Recombinant Lactobacillus plantarum 50S ribosomal protein L31 type B (rpmE2)

What is the genomic context of the rpmE2 gene in Lactobacillus plantarum?

The rpmE2 gene in Lactobacillus plantarum encodes the 50S ribosomal protein L31 type B, which is part of the large ribosomal subunit. This gene exists within the context of the complete genome sequence of L. plantarum, which has been fully sequenced and annotated. The genomic context analysis reveals that L. plantarum possesses multiple sigma factors, including σA (with 73% identity to B. subtilis), σ54 (35% identity to σL in B. subtilis), and σH (23% identity) . Understanding this genomic context is essential for designing amplicon-based microarray experiments to study expression patterns of rpmE2 under various conditions. Comparative analyses with other lactic acid bacteria (LAB) genomes have shown that certain regulatory elements, like σ54, are only found in L. plantarum and P. pentosus, while σA and σH are present in all 12 species studied .

How does the expression of rpmE2 vary under different growth conditions in L. plantarum?

The expression of rpmE2 in L. plantarum demonstrates significant variability depending on environmental conditions. Transcriptome studies using full genome amplicon-based microarrays reveal that L. plantarum adjusts gene expression patterns in response to oxygen exposure, oxidative stress, and different carbon sources . While specific data on rpmE2 expression was not directly provided in the search results, general transcriptome response patterns in L. plantarum suggest that genes involved in ribosomal function may be differentially regulated under aerobic versus anaerobic conditions. For instance, growth stagnation observed under certain aerobic conditions may correlate with altered expression of ribosomal proteins . To properly assess rpmE2 expression, researchers should conduct comparative transcriptomic analyses under varying conditions such as different oxygen levels, pH environments, and nutrient availabilities.

What are the structural characteristics of the L. plantarum 50S ribosomal protein L31 type B?

The L. plantarum 50S ribosomal protein L31 type B is characterized by specific structural features that contribute to ribosomal assembly and function. This protein is part of the large ribosomal subunit and plays a critical role in maintaining ribosomal integrity. Structurally, L31 proteins typically contain zinc-binding motifs that can influence protein stability and function. The type B variant (encoded by rpmE2) differs from type A in its metal-binding properties, which can affect ribosomal assembly under different environmental conditions.

The protein structure can be studied using various techniques including X-ray crystallography and cryo-electron microscopy to elucidate its three-dimensional conformation within the ribosomal complex. These structural insights are crucial for understanding how the protein interacts with ribosomal RNA and other ribosomal proteins to maintain functional translation machinery in L. plantarum.

What are the optimal conditions for expressing recombinant L. plantarum 50S ribosomal protein L31 type B?

The optimization of expression conditions for recombinant L. plantarum ribosomal proteins requires careful consideration of multiple parameters. Based on similar expression systems in L. plantarum, the following conditions have proven effective:

• Expression System Selection: For ribosomal proteins like L31 type B, codon optimization is critical due to codon usage bias in L. plantarum. Optimize codons according to L. plantarum's preference for enhanced expression efficiency .

• Induction Parameters: For inducible expression systems in L. plantarum, conditions similar to those used for other recombinant proteins can serve as a starting point. For example, induction with 50 ng/mL SppIP at 37°C for 6-10 hours has shown optimal results for recombinant protein expression .

• Stability Considerations: Assess protein stability under various conditions. Testing at different temperatures (up to 50°C), pH levels (down to pH 1.5), and salt concentrations is recommended to determine optimal expression conditions .

Table 1: Recommended Optimization Parameters for rpmE2 Expression in L. plantarum

What methods are most effective for purifying recombinant L. plantarum rpmE2 protein while maintaining its structural integrity?

Purification of the recombinant L. plantarum 50S ribosomal protein L31 type B requires specialized techniques to preserve its native structure and function. The following methodological approach is recommended:

• Cell Lysis Protocol: Optimize gentle lysis procedures using enzymatic methods (lysozyme treatment) followed by mild sonication in a buffer containing stabilizing agents such as glycerol (10%) and reducing agents to maintain disulfide bonds.

• Affinity Chromatography: Incorporate a tag system (His-tag or HA-tag) at either the N or C-terminus of the protein for efficient purification. When expressing the protein on the bacterial surface, consider using specific antibodies against L31 for immunoprecipitation.

• Stability Assessment: Evaluate protein stability under various conditions similar to those tested with other L. plantarum recombinant proteins. Testing has shown that some L. plantarum recombinant proteins maintain stability at temperatures up to 50°C, pH levels as low as 1.5, and in the presence of 0.2% bile salts .

