

  • Cryo-electron microscopy or X-ray crystallography for high-resolution structural determination

• Functional Assays:

  • Reconstitution in liposomes to test transport activity

  • Fluorescence-based assays to monitor ion flux

  • Binding assays to identify interaction partners

• In Planta Validation:

  • Complementation studies with CrcB mutants

  • Localization studies using fluorescently tagged CrcB variants

  • Co-immunoprecipitation to identify in vivo interaction partners

These methodological approaches provide a comprehensive framework for elucidating the biochemical properties and biological functions of CrcB in both in vitro systems and plant-microbe interaction contexts.

How should researchers interpret comparative transcriptomic data between different Burkholderia strains in plant colonization studies?

When interpreting comparative transcriptomic data of different Burkholderia strains in plant colonization studies, researchers should:

• Consider Host Specificity Effects: Recognize that non-native strains like B. phytofirmans PsJN may elicit different transcriptional responses compared to host-adapted strains. Research shows markedly different responses to rice-native vs. non-native Burkholderia strains .

• Examine Tissue-Specific Patterns: Analyze tissues separately, as studies indicate more conserved responses to endophytes in leaves compared to roots . This tissue-specific variability may reflect different colonization strategies or plant defense responses.

• Focus on Key Functional Categories: Pay particular attention to genes related to:

  • Secondary metabolism

  • Immunity

  • Phytohormone signaling

  These categories appear to be markers of strain-specific responses and may provide insights into CrcB function in the context of plant-microbe interaction.

• Normalize Bacterial Gene Expression: When analyzing bacterial gene expression in planta, use appropriate reference genes (e.g., 16S rRNA) and consider the relative abundance of bacterial cells in different plant tissues.

• Integrate Multi-Omics Data: Complement transcriptomic findings with proteomics, metabolomics, or phenotypic data to build a comprehensive understanding of the functional implications of differential gene expression.

This systematic analytical approach will help distinguish general plant responses to bacterial colonization from specific effects potentially related to CrcB function.

What statistical approaches are most appropriate for analyzing the impact of B. phytofirmans colonization on plant iron homeostasis?

When analyzing the impact of B. phytofirmans colonization on plant iron homeostasis, the following statistical approaches are recommended:

• Multivariate Analysis: Apply principal component analysis (PCA) or partial least squares discriminant analysis (PLS-DA) to identify patterns in multi-element data sets from techniques like ICP-MS that measure multiple metals simultaneously.

• Correlation Analysis: Use Pearson or Spearman correlation to identify relationships between:

  • Expression levels of bacterial iron-related genes (ferritins, siderophore receptors)

  • Plant iron content

  • Expression of plant iron homeostasis genes

• ANOVA with Post-hoc Tests: For comparing multiple experimental conditions:

  • One-way ANOVA with Tukey's HSD for comparing metal content across different treatments

  • Two-way ANOVA to assess interaction effects between bacterial colonization and environmental factors (e.g., iron availability)

• Linear Mixed Models: For time-course experiments or studies with nested experimental designs, to account for random effects and repeated measurements.

• Meta-Analysis Approaches: When comparing results across different plant species or experimental systems to identify conserved patterns of B. phytofirmans effects on plant iron homeostasis.

These statistical methods, when properly applied with appropriate controls and sample sizes, will enable robust analysis of the complex interactions between B. phytofirmans colonization, CrcB function, and plant iron homeostasis.

How can researchers differentiate between direct effects of CrcB and indirect effects mediated through other bacterial factors?

Differentiating between direct effects of CrcB and indirect effects mediated through other bacterial factors requires a multi-faceted experimental approach:

• Genetic Complementation Studies:

  • Generate CrcB deletion mutants of B. phytofirmans

  • Conduct complementation with wild-type CrcB

  • Create point mutations in functional domains to identify critical residues

  • Compare phenotypes to isolate CrcB-specific effects

• Temporal Analysis:

  • Implement time-course experiments to establish the sequence of molecular events

  • Determine whether CrcB expression precedes or follows changes in other factors

  • Use inducible expression systems to control the timing of CrcB expression

• Spatial Localization:

  • Use fluorescently tagged CrcB to determine its subcellular localization in bacteria

  • Track localization during different stages of plant colonization

  • Correlate localization patterns with observed plant responses

• Protein-Protein Interaction Studies:

  • Conduct pull-down assays or yeast two-hybrid screens to identify direct interaction partners

  • Validate interactions using techniques like bimolecular fluorescence complementation

  • Map the interactome to place CrcB within cellular pathways

• Heterologous Expression:

  • Express CrcB in non-Burkholderia bacterial species

  • Determine if CrcB alone can confer specific phenotypes

  • Evaluate if co-expression with other B. phytofirmans factors enhances effects

By integrating data from these complementary approaches, researchers can build a comprehensive understanding of CrcB's direct functions versus its roles within broader bacterial response networks.

