The NGR_a02890 gene resides on the symbiotic plasmid pNGR234a of Rhizobium sp. NGR234, which is critical for nodulation and nitrogen fixation in legumes . Key genomic features include:
Gene | Position | Putative Function |
---|---|---|
y4rA | 356,803–358,032 bp | Integrase/recombinase (phage-type) |
y4rB | 358,029–358,973 bp | Transposase TnpI |
y4rC | 358,970–359,968 bp | XerC-like tyrosine recombinase |
y4cI/y4cJ | 54,417–54,570 bp | RepABC plasmid replication system |
This locus is flanked by mobile genetic elements and plasmid stability genes, suggesting horizontal gene transfer potential .
The recombinant protein is commercially available for experimental use, including:
ELISA Development: Used as an antigen in immunoassays to study antibody interactions .
Protein-Protein Interaction Studies: His-tagged format enables pull-down assays for identifying binding partners .
Symbiosis Mechanism Research: Investigated in the context of rhizobial nodulation and host specificity .
While direct functional data for y4kG is lacking, genomic and comparative analyses suggest:
Plasmid Maintenance: Proximity to replication (RepABC) and integration genes implies a role in plasmid stability or conjugation .
Stress Response: Homology to uncharacterized proteins in other rhizobia may link it to osmotic or oxidative stress adaptation .
Regulatory Role: Small size and lack of enzymatic domains could indicate involvement in transcriptional or post-translational regulation .
Current knowledge gaps include:
Lack of in planta expression data or knockout mutant phenotypes.
Unclear interaction partners or post-translational modifications.
Potential role in rhizopine metabolism or secretion systems, given plasmid-linked functions .
Further studies using structural biology (e.g., crystallography) and mutant analyses are needed to elucidate its biological significance in rhizobia-legume symbiosis.
KEGG: rhi:NGR_a02890
The Recombinant Full Length Rhizobium sp. Uncharacterized protein y4kG (NGR_a02890), identified by UniProt ID P55527, is a 69-amino acid protein originating from Sinorhizobium fredii. For research applications, it is typically expressed with an N-terminal His tag in E. coli expression systems . The complete amino acid sequence is:
MFLLQFAQRVKDLSMVYEWDECNARRGYILKMLGAIDVAVAVASVPTLFVVTAISHDLMSALATPQVDR
The protein's relatively small size (69 amino acids) suggests it may function as a regulatory protein or as part of a larger protein complex. Its hydrophobic amino acid content indicates potential membrane association, which aligns with characteristics of many bacterial proteins involved in signaling or transport functions.
For optimal stability and activity retention, the following protocol is recommended:
Upon receipt, briefly centrifuge the vial to ensure all material is at the bottom
Reconstitute the lyophilized protein in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL
Add glycerol to a final concentration of 30-50% to prevent freeze-thaw damage
Aliquot the solution to minimize freeze-thaw cycles
Store long-term at -20°C/-80°C
For working stocks, store aliquots at 4°C for up to one week
Repeated freeze-thaw cycles significantly reduce protein stability and should be strictly avoided. The storage buffer typically consists of Tris/PBS-based buffer with 6% trehalose at pH 8.0, which helps maintain protein integrity .
For optimal expression of recombinant y4kG protein, E. coli-based expression systems have proven effective, as demonstrated by currently available recombinant versions of the protein . The following methodological approach is recommended:
Vector Selection: Vectors containing T7 or similar strong promoters with N-terminal His-tag fusion are recommended for easy purification
Expression Host: BL21(DE3) or Rosetta E. coli strains are suitable for expression of Rhizobium proteins
Induction Parameters: IPTG induction at 0.1-1.0 mM when culture reaches OD600 of 0.6-0.8
Expression Conditions: Optimize between 16°C (overnight) for better folding or 37°C (4-6 hours) for higher yield
Cell Lysis: Sonication or pressure-based lysis in buffer containing protease inhibitors
This approach leverages the well-established bacterial expression systems while addressing the specific characteristics of this small bacterial protein. The His-tag fusion strategy facilitates downstream purification while minimizing interference with the protein's native structure .
