Recombinant Bradyrhizobium japonicum 50S ribosomal protein L19 (rplS)

How is the rplS gene organized in the Bradyrhizobium japonicum genome?

The rplS gene in B. japonicum is typically arranged within a ribosomal protein operon, consistent with the genomic organization observed in many prokaryotes. This arrangement facilitates coordinated expression of ribosomal components. In B. japonicum USDA110, the genome has been extensively characterized through proteogenomic analysis, which has refined gene models for numerous proteins .

Genome analysis of B. japonicum has revealed complex operon structures, with some operons showing differential expression under symbiotic conditions. For instance, proteogenomic analysis discovered "a novel protein which redefined the boundary of a crucial cytochrome P450 system related operon was discovered, known to be highly expressed in the anaerobic symbiotic bacteroids" . Similar detailed analysis could clarify the precise genomic context of rplS and any potential co-regulated genes that might influence its expression during different physiological states.

Accurate determination of translation initiation sites (TIS) has been challenging in B. japonicum, with proteogenomic approaches identifying numerous refinements to predicted gene models . This suggests that careful analysis is needed to confirm the precise boundaries of the rplS gene and its regulatory elements.

How does rplS expression change during different growth phases and symbiotic stages?

Expression analysis of B. japonicum genes has shown that certain genes have dual expression patterns, being active in both free-living cells and symbiotic nodules. For example, genes like "bll6451 or bll7010 are also expressed in the symbiotic nodules" , suggesting that some cellular machinery remains active across different life stages. Analysis of bacteroids has revealed "significant sulfatase activity" , indicating metabolic adaptations during symbiosis that may affect ribosomal composition and activity.

The most effective methods for tracking rplS expression include qRT-PCR for transcript levels, ribosome profiling for translation efficiency, and quantitative proteomics for protein abundance. Each approach provides complementary information about how rplS regulation responds to environmental cues and developmental transitions.

What is the evolutionary conservation pattern of rplS across Bradyrhizobium species?

The rplS gene exhibits strong conservation across Bradyrhizobium species, reflecting its essential role in ribosome function. Comparative genomic analyses between B. japonicum strains such as USDA110 and USDA6T have revealed both highly conserved core genes and strain-specific adaptations . As a component of the essential protein synthesis machinery, rplS likely belongs to the core genome with high sequence similarity across species.

The most informative approach to studying rplS conservation involves comparative genomics combined with structural modeling to identify functionally significant variations. "Ortho-proteogenomic analysis" approaches, which have successfully revealed novel genes across Bradyrhizobium strains , could similarly identify structural or regulatory variations in rplS that might influence its function across the genus.

What role does rplS play in translation efficiency and accuracy?

• Stabilization of rRNA tertiary structure within the ribosome

• Facilitation of interactions with translation factors

• Contribution to proper tRNA positioning in the A and P sites

• Potential involvement in ribosomal subunit joining

Changes in rplS expression or structure could affect translation rates, particularly of specific mRNA transcripts that might be more sensitive to ribosomal composition. This could have downstream effects on growth rate, stress responses, and symbiotic efficiency.

Proteogenomic approaches have demonstrated that refined gene models, including correct identification of translation initiation sites, are essential for understanding protein function in B. japonicum . Similar refined analysis of rplS structure and interactions would clarify its specific contributions to translation in this agriculturally important symbiont.

What are optimal expression systems for producing recombinant B. japonicum rplS?

Producing well-folded, soluble recombinant B. japonicum rplS requires careful optimization of expression systems. Based on experiences with other bacterial ribosomal proteins and molecular biology techniques used with B. japonicum genes, the following approaches are recommended:

For cloning B. japonicum genes into expression vectors, established protocols involve PCR amplification followed by appropriate restriction digestion and ligation steps. For example, B. japonicum genes have been successfully cloned by creating fragments with appropriate restriction sites, followed by "digestion with BglII and EcoRI, the fragment was cloned into the BamHI-EcoRI site of pRSETB" .

Expression levels should be verified by SDS-PAGE and Western blotting, with optimization focusing on the soluble fraction rather than total protein yield. Co-expression with bacterial chaperones (GroEL/ES) may improve folding and solubility in challenging cases.

How can post-translational modifications of rplS be analyzed during symbiosis?

