Recombinant Bradyrhizobium japonicum Protein-export protein SecB (secB)

What is Bradyrhizobium japonicum and why is it important for nitrogen fixation research?

Bradyrhizobium japonicum is an aerobic, mesophilic, Gram-negative soil bacterium that forms symbiotic relationships with legumes, particularly soybeans (Glycine hispida) . This bacterium is significant in agricultural research because it establishes mutually beneficial relationships with legume plants by forming nodules on their roots, where it converts atmospheric nitrogen into a form usable by plants .

The bacterium has a 9.1 Mb genome, one of the longest among bacteria, with 64.1% GC content . This symbiotic relationship is crucial for sustainable agriculture as it reduces the need for nitrogen fertilizers . Multiple strains of B. japonicum have been studied and field-tested, including USDA110, which serves as a model organism for studying rhizobium-legume symbiosis .

Notably, protein synthesis in B. japonicum bacteroids (the form of the bacterium inside plant nodules) has been found to decline about 60% between 14 and 20 days after planting, correlating with a rapid increase in nitrogen fixation activity. This suggests a metabolic mechanism where cellular energy is diverted from protein synthesis to nitrogen fixation .

What is the Sec pathway and what role does SecB play in protein export?

The Sec pathway is a protein translocation system that exports unfolded preproteins across the cytoplasmic membrane. In this system, SecB functions as a molecular chaperone that binds to preproteins, maintaining them in an unfolded, translocation-competent state before they are secreted .

For example, when carrier proteins like ACP (acyl carrier protein) or BCCP (biotin carboxyl carrier protein) domains are fused to secretory proteins such as YebF or MBP, the resulting fusion proteins can be modified by enzymes AcpS or BirA respectively and then successfully secreted via the Sec pathway . This finding expands our understanding of the flexibility of the Sec machinery and provides new opportunities for biotechnological applications.

How does protein synthesis occur in B. japonicum bacteroids and what are its unique characteristics?

Protein synthesis in B. japonicum bacteroids demonstrates several distinctive features:

• Amino Acid Accumulation: Isolated bacteroids of B. japonicum can accumulate exogenously supplied amino acids such as [(sup35)S]methionine or [(sup3)H]leucine, with this accumulation being inhibited by azide .

• Limited Incorporation: Only 3-6% of accumulated labeled amino acids are actually incorporated into cytosolic proteins, suggesting a selective or regulated protein synthesis process .

• Regulation Factors: Protein synthesis in bacteroids is not stimulated by potassium salts, malate, or amino acids, but is inhibited by azide, chloramphenicol, and acridine .

• Developmental Regulation: Both protein synthesis and protein turnover rates decline during nodule development. Specifically, protein synthesis declines approximately 60% between 14 and 20 days after planting, coinciding with the period of rapid increase in acetylene reduction activity (a measure of nitrogen fixation) .

This correlation between decreased protein synthesis and increased nitrogen fixation suggests a metabolic mechanism where significant amounts of cellular energy are diverted from protein synthesis to support the energy-intensive nitrogen fixation process .

What are the optimal experimental approaches for studying SecB-dependent protein export in B. japonicum?

Based on previous successful research methodologies in similar systems, the following experimental approaches are recommended for studying SecB-dependent protein export in B. japonicum:

• Fusion Protein Construction: Design experiments using fusion proteins where the N-terminus contains a signal peptide (SP) from a known B. japonicum secretory protein, followed by the protein of interest. This approach has been successfully used with YebF and MBP as carrier proteins in E. coli systems .

• Expression Analysis: Compare expression in total cell contents (W), supernatant (S), and periplasm samples (P) after centrifugation and osmotic shock treatment to track protein localization .

• Modification Tracking: Co-express the fusion protein with cytosolic modification enzymes (like AcpS or BirA) to assess whether modification occurs before translocation. Addition of sodium azide (1 mM) can retard translocation, potentially enhancing modification efficiency .

• Quantification Protocol: Quantify target proteins using densitometric analysis, setting the value of the unmodified exported fusion protein as a reference point (value of 1) for comparative analysis. This allows for clear visualization of the effects of co-expression with modification enzymes and translocation inhibitors .

This methodology has been validated in E. coli and can be adapted for B. japonicum, though researchers should be aware that species-specific optimizations may be necessary due to differences in codon usage, expression systems, and growth conditions.

How can proteogenomic analysis be applied to validate and characterize SecB in B. japonicum?

Proteogenomic analysis offers a powerful approach to validate and characterize proteins like SecB in B. japonicum, as demonstrated by previous studies using the GenoSuite integrated pipeline . The recommended methodological framework includes:

• Mass Spectrometry Data Collection: Collect high-throughput MS data from B. japonicum under various growth conditions, particularly focusing on conditions where protein export would be active. Previous studies have successfully analyzed 621,176 MS/MS spectra across nine datasets from three different host systems .