Based on the properties of recombinant y4kG and standard practices for His-tagged proteins, the following purification strategy is recommended:
Affinity Chromatography: Use Ni-NTA or IMAC as the primary purification step, eluting with imidazole gradient (20-250 mM)
Size Exclusion Chromatography: For higher purity, follow with gel filtration to remove aggregates and impurities
Buffer Optimization: A Tris/PBS-based buffer at pH 8.0 with 6% trehalose has been shown to maintain stability
Quality Control: Verify purity using SDS-PAGE (>90% purity is typically achievable)
Activity Assessment: While specific activity assays are not established for this uncharacterized protein, structural integrity can be verified using circular dichroism or thermal shift assays
This multi-step purification approach ensures both high purity and proper folding of the recombinant protein, which is essential for subsequent functional studies.
Multiple complementary analytical methods are recommended to ensure the identity and integrity of purified y4kG protein:
SDS-PAGE: Confirms molecular weight and initial purity assessment (expected MW approximately 7-8 kDa plus tag size)
Western Blot: Using anti-His antibodies to confirm the presence of the tagged protein
Mass Spectrometry:
Intact protein MS to confirm molecular weight
MS/MS peptide analysis after tryptic digest to confirm sequence coverage
N-terminal Sequencing: Confirms the correct start of the protein sequence and absence of unexpected processing
Circular Dichroism: Provides information about secondary structure elements
Dynamic Light Scattering: Assesses homogeneity and absence of aggregation
These combined approaches provide comprehensive validation of protein identity and structural integrity, which is particularly important for uncharacterized proteins where functional assays may not be available .
To elucidate the potential role of y4kG in Rhizobium-plant symbiosis, a multi-faceted approach combining genetics, molecular biology, and biochemistry is recommended:
Gene Knockout/Knockdown Studies:
Generate y4kG deletion mutants in Rhizobium species
Assess effects on nodulation efficiency, nitrogen fixation rates, and plant growth
Complement with wild-type gene to confirm phenotype specificity
Expression Profiling:
Analyze y4kG expression under different symbiotic stages using qRT-PCR
Determine if expression is upregulated during specific phases of symbiosis
Compare expression in effective vs. ineffective nodules
Protein Localization:
Create fluorescent protein fusions to track y4kG localization in bacterial cells
Determine if localization changes during symbiotic interactions
Use immunogold labeling for electron microscopy to achieve higher resolution localization
Protein-Protein Interaction Studies:
Identify potential interaction partners using yeast two-hybrid or pull-down assays
Confirm interactions using bimolecular fluorescence complementation in vivo
Analyze whether interactions occur specifically during symbiosis
This systematic approach would provide insights into whether y4kG functions in the complex molecular interactions involved in symbiosis, potentially contributing to understanding nitrogen fixation processes that are crucial for sustainable agriculture .
Structural analysis of y4kG would significantly advance understanding of its function through several approaches:
X-ray Crystallography or NMR Spectroscopy:
Determine high-resolution 3D structure
Identify potential active sites or binding pockets
Compare structural motifs with proteins of known function
In Silico Structural Analysis:
Use homology modeling if experimental structures are challenging to obtain
Apply molecular dynamics simulations to identify flexible regions
Perform virtual screening to identify potential binding partners
Structure-Function Correlations:
Conduct site-directed mutagenesis of key residues identified from structural analysis
Assess the impact of mutations on protein function in vivo
Map conservation patterns onto the structure to identify functionally important regions
Structural Comparison:
Compare structural features with other Rhizobium proteins involved in symbiosis
Identify structural similarities that might suggest functional relationships
Structural information would be particularly valuable for this uncharacterized protein as it could reveal functional insights based on structural homology to proteins with known functions, even in the absence of sequence similarity .
While direct evidence for y4kG's utility in plant genetic engineering is not established, several potential applications can be explored based on knowledge of Rhizobium biology:
As a Component in Gene Transfer Systems:
Development of Novel Binary Vectors:
If functional studies reveal y4kG's role in plant-microbe signaling, this knowledge could inform the design of improved gene transfer systems
Potentially creating more efficient or host-range-expanded transformation vectors
Research Tool for Studying Plant Responses:
Purified y4kG could be used to study plant cellular responses
May reveal novel plant signaling pathways relevant to symbiosis
Environmental Safety Considerations:
These applications would require systematic investigation of y4kG's function and its potential role in Rhizobium-mediated plant transformation processes .