Post-translational modifications (PTMs) of rplS may play important regulatory roles during B. japonicum's transition to symbiosis. Analysis of these modifications requires specialized approaches:

• Sample preparation considerations:

  • Careful isolation of bacteroids from nodules while preventing artificial modifications

  • Immediate protein extraction in buffers containing PTM-preserving inhibitors

  • Enrichment for ribosomal proteins to increase detection sensitivity

• Mass spectrometry approaches:

  • Tandem MS/MS with higher-energy collisional dissociation (HCD) for comprehensive PTM mapping

  • Parallel reaction monitoring (PRM) for targeted quantification of known modifications

  • Electron transfer dissociation (ETD) for preserving labile modifications

• Comparative analysis strategies:

  • Direct comparison between free-living bacteria and bacteroids

  • Time-course analysis during nodule development

  • Comparison across different host plant species

Focused analysis of N-terminal acetylation has already proven valuable in refining gene models in B. japonicum, indicating "downstream TIS for gene blr0594" . Similar approaches could identify modifications on rplS that affect its function during symbiosis.

Potential PTMs to investigate include phosphorylation (affecting protein-protein interactions), methylation (modulating rRNA binding), and acetylation (altering protein stability). Correlating these modifications with symbiotic efficiency could provide insights into how ribosome function is regulated during this critical biological process.

What experimental approaches can determine the structure of B. japonicum rplS?

Determining the three-dimensional structure of B. japonicum rplS presents several challenges but is achievable through complementary approaches:

• X-ray crystallography strategies:

  • Co-crystallization with binding partners (rRNA fragments or adjacent ribosomal proteins)

  • Surface entropy reduction (replacing flexible, charged residues with alanines)

  • Crystallization screening with varied pH, salt, and precipitant conditions

  • Use of crystallization chaperones or antibody fragments

• Cryo-electron microscopy (cryo-EM):

  • Analysis of intact B. japonicum ribosomes to visualize rplS in its native context

  • Sub-particle classification to capture different conformational states

  • Local refinement focused on the rplS region of the ribosome

• NMR spectroscopy:

  • Suitable for dynamic regions and smaller domains of rplS

  • 15N,13C-labeling for complete structure determination

  • Residual dipolar coupling to improve structural precision

• Integrative structural biology:

  • Combining data from multiple structural techniques

  • Molecular dynamics simulations based on experimental constraints

  • Homology modeling leveraging structures from related organisms

The proteogenomic approaches that have successfully refined gene models in B. japonicum could be combined with structural analysis to ensure accurate protein boundaries prior to structural studies. This integrated approach would provide the most reliable structural information about this important ribosomal protein.

How do mutations in rplS affect ribosome assembly and symbiotic efficiency?

Mutations in rplS can have wide-ranging effects on ribosome assembly and function, with implications for B. japonicum's symbiotic efficiency. Different types of mutations may produce distinct phenotypes:

• Mutations affecting rRNA interactions:

  • May destabilize the 50S subunit

  • Could alter translation efficiency or accuracy

  • Potentially temperature-sensitive phenotypes

• Mutations at protein-protein interfaces:

  • May disrupt ribosome assembly pathways

  • Could affect recruitment of translation factors

  • Potential cold-sensitive phenotypes

• Core structural mutations:

  • May cause protein misfolding and degradation

  • Likely to produce severe growth defects

  • Potential complete loss of function

Careful phenotyping is essential to reveal the true impact of rplS mutations, as different growth conditions often reveal distinct phenotypes. For instance, B. japonicum mutants in sulfonate utilization operons "were not affected for symbiosis with soybean, indicating the functional redundancy or availability of other sulfur sources in planta" , demonstrating that apparent redundancy under standard conditions may mask phenotypes relevant to specific ecological contexts.

Assessing the impact of rplS mutations should include measurements of growth rates, stress responses, ribosome profiles, translation accuracy, and most importantly, symbiotic performance metrics such as nodulation efficiency and nitrogen fixation rates with host legumes.

What methods can identify proteins that interact with rplS during different growth states?