• Database Construction and Search Parameters:

  • Create a six-frame translated database of the B. japonicum genome

  • Include translation products of 50 amino acids or longer

  • Use multiple search algorithms with the following parameters:

    • 20 ppm precursor ion tolerance

    • 0.6 Da product ion tolerance

    • Trypsin as the protease with one missed cleavage

    • Carbamidomethylation of cysteine (fixed modification)

    • Methionine oxidation (variable modification)

• Stringent Validation: Apply a False Discovery Rate (FDR) threshold of ≤1% to peptide-spectrum matches (PSMs) to ensure high confidence in identifications .

• Translation Initiation Site (TIS) Search: Perform a specialized search using semitryptic enzyme specificity and N-terminal modifications (acetylation and formylated methionine) to accurately determine protein start sites .

• Gene Prediction and Comparative Analysis: Use multiple gene prediction algorithms (GeneMark.hmm, Glimmer3, Prodigal, FGENESB) to define boundaries of novel translations and compare with observed peptides .

• Functional Annotation: Apply domain assignment methods (Pfam), gene ontology assignments (GOANNA), and specialized annotation tools (RAST) to characterize the functions of identified proteins .

This comprehensive approach has successfully identified 59 novel protein coding regions and refined 49 gene models in B. japonicum USDA110, demonstrating its effectiveness for characterizing proteins that may be missed by traditional annotation methods .

What factors affect the efficiency of recombinant B. japonicum SecB expression and purification?

The expression and purification of recombinant B. japonicum SecB requires careful consideration of several critical factors:

• Expression Systems: While E. coli is commonly used for heterologous protein expression, its SecB may interact differentially with B. japonicum signal sequences. Research has shown that preproteins can be efficiently modified in the cytosol prior to translocation , suggesting that E. coli expression systems can accommodate B. japonicum proteins if properly designed.

• Fusion Tags and Constructs: Data suggests that His6-tags with flexible peptide linkers (ASGGGGA) and protease recognition sites (TEV: ENLYFQ) generate functional fusion proteins with minimal interference in SecB activity . The calculated molecular weights of fusion proteins should be carefully considered; for example, YebF-BCCP87 is 23.2 kDa, while YebF-ACP is 22.1 kDa .

• Co-expression Strategies: Co-expression with cytosolic modification enzymes significantly enhances the yield of properly modified proteins. When fusion proteins are co-expressed with their corresponding modification enzymes, production can increase by approximately 2-fold .

• Translocation Rate Modulation: Adding sodium azide (1 mM) to slow translocation can increase the yield of modified proteins by allowing more time for cytosolic modification. This approach has been shown to further increase production by approximately 3-fold when combined with co-expression of modification enzymes .

• Strain Selection: Different E. coli strains show variable expression efficiency for the same construct. Comparative analysis of expression in strains like JM109 and BL21(DE3) is recommended to determine optimal host conditions .

• Protein Turnover Considerations: The rate of protein synthesis and turnover in B. japonicum declines during development, with protein synthesis declining about 60% between days 14-20 . Understanding this native regulation pattern may help design expression strategies that account for potential instability or regulatory mechanisms.

These methodological considerations provide a framework for optimizing recombinant B. japonicum SecB expression while maintaining functional integrity.

How can B. japonicum SecB be utilized in agricultural applications to enhance nitrogen fixation?

B. japonicum strains optimized for SecB-mediated protein export have significant potential for agricultural applications. Based on research findings, the following methodological approach is recommended:

This methodological framework is based on previous successful applications with modified B. japonicum strains and could be adapted specifically for strains with enhanced SecB functionality .

What experimental methods can detect the interaction between SecB and its target proteins in B. japonicum?

To effectively study the interactions between SecB and its target preproteins in B. japonicum, researchers should employ a multi-faceted experimental approach:

• In vitro Binding Assays:

  • Purify recombinant B. japonicum SecB protein using affinity chromatography

  • Express potential target preproteins with fusion tags

  • Perform pull-down assays to detect direct interactions

  • Quantify binding affinities using isothermal titration calorimetry or surface plasmon resonance

• In vivo Crosslinking:

  • Treat B. japonicum cultures with crosslinking agents to stabilize transient protein-protein interactions

  • Immunoprecipitate SecB complexes using anti-SecB antibodies

  • Identify interacting partners through mass spectrometry analysis

  • This approach has proven successful in identifying novel protein interactions in B. japonicum, as demonstrated in proteogenomic studies

• Fluorescence-based Interaction Assays:

  • Generate fluorescently labeled SecB and candidate preproteins

  • Monitor interactions using FRET (Förster Resonance Energy Transfer)

  • Track the dynamics of these interactions in real-time

• Translocation Assays:

  • Design fusion proteins containing potential SecB targets

  • Compare translocation efficiency in wild-type and SecB-deficient strains

  • Analyze protein accumulation in cytosolic, periplasmic, and extracellular fractions

  • This method has been validated for studying protein export mechanisms

• Structural Analysis:

  • Use X-ray crystallography or cryo-EM to determine the structure of B. japonicum SecB alone and in complex with substrate proteins

  • Compare with known structures from model organisms to identify conserved and divergent features

• Mutational Analysis:

  • Generate SecB variants with mutations in potential binding sites

  • Assess impact on protein export efficiency and target recognition

  • Correlate with nitrogen fixation efficiency

By combining these methodological approaches, researchers can develop a comprehensive understanding of SecB interactions in B. japonicum, potentially revealing unique features related to its role in symbiotic nitrogen fixation.

How does the protein synthesis and export machinery in B. japonicum bacteroids differ from free-living cells?

The protein synthesis and export machinery in B. japonicum demonstrates significant differences between bacteroids and free-living cells, with important implications for research design:

These findings highlight the importance of considering the physiological state of B. japonicum when designing experiments involving protein export and the SecB chaperone. The bacteroid state represents a specialized condition with unique metabolic priorities that differ substantially from free-living cells.

What are the key methodological challenges in studying B. japonicum SecB and how can they be addressed?

Researchers face several significant methodological challenges when studying B. japonicum SecB, requiring carefully designed experimental strategies:

• Complex Genome and Annotation Challenges:

  • B. japonicum has a 9.1 Mb genome with 64.1% GC content, making gene prediction and annotation difficult

  • Challenge: Traditional annotation methods miss many protein-coding genes

  • Solution: Apply proteogenomic approaches using GenoSuite or similar integrated pipelines that combine multiple search algorithms and statistical validation

• Expression System Limitations:

  • Challenge: Native expression levels of SecB may be low, particularly during nodule development when protein synthesis declines

  • Solution: Develop regulated expression systems with inducible promoters calibrated to B. japonicum's codon usage preferences

  • Alternative: Use the methodology validated for fusion proteins, where sodium azide (1 mM) retards translocation, enhancing cytosolic modification and potentially yielding more functional protein

• Protein Modification and Translocation Balance:

  • Challenge: Only 3-6% of accumulated amino acids are incorporated into proteins in bacteroids

  • Solution: Implement a dual-expression system where SecB and target preproteins are co-expressed with appropriate modification enzymes

  • Experimental approach: Compare results using the quantification protocol where densitometric values are normalized to unmodified protein expression (set at value of 1)

• Host-Specific Expression Patterns:

  • Challenge: Many proteins show host-specific expression, with 13 novel proteins specific to soybean bacteroids

  • Solution: Study SecB function across multiple host systems (soybean, siratro, cowpea) to account for potential regulatory differences

  • Experimental design: Apply the same proteogenomic analysis pipeline across samples from different hosts to enable direct comparisons

• Developmental Regulation:

  • Challenge: Protein synthesis declines approximately 60% between 14-20 days after planting

  • Solution: Design time-course experiments with precise sampling at key developmental stages

  • Analytical approach: Apply statistical methods that account for temporal changes in baseline expression

By addressing these methodological challenges with the recommended solutions, researchers can develop more robust experimental designs for studying B. japonicum SecB and its role in protein export during symbiotic nitrogen fixation.

What are the future research directions for improving recombinant protein production using B. japonicum SecB?

Based on current research findings, several promising future research directions can enhance recombinant protein production using B. japonicum SecB:

• Engineered SecB Variants:

  • Develop modified SecB proteins with enhanced chaperone activity through site-directed mutagenesis

  • Create chimeric SecB proteins incorporating functional domains from homologs in other species

  • Methodology: Apply proteogenomic analysis to validate these modifications and quantify improvements in export efficiency

• Synthetic Biology Approaches:

  • Design synthetic operons that coordinate expression of SecB, target preproteins, and modification enzymes

  • Develop genetic circuits that respond to nodulation signals to optimize protein export during symbiosis

  • Experimental design: Field test modified strains on multiple legume hosts to assess performance under agricultural conditions

• Novel Fusion Systems:

  • Expand on the YebF and MBP fusion systems to develop B. japonicum-specific fusion partners

  • Optimize flexible peptide linkers (beyond ASGGGGA) for improved folding and translocation

  • Quantification approach: Apply the established methodology of comparing expression levels with a baseline value set at 1 for unmodified proteins

• Integration with Nitrogen Fixation Machinery:

  • Investigate the relationship between SecB-mediated protein export and nitrogen fixation efficiency

  • Develop co-expression systems that balance energy allocation between protein export and nitrogen fixation

  • Analysis framework: Correlate acetylene reduction activity with protein export metrics across developmental stages