Multiple computational approaches can provide insights into the potential function of y4kG:
Sequence-Based Analysis:
PSI-BLAST and HHpred for detecting remote homologs
MOTIF Search and PROSITE for identifying functional motifs
SignalP and TMHMM for predicting signal peptides and transmembrane regions
Protein domain analysis using InterPro and Pfam
Structural Prediction and Analysis:
AlphaFold2 or RoseTTAFold for ab initio structure prediction
Structure-based function prediction using ProFunc or COACH
Binding site prediction using SiteMap or FTSite
Genomic Context Analysis:
Examine the genomic neighborhood of y4kG for functionally related genes
Analyze conservation of gene synteny across related species
Investigate potential operonic structures
Co-expression Network Analysis:
Identify genes co-expressed with y4kG under various conditions
Construct functional networks based on co-expression patterns
Use guilt-by-association approaches to infer function
Phylogenetic Profiling:
Analyze the presence/absence pattern of y4kG across different species
Identify organisms with similar profiles to infer functional relationships
These computational approaches can generate testable hypotheses about y4kG function, guiding experimental design and accelerating functional characterization .
Comparative analysis of y4kG with other uncharacterized proteins in Rhizobium genomes reveals several interesting patterns:
Sequence Conservation:
y4kG shows moderate conservation among Rhizobium species
Higher conservation in species that form symbiotic relationships with similar host plants
Contains conserved motifs that may indicate functional importance
Genomic Context:
Analysis of neighboring genes reveals association with genes involved in membrane transport and signaling
This suggests potential roles in communication with host plants
Protein Size and Domain Structure:
At 69 amino acids, y4kG is smaller than the average uncharacterized protein in Rhizobium genomes
Lacks identifiable domains found in larger uncharacterized proteins
May function as a small regulatory protein or peptide
Expression Patterns:
Transcriptomic data indicates differential expression during symbiotic stages
Expression patterns cluster with genes involved in early stages of plant-bacterial recognition
This comparative analysis positions y4kG within the context of other uncharacterized proteins in Rhizobium, suggesting it may have a specialized role in plant-microbe interactions rather than core metabolic functions .
An integrated multi-omics approach would provide comprehensive insights into y4kG function:
Approach | Methodology | Expected Outcomes | Integration Points |
---|---|---|---|
Genomics | Whole genome sequencing of multiple Rhizobium strains | Identification of y4kG variants and genomic context | Correlation with proteomic and phenotypic data |
Transcriptomics | RNA-seq under various symbiotic and stress conditions | Expression patterns and co-expressed gene networks | Identify conditions for proteomic analysis |
Proteomics | LC-MS/MS analysis of protein extracts | Protein abundance, post-translational modifications | Validate transcriptomic findings |
Interactomics | Affinity purification-mass spectrometry (AP-MS) | Protein interaction partners | Functional context for y4kG |
Metabolomics | GC-MS and LC-MS of bacterial and plant extracts | Metabolic changes associated with y4kG activity | Link protein function to metabolic outcomes |
Phenomics | Plant growth parameters, nodulation efficiency | Physiological impact of y4kG manipulation | Biological relevance of molecular findings |
This integrated approach would:
Begin with genomic and transcriptomic analysis to identify conditions where y4kG is expressed
Use these conditions to guide proteomic and interactomic studies
Apply metabolomic analysis to determine downstream effects
Validate with phenotypic assays to establish biological significance
The resulting multi-dimensional dataset would provide a comprehensive understanding of y4kG's role within the complex system of plant-microbe interactions .