Identifying the protein interaction network of rplS across different growth states of B. japonicum requires specialized approaches:

• Co-immunoprecipitation (Co-IP) strategies:

  • Generation of tagged rplS constructs that maintain functionality

  • Expression under native promoter to preserve physiological interactions

  • Careful buffer optimization to preserve weak or transient interactions

  • Mass spectrometry identification of co-precipitated proteins

• Crosslinking mass spectrometry (XL-MS):

  • In vivo crosslinking with membrane-permeable reagents

  • Analysis of crosslinked peptides to identify spatial proximity

  • Direct detection of ribosome assembly intermediates

  • Comparison between free-living and symbiotic states

• Proximity labeling approaches:

  • BioID or APEX2 fusion to rplS

  • Controlled expression in different growth phases

  • Identification of proteins that come into proximity with rplS

  • Temporal mapping of interaction changes during differentiation

The proteogenomic pipeline "GenoSuite" used for analyzing B. japonicum could be adapted to process interaction data, potentially revealing novel associating partners during different growth states. Particular attention should be paid to interactions that change during the transition to symbiosis, as these may reveal regulatory mechanisms specific to the nitrogen-fixing state.

How does rplS expression respond to environmental stresses relevant to the rhizosphere?

The rhizosphere presents multiple environmental stresses that likely affect rplS expression in B. japonicum. Predicting and measuring these responses provides insights into ribosome adaptation mechanisms:

B. japonicum demonstrates remarkable metabolic flexibility, capable of "utilizing sulfate, cysteine, sulfonates, and sulfur-ester compounds as sole sulfur sources for growth" , suggesting sophisticated stress response systems that likely include ribosomal adaptations. Studying rplS regulation under these conditions would reveal how ribosome composition adjusts to environmental challenges.

Gene expression during symbiosis often differs from free-living conditions, as evidenced by genes like "bll6451 or bll7010 [which] are also expressed in the symbiotic nodules" . Similar analysis of rplS expression across conditions would provide insights into its regulation during different physiological states.

How might rplS variants influence antibiotic susceptibility in B. japonicum?

Variants in rplS could potentially influence B. japonicum's susceptibility to antibiotics that target the ribosome, with several mechanisms possible:

Proteogenomic analyses that have successfully "refined 49 gene models for their translation initiation site (TIS)" could be applied to identify natural variations in rplS across Bradyrhizobium strains. These variations could then be correlated with antibiotic susceptibility profiles to identify potentially causative relationships.

Understanding these relationships has practical implications for agricultural applications, where soil antibiotic residues might affect rhizobial populations and subsequently impact legume nodulation efficiency.

What CRISPR-Cas9 strategies are most effective for modifying the rplS gene?

Developing effective CRISPR-Cas9 strategies for modifying rplS in B. japonicum requires careful consideration of several factors:

• sgRNA design optimization:

  • Targeting efficiency based on GC content (40-60%)

  • Minimizing off-target effects through thorough genome analysis

  • Consideration of secondary structure in the target region

  • Testing multiple sgRNAs targeting different regions of rplS

• Delivery system optimization:

  • Conjugation-based plasmid transfer from E. coli donors

  • Electroporation protocols optimized for B. japonicum

  • Transient vs. stable Cas9 expression strategies

• Homology-directed repair (HDR) template design:

  • Arm lengths of 750-1000bp for efficient recombination

  • Incorporation of silent mutations in PAM sites

  • Selection markers flanked by FRT sites for marker removal

• Editing verification strategies:

  • PCR-based screening of transformants

  • Sequencing confirmation of expected edits

  • Phenotypic validation of mutants

What proteomics approaches best quantify rplS across different cellular states?

Accurate quantification of rplS across different cellular states of B. japonicum requires specialized proteomics approaches:

The proteogenomic approach described for B. japonicum using "GenoSuite, an integrated proteogenomic pipeline to validate, refine and discover protein coding genes using high-throughput mass spectrometry" provides a solid foundation for quantitative analysis of rplS. This approach could be extended to compare rplS abundance across different growth conditions, particularly comparing free-living bacteria with bacteroids.

Special attention should be paid to potential post-translational modifications of rplS, which might vary between conditions and affect both function and detection by proteomics methods.

How does rplS function change during the transition to symbiotic bacteroids?