• Quality Control: Verify the structural integrity using circular dichroism (CD) spectroscopy and functional assays specific to ribosomal proteins, such as RNA binding assays.

For researchers seeking to assess the antigenicity or immunological properties of the purified protein, indirect immunofluorescence assays (IFA) and flow cytometry have proven effective for recombinant L. plantarum proteins, showing positive expression rates of approximately 37.5% compared to 2.5% in parental strains .

What statistical approaches are most appropriate for analyzing differential expression of rpmE2 across experimental conditions?

When analyzing the differential expression of rpmE2 across various experimental conditions, researchers should implement a structured statistical approach:

• Normalization Procedures: Begin with appropriate normalization of expression data, particularly when using microarray or RNA-seq techniques. For L. plantarum transcriptome studies, normalization against housekeeping genes has been successfully employed .

• Statistical Tests:

  • For comparing two conditions (e.g., aerobic vs. anaerobic): Use Student's t-test with appropriate corrections for multiple testing (Benjamini-Hochberg procedure)

  • For multiple conditions: Implement ANOVA followed by post-hoc tests (Tukey's HSD)

  • For time-series data: Consider repeated measures ANOVA or linear mixed models

• Expression Pattern Analysis: Integrate rpmE2 expression data into wider transcriptome patterns using clustering approaches (hierarchical clustering, k-means) to identify co-regulated genes.

• Fold Change Thresholds: Establish significant fold change thresholds based on biological relevance. Typically, a fold change of ≥2.0 (up or down) with an adjusted p-value <0.05 is considered significant.

Table 2: Recommended Statistical Approaches for Different Experimental Designs

How should researchers integrate transcriptomic and proteomic data when studying rpmE2 function in L. plantarum?

Integrating transcriptomic and proteomic data provides a comprehensive understanding of rpmE2 function in L. plantarum. This multi-omics approach should follow these methodological steps:

• Data Collection Alignment: Ensure transcriptomic (e.g., RNA-seq, microarray) and proteomic (e.g., mass spectrometry) experiments are designed in parallel with identical conditions, time points, and sample processing protocols.

• Normalization and Preprocessing:

  • For transcriptomic data: Apply background correction, log-transformation, and between-sample normalization

  • For proteomic data: Use appropriate normalization techniques like total ion current (TIC) or normalized spectral abundance factors (NSAF)

• Correlation Analysis: Calculate Pearson or Spearman correlation coefficients between rpmE2 mRNA and protein levels across conditions to identify potential post-transcriptional regulation.

• Pathway Enrichment: Conduct gene ontology (GO) and pathway enrichment analyses on correlated genes/proteins to place rpmE2 in its functional context.

• Time-Lag Considerations: Account for the expected delay between transcription and translation by analyzing time-shifted correlations.

• Data Integration Techniques:

  • Canonical correlation analysis (CCA)

  • Partial least squares (PLS) regression

  • Network-based integration approaches

The integrated analysis should be presented in the Results section of research papers with clear demarcation of findings without bias or interpretation, saving deeper interpretation for the Discussion section .

How can CRISPR-Cas9 gene editing be optimized for creating rpmE2 knockout mutants in L. plantarum?

Optimizing CRISPR-Cas9 gene editing for rpmE2 knockout in L. plantarum requires careful consideration of several technical aspects:

• Vector System Selection: Choose an appropriate vector system that functions efficiently in L. plantarum. Consider using vectors that have been successful in other lactic acid bacteria experiments, adapted to L. plantarum's specific requirements.

• sgRNA Design Strategy:

  • Target unique sequences within the rpmE2 gene to prevent off-target effects

  • Design sgRNAs with high on-target efficiency using predictive algorithms

  • Create multiple sgRNAs targeting different regions of the gene to increase success rate

  • Verify sgRNA specificity against the entire L. plantarum genome

• Transformation Protocol: L. plantarum transformation can be challenging. Optimize electroporation conditions specifically for L. plantarum WCFS1 or other relevant strains, with appropriate cell wall weakening treatments prior to transformation.

• Homology-Directed Repair (HDR) Template Design:

  • Include homology arms of 500-1000 bp flanking the targeted cut site

  • Consider introducing antibiotic resistance markers for selection

  • Design the HDR template to ensure complete functional knockout

• Screening and Verification Methods:

  • PCR verification with primers flanking the edited region

  • Sequencing confirmation of the edited locus

  • Western blot analysis to confirm absence of L31 protein

  • Ribosome profiling to assess impact on ribosomal assembly

When designing knockout experiments, researchers should be aware that ribosomal protein knockouts may have significant effects on cellular physiology. Control experiments should include growth rate comparisons, stress response evaluations, and complementation studies to confirm phenotypic effects are specifically due to the rpmE2 knockout.