• Cross-Species Translocation Studies:

  • Compare the efficiency of B. japonicum SecB in handling preproteins from different bacterial species

  • Identify unique features that may make B. japonicum SecB advantageous for specific biotechnological applications

  • Methodology: Utilize orthologous gene identification approaches similar to those used in ortho-proteogenomic analysis of rhizobial genomes

• Translational Research Applications:

  • Develop B. japonicum strains with enhanced SecB function for agricultural applications

  • Design field trial protocols following established EPA guidelines for testing modified strains

  • Evaluation metrics: Assess improvements in nodulation, nitrogen fixation capability, and crop yield using standardized measurements

These research directions leverage current methodological advances while addressing gaps in our understanding of B. japonicum SecB function, potentially leading to significant improvements in both agricultural applications and biotechnology platforms.

What are the most effective experimental protocols for studying SecB-dependent protein export in B. japonicum?

Based on the compilation of available research, the following experimental protocol framework is recommended for studying SecB-dependent protein export in B. japonicum:

• Strain Development and Validation:

  • Generate B. japonicum strains with tagged SecB (His6-tag recommended)

  • Validate strains using proteogenomic analysis with ≤1% FDR threshold for peptide identifications

  • Compare expression across different growth phases and symbiotic states

• Fusion Protein Construction Protocol:

  • Design fusion constructs following the validated pattern:
    Signal Peptide - His6-Tag - Flexible Peptide (ASGGGGA) - TEV Recognition Site (ENLYFQ) - Target Protein

  • Express constructs in both E. coli and B. japonicum to compare efficiency

  • Co-express with modification enzymes when appropriate

• Translocation Analysis Workflow:

  • Fractionate cells to isolate cytoplasmic, periplasmic, and extracellular components

  • Use sodium azide (1 mM) as a translocation inhibitor to enhance cytosolic modification

  • Quantify protein distributions using densitometric analysis with unmodified protein as baseline

• Mass Spectrometry-Based Detection:

  • Process samples following established protocols:

    • 20 ppm precursor ion tolerance

    • 0.6 Da product ion tolerance

    • Carbamidomethylation of cysteine (fixed modification)

    • Methionine oxidation (variable modification)

  • Analyze data using multiple search algorithms for robust identification

• Comparative Analysis Framework:

  • Study SecB function across multiple developmental stages, noting that protein synthesis declines about 60% between 14-20 days after planting

  • Compare expression patterns across different host plants (soybean, siratro, cowpea)

  • Correlate SecB function with nitrogen fixation efficiency measured by acetylene reduction assays

• Data Validation Protocol:

  • Apply stringent statistical validation (≤1% FDR)

  • Use multiple gene prediction algorithms to validate novel findings

  • Implement orthology-based validation by comparing with related species

This comprehensive experimental protocol integrates methodologies validated across multiple studies and provides a robust framework for investigating SecB-dependent protein export in B. japonicum.

What are the key considerations for designing field trials with modified B. japonicum strains expressing recombinant SecB?

Researchers planning field trials with modified B. japonicum strains expressing recombinant SecB should adhere to the following evidence-based framework:

• Regulatory Compliance Protocol:

  • Submit a TSCA Environmental Release Application (TERA) following established precedents

  • Provide detailed information on the genetic modifications

  • Plan for multi-year testing (typically three years) to assess long-term performance and safety

• Site Selection Criteria:

  • Establish multiple test sites (typically 0.25-0.5 acres each)

  • Include diverse geographical locations to assess performance under different environmental conditions

  • Implement appropriate containment measures to prevent unintended spread

• Experimental Design Parameters:

  • Use a randomized complete block design (RCBD) with at least three replications

  • Include both positive controls (commercial inoculant strains) and negative controls (uninoculated)

  • Implement split-plot designs to test multiple variables simultaneously

• Inoculation Protocol:

  • Prepare bacterial suspensions at standardized optical densities

  • Use gum Arabic 20% (w/v) as an adhesive agent for seed coating

  • Apply the mixture to seeds, ensure proper coating, and allow to dry before planting

• Performance Assessment Metrics:

  • Evaluate nodulation patterns (count, size, distribution)

  • Measure nitrogen fixation using acetylene reduction assays

  • Assess plant growth parameters (height, biomass, yield)

  • Monitor soil nitrogen levels before and after the growing season

• Data Collection Timeline:

  • Conduct assessments at multiple time points throughout the growing season

  • Pay particular attention to the period between 14-20 days after planting, when protein synthesis naturally declines and nitrogen fixation increases

  • Continue monitoring through harvest to assess yield impacts

• Biosafety Monitoring Protocol:

  • Implement perimeter sampling to detect potential spread beyond test plots

  • Assess impacts on non-target organisms in the surrounding ecosystem

  • Maintain detailed records for regulatory compliance