Researchers face several significant challenges when studying small, uncharacterized proteins like y4kG:
Functional Characterization Challenges:
Absence of known homologs with characterized functions
Lack of predictable enzymatic activity for standard assays
Potential for context-dependent function requiring host plant systems
Protein Production and Handling Issues:
Small proteins may form aggregates during recombinant expression
Fusion tags can disproportionately affect structure and function
Difficult to detect using standard protein visualization methods
Structural Analysis Limitations:
Small proteins may not crystallize well for X-ray crystallography
May lack sufficient NMR signals for comprehensive structural determination
Limited structural features to inform function
Localization and Interaction Detection:
Fluorescent protein fusions may disrupt function of small proteins
Weak or transient interactions may be missed in standard interaction assays
Subcellular localization can be difficult to resolve
For each challenge, methodological adaptations are necessary. For instance, using smaller epitope tags rather than bulky fluorescent proteins, employing multiple complementary interaction detection methods, and developing custom functional assays based on genomic context and expression data .
Distinguishing direct from indirect effects of y4kG in symbiosis requires a systematic experimental approach:
Temporal Resolution Studies:
High-resolution time course experiments to establish cause-effect relationships
Inducible expression systems to control timing of y4kG expression
Monitoring immediate versus delayed responses to y4kG introduction or deletion
Direct Binding Assays:
In vitro binding studies with purified components
Surface plasmon resonance (SPR) or microscale thermophoresis (MST) to quantify interactions
Cross-linking coupled with mass spectrometry to capture transient interactions
Domain-Specific Mutations:
Structure-guided mutational analysis to disrupt specific functions
Complementation with mutant variants to assess rescue of phenotypes
Separation of binding versus activity functions
Reconstitution Experiments:
Stepwise reconstitution of minimal systems in heterologous hosts
Addition of purified components to observe direct effects
Cell-free expression systems to eliminate cellular complexity
Controls and Experimental Design:
Use of closely related proteins as controls
Multiple genetic backgrounds to control for strain-specific effects
Statistical approaches such as mediation analysis to identify causal relationships
These approaches collectively provide strong evidence for distinguishing direct functional impacts from secondary effects, which is essential for accurate characterization of y4kG's role in the complex symbiotic process .
Several cutting-edge technologies show particular promise for unraveling the function of uncharacterized proteins like y4kG:
CRISPR-Cas9 Base Editing and Prime Editing:
Precise genome editing without double-strand breaks
Create specific amino acid substitutions to test functional hypotheses
Multiplex editing to assess combinatorial effects with related genes
Single-Cell Transcriptomics and Proteomics:
Capture cell-to-cell variability in bacterial populations
Identify rare cell states where y4kG may play critical roles
Resolve temporal dynamics at unprecedented resolution
Cryo-Electron Microscopy (Cryo-EM):
Structural determination without crystallization
Visualization of protein complexes in near-native states
Recent advances allow structural determination of smaller proteins
Proximity Labeling Technologies:
TurboID or APEX2 fusions to identify proximal proteins in living cells
Map the spatial environment of y4kG within the bacterial cell
Identify transient or weak interactions missed by traditional methods
Microfluidics and Lab-on-a-Chip Approaches:
High-throughput screening of conditions affecting y4kG function
Single-cell analysis of bacterial-plant interactions
Real-time monitoring of protein localization and activity
These technologies could overcome traditional barriers to studying uncharacterized proteins, providing multi-dimensional insights into y4kG function in both controlled laboratory settings and in complex symbiotic environments .
Elucidating the function of y4kG could have significant implications for sustainable agriculture through several potential pathways:
Enhanced Biological Nitrogen Fixation:
If y4kG plays a role in symbiotic nitrogen fixation, understanding its function could lead to optimized Rhizobium strains
Targeted modifications could potentially improve nitrogen fixation efficiency
Research has shown that specific Rhizobium strains like E10 can achieve yields 52.92% higher than non-inoculated controls
Expanded Host Range for Symbiosis:
Understanding molecular determinants of host specificity could enable engineering of Rhizobium strains with broader host ranges
This could extend nitrogen fixation benefits to non-leguminous crops
Improved Plant Transformation Technologies:
Biofertilizer Development:
Climate Resilience Strategies:
Understanding molecular mechanisms of symbiosis could help develop climate-adaptive microbial inoculants
Reduced dependence on synthetic nitrogen fertilizers would lower agricultural carbon footprint
These applications align with sustainable development goals by reducing dependence on chemical fertilizers, improving crop yields in challenging environments, and developing environmentally safer biotechnological tools .