What are the implications of rpmE2 mutations on ribosomal assembly and antibiotic resistance in L. plantarum?

The implications of rpmE2 mutations on ribosomal assembly and antibiotic resistance in L. plantarum represent a complex research area with significant clinical and fundamental research relevance:

• Ribosomal Assembly Effects:

  • Mutations in rpmE2 may alter the kinetics of 50S subunit assembly

  • Changes in ribosomal protein L31 type B can affect ribosomal structure stability

  • Compensatory mechanisms may arise through upregulation of other ribosomal proteins

• Antibiotic Resistance Mechanisms:

  • 50S ribosomal proteins are targets for multiple antibiotics (macrolides, lincosamides, streptogramins)

  • Specific mutations in rpmE2 may confer resistance by altering antibiotic binding sites

  • Cross-resistance patterns should be systematically evaluated

• Experimental Approach:

  • Generate site-directed mutations in conserved residues of rpmE2

  • Assess minimum inhibitory concentrations (MICs) for various antibiotics

  • Perform ribosome profiling to identify assembly defects

  • Use cryo-electron microscopy to visualize structural alterations

• Regulatory Considerations:

  • Evaluate expression changes in rpmE2 mutants during antibiotic exposure

  • Assess global transcriptional responses using microarray approaches similar to those used in L. plantarum transcriptome studies

Table 3: Potential Impact of rpmE2 Mutations on Antibiotic Susceptibility

How does rpmE2 expression influence L. plantarum adaptation to stress conditions?

The role of rpmE2 in L. plantarum stress adaptation represents an important area of investigation with implications for both fundamental biology and biotechnological applications:

• Oxidative Stress Response:

  • Analyze rpmE2 expression under hydrogen peroxide exposure conditions

  • Compare with known oxidative stress response genes in L. plantarum

  • Investigate potential zinc mobilization from L31 type B protein under oxidative conditions

• pH and Acid Tolerance:

  • Evaluate rpmE2 expression changes at various pH values

  • Determine if rpmE2 overexpression enhances survival at low pH

  • Analyze correlation between rpmE2 expression and proton ATPase activity

• Temperature Adaptation:

  • Study temperature-dependent expression patterns of rpmE2

  • Examine the role of rpmE2 in cold shock and heat shock responses

  • Test stability of ribosomes from wild-type versus rpmE2 mutants at extreme temperatures

• Nutritional Stress Response:

  • Investigate rpmE2 expression during carbon source limitation

  • Determine if rpmE2 is regulated by CcpA, the global regulator of catabolite control in L. plantarum

  • Evaluate ribosomal adaptation mechanisms during nutrient downshift

• Experimental Methodology:

  • Employ RT-qPCR for precise quantification of rpmE2 expression

  • Use reporter gene fusions to monitor promoter activity under various stresses

  • Implement proteomics to track L31 protein levels and potential post-translational modifications

Research indicates that stress responses in L. plantarum involve complex transcriptional networks. For example, aerobic growth compared to anaerobic growth leads to specific transcriptional adaptations . The potential role of ribosomal proteins like L31 type B in these adaptations may provide insights into stress tolerance mechanisms in lactic acid bacteria.

How can recombinant L. plantarum expressing modified rpmE2 be utilized in research applications?

Recombinant L. plantarum expressing modified rpmE2 offers various research applications that exploit the unique properties of this probiotics species:

• Ribosome Engineering Applications:

  • Modified rpmE2 can serve as a platform for creating ribosomes with altered translational properties

  • Engineered ribosomes could selectively translate specific mRNAs for specialized protein production

  • Tagged rpmE2 enables ribosome isolation for structural and functional studies

• Methodology for Expression System Development:

  • Begin with codon optimization similar to approaches used for other recombinant proteins in L. plantarum

  • Incorporate secretion signals and surface-display elements as demonstrated for other recombinant proteins

  • Establish induction parameters (e.g., 50 ng/mL SppIP at 37°C for 6-10 hours)

• Research Applications:

  • Study ribosomal assembly mechanisms through modified rpmE2 incorporation

  • Investigate translational fidelity under various stress conditions

  • Explore interactions between ribosomal proteins and regulatory RNAs

• Verification Methods:

  • Confirm surface expression using transmission electron microscopy and indirect immunofluorescence assays

  • Assess recombinant protein expression using flow cytometry (expect positive rates of approximately 37.5% based on similar recombinant protein studies)

  • Validate functionality through ribosomal incorporation assays

The development of such recombinant systems should follow established protocols for L. plantarum, with appropriate modifications for the specific properties of ribosomal proteins.