The transition of B. japonicum from free-living cells to symbiotic bacteroids involves profound physiological changes that likely affect rplS function:

• Expression level changes:

  • Potential initial downregulation during infection thread development

  • Possible upregulation during bacteroid differentiation

  • Establishment of a new steady-state expression level in mature bacteroids

• Functional adaptations:

  • Possible post-translational modifications specific to the symbiotic state

  • Potential differential association with bacteroid-specific proteins

  • Adaptation to the altered metabolic demands of nitrogen fixation

• Regulatory mechanisms:

  • Transcriptional control through symbiosis-specific regulatory networks

  • Post-transcriptional regulation through sRNAs

  • Translational regulation affecting ribosome composition

Studies of gene expression in B. japonicum have shown that many genes maintain activity across both free-living and symbiotic states, with genes like "bll6451 or bll7010 also expressed in the symbiotic nodules" . Similar analysis of rplS expression patterns would clarify its regulation during symbiosis.

Proteogenomic approaches have identified "a novel protein which redefined the boundary of a crucial cytochrome P450 system related operon was discovered, known to be highly expressed in the anaerobic symbiotic bacteroids" . Similar approaches focused on ribosomal proteins could reveal how translation machinery adapts during symbiosis.

What purification protocol best maintains native rplS structure?

Purifying recombinant B. japonicum rplS while preserving its native structure requires an optimized protocol addressing the specific challenges of ribosomal proteins:

• Expression optimization:

  • Vector: pET28a with N-terminal His6-tag and TEV cleavage site

  • Host: E. coli BL21(DE3) or Rosetta(DE3) for rare codons

  • Induction: 0.3 mM IPTG at OD600 = 0.6-0.8

  • Temperature: 20°C for 16-18 hours post-induction

• Lysis and initial purification:

  • Buffer: 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5% glycerol, 1 mM DTT

  • Protease inhibitors (PMSF, leupeptin, pepstatin A)

  • Lysis by sonication with cooling intervals

  • Clarification by centrifugation at 40,000 × g, 30 min, 4°C

  • Ni-NTA affinity chromatography with gradient elution

• Tag removal and secondary purification:

  • TEV protease cleavage (1:100 ratio) at 4°C overnight

  • Removal of uncleaved protein by reverse Ni-NTA

  • Ion exchange chromatography (typically SP Sepharose)

  • Size exclusion chromatography in final buffer (20 mM HEPES pH 7.5, 150 mM KCl, 5 mM MgCl2)

• Quality assessment:

  • SDS-PAGE for purity (>95%)

  • Circular dichroism for secondary structure verification

  • Thermal shift assay for stability assessment

  • RNA binding assay to confirm functionality

Experience with expression of B. japonicum proteins has established effective approaches, including methods where "The amplified fragment was isolated, blunt ended with Klenow DNA polymerase, phosphorylated with T4 polynucleotide kinase, and blunt-end ligated with T4 ligase to form concatemers. Following digestion with BglII and EcoRI, the fragment was cloned into the BamHI-EcoRI site of pRSETB" .

How can isotope labeling track rplS incorporation into ribosomes?

Isotope labeling provides powerful methods to track rplS incorporation into ribosomes and study ribosome assembly dynamics in B. japonicum:

• Pulse-chase labeling approach:

  • Cultivate B. japonicum in minimal media with 14N sources

  • Shift to media containing 15N-labeled amino acids

  • Sample at defined intervals (10 min, 30 min, 1 h, 2 h, 4 h)

  • Isolate ribosomes via sucrose density gradient ultracentrifugation

  • Analyze ribosomal proteins by mass spectrometry

  • Quantify 14N/15N ratios to determine turnover rates

• SILAC for comparative incorporation studies:

  • Grow cells under different conditions with distinct isotope labels

  • Culture 1: 14N/12C (normal) – free-living aerobic

  • Culture 2: 15N/12C – microaerobic

  • Culture 3: 14N/13C – symbiotic bacteroids

  • Compare incorporation rates across conditions

• In vivo ribosome assembly analysis:

  • Use runoff translation to generate ribosomes with defined isotopic composition

  • Add labeled precursors and follow assembly intermediates

  • Combine with time-resolved structural studies

This approach complements proteogenomic analyses that have been successfully applied to B. japonicum, which used "high-throughput mass spectrometry (MS) data from prokaryotes" to refine gene models and characterize protein expression. Mass spectrometry techniques can be adapted to specifically track isotope-labeled rplS through ribosome assembly pathways.

What methods best analyze site-directed rplS mutants?