What are the most promising approaches for elucidating the molecular function of CrcB in B. phytofirmans?

The molecular function of CrcB in B. phytofirmans could be effectively elucidated through:

• Comparative Genomics and Structural Biology:

  • Conduct detailed sequence and structural comparisons with CrcB homologs of known function

  • Apply advanced structural prediction tools like AlphaFold2 to model protein structure

  • Use molecular dynamics simulations to predict functional domains and binding sites

• Advanced Genetic Engineering:

  • Implement CRISPR-Cas9 techniques for precise genomic modifications

  • Create conditional knockdowns to study essential functions

  • Develop reporter fusions to monitor CrcB expression under various conditions

• Systems Biology Integration:

  • Apply network analysis to position CrcB within bacterial response networks

  • Develop mathematical models predicting CrcB function based on -omics data

  • Use machine learning approaches to identify patterns in multi-omics datasets that correlate with CrcB expression

• Single-Cell Technologies:

  • Apply single-cell RNA-seq to capture heterogeneity in bacterial populations

  • Use time-lapse microscopy with fluorescent reporters to track dynamic changes

  • Implement spatial transcriptomics to map expression patterns during colonization

• Cross-Species Validation:

  • Compare CrcB function across different Burkholderia species

  • Examine conservation of function in diverse plant hosts

  • Explore potential analogous functions in non-plant-associated bacteria

These approaches, particularly when used in combination, offer promising avenues for uncovering the precise molecular function of CrcB in B. phytofirmans and its role in plant-microbe interactions.

How might understanding CrcB function contribute to applications in sustainable agriculture?

Understanding CrcB function in B. phytofirmans could contribute to sustainable agriculture through:

• Enhanced Biofertilizer Development:

  • If CrcB plays a role in nutrient acquisition or metabolism, this knowledge could be used to optimize bacterial strains for improved plant nutrition

  • Potential development of rationally designed bacterial consortia with complementary functions

• Improved Plant Stress Tolerance:

  • Research indicates B. phytofirmans influences iron accumulation in plants , which is critical for stress responses

  • Understanding CrcB's potential role in this process could lead to bacterial inoculants tailored for specific environmental stresses

• Optimized Host-Microbe Compatibility:

  • Insights from comparative responses between rice-native and non-native Burkholderia strains could inform selection or engineering of strains with enhanced colonization efficiency

  • Development of plant varieties with improved receptivity to beneficial microbes

• Precision Agriculture Applications:

  • Knowledge of specific bacterial genes like CrcB that influence plant processes could enable biomarker development for monitoring soil microbial function

  • Creation of diagnostic tools to assess potential benefits of microbial applications

• Biomass Enhancement Strategies:

  • B. phytofirmans colonization affects plant cell anatomy in ways that can benefit downstream biomass deconstruction

  • Understanding CrcB's potential contribution to these effects could lead to applications in bioenergy crop improvement

These potential applications highlight how fundamental research on bacterial proteins like CrcB can translate into practical agricultural innovations that reduce reliance on chemical inputs while enhancing crop resilience and productivity.

How do key characteristics of CrcB homologs compare across different Burkholderia species?

The following table presents a comparative analysis of CrcB homologs across different Burkholderia species:

This comparative analysis reveals both conservation and divergence in CrcB homologs across Burkholderia species, suggesting potential adaptation to different ecological niches while maintaining core structural features .

What experimental considerations are needed when working with different recombinant Burkholderia proteins?

When working with different recombinant Burkholderia proteins, researchers should consider the following experimental variables:

This systematic approach to experimental design ensures reliable and reproducible results when working with recombinant Burkholderia proteins, particularly membrane proteins like CrcB homologs .