Comprehensive analysis of site-directed rplS mutants requires multiple complementary approaches to understand functional implications:

• Structural and biophysical characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Thermal shift assays to evaluate stability changes

  • Isothermal titration calorimetry (ITC) for rRNA binding kinetics

  • Small-angle X-ray scattering (SAXS) for solution structure

• Functional in vitro assays:

  • Reconstitution of 50S subunits with wild-type vs. mutant rplS

  • In vitro translation assays measuring efficiency and fidelity

  • Ribosome assembly kinetics using sucrose gradient analysis

  • RNA binding assays with specific rRNA fragments

• In vivo characterization:

  • Growth curve analysis under various conditions

  • Polysome profiling to assess translation efficiency

  • Reporter systems for translation accuracy (nonsense suppression)

  • Stress response activation measurements

• Symbiosis-specific assessments:

  • Nodulation efficiency on host legumes

  • Bacteroid differentiation analysis

  • Nitrogen fixation activity measurements

  • Competitive fitness in mixed inoculation experiments

Site-directed mutagenesis approaches used for other B. japonicum genes provide established methods, where "Derivatives of pJLDA containing mutations to ATG1, ATG2, or ATG3 were obtained by replacing the BamHI wild-type fragment of pJLDA with the corresponding BamHI fragment of the pAlt plasmids harboring mutations" . Similar approaches can be adapted for systematic mutation of rplS to analyze structure-function relationships.

What experimental design best resolves contradictory findings about rplS?

When faced with contradictory findings regarding rplS function in B. japonicum, a systematic experimental design can help resolve discrepancies:

• Strain and condition standardization:

  • Establish a reference strain set with verified genotypes

  • Define standardized growth conditions (media, temperature, aeration)

  • Create a shared protocol database with detailed methodologies

  • Implement quality control metrics for each experimental step

• Multi-laboratory validation approach:

  • Design experiments with technical replicates across laboratories

  • Standardize key reagents and biological materials

  • Implement blinded analysis of critical measurements

  • Use statistical methods appropriate for multi-site studies

• Orthogonal methodologies:

  • Apply multiple independent techniques to address the same question

  • Combine genetic, biochemical, and structural approaches

  • Integrate in vitro and in vivo data

  • Use both targeted and systems-level analyses

• Sequential hypothesis refinement:

  • Start with simplified conditions to establish baseline findings

  • Systematically introduce complexity to identify condition-dependent effects

  • Develop mathematical models to predict outcomes under different conditions

  • Iteratively test and refine these models

The proteogenomic approach described for B. japonicum, using "GenoSuite, an integrated proteogenomic pipeline" , exemplifies how integrated data analysis can resolve conflicting gene models. Similar multi-faceted approaches could resolve contradictions about rplS function by integrating genomic, transcriptomic, proteomic, and phenotypic data.

How can cryo-EM be optimized for studying B. japonicum ribosomes?

Cryo-electron microscopy (cryo-EM) offers powerful approaches for studying B. japonicum ribosomes, but requires optimization for this specific organism:

• Sample preparation optimization:

  • Ribosome isolation through gentle lysis and differential centrifugation

  • Sucrose density gradient purification with buffer optimization

  • Concentration adjustment (typically 50-100 nM for ribosomes)

  • Grid type selection and glow discharge parameters

  • Vitrification conditions (blot time, humidity control)

• Data collection strategies:

  • Motion correction parameters for beam-induced movement

  • Dose fractionation approach (typically 40-50 frames total)

  • Total dose limitation (60-80 e-/Å2) to minimize radiation damage

  • Defocus range selection (-0.8 to -2.5 μm)

  • Automated data collection with image quality filtering

• Image processing considerations:

  • 2D classification to eliminate non-ribosomal particles

  • 3D classification to separate conformational states

  • Focused refinement on regions containing rplS

  • Local resolution estimation for structure interpretation

  • Multiple body refinement for flexible regions

• Functional state capture:

  • Preparation of stalled translation complexes

  • Capture of initiation, elongation, or termination states

  • Complex formation with translation factors

  • Comparison between free-living and bacteroid ribosomes

This approach complements proteogenomic analyses that have identified novel proteins and refined gene models in B. japonicum . Cryo-EM would provide structural context for these findings, showing how newly discovered or refined proteins like rplS interact within the ribosome's three-dimensional architecture.