Recombinant Lactobacillus plantarum 50S ribosomal protein L13 (rplM)

Basic Research Questions

• What is the function of the 50S ribosomal protein L13 (rplM) in Lactobacillus plantarum?

  The 50S ribosomal protein L13 (rplM) in Lactobacillus plantarum is one of the early assembly proteins of the 50S ribosomal subunit. While it does not directly bind to rRNA by itself, it plays a crucial role during the early stages of 50S ribosomal subunit assembly. This protein contributes to the structural integrity of the ribosome and is essential for proper protein synthesis in this lactic acid bacterium .

• What expression systems are commonly used for recombinant L. plantarum proteins?

  Several expression systems have been developed for L. plantarum, with the pSIP expression system being one of the most commonly used. This methodological approach utilizes:

  • Inducible promoters (e.g., sakacin P promoters)

  • Various signal peptides for secretion

  • Different host strains (e.g., L. plantarum WCFS1)

  For optimal expression, researchers typically use the following methodology:

  1. Clone the target gene into a suitable vector (e.g., pSIP401)

  2. Transform into L. plantarum using electroporation

  3. Induce expression using inducing peptides (e.g., IP-673)

  4. Harvest cells at appropriate time points post-induction

• How can I optimize codon usage for recombinant rplM expression in L. plantarum?

  To optimize codon usage for recombinant rplM expression in L. plantarum, implement the following methodological approach:

  1. Analyze the codon usage bias in highly expressed L. plantarum genes

  2. Use codon optimization software to adapt the rplM sequence accordingly

  3. Consider GC content and avoid rare codons

  4. Remove potential mRNA secondary structures that might impede translation

  5. Synthesize the optimized gene commercially

  Research has shown that proper codon optimization can significantly increase expression levels of recombinant proteins in L. plantarum .

Advanced Research Questions

• How does signal peptide selection affect the expression efficiency of recombinant proteins in L. plantarum?

  Signal peptide selection dramatically impacts both expression levels and secretion efficiency of recombinant proteins in L. plantarum. Research comparing different signal peptides for α-amylase expression showed:

  Signal Peptide	Total Activity (kU/L)	Extracellular Activity (kU/L)	Secretion Efficiency (%)
  Lp_2145	13.1	8.1	Lowest
  Lp_0373	Lower than Lp_2145	Lower than Lp_2145	Highest
  SP_AmyL (native)	2.1 (6.2-fold lower)	1.5 (5.4-fold lower)	Medium

  Interestingly, the signal peptide affecting the highest total expression (Lp_2145) was not the most efficient for secretion. RT-qPCR analysis revealed that different signal peptides led to varying mRNA levels, suggesting that the choice of signal peptide affects transcription, not just secretion .

• What reference genes should be used for RT-qPCR studies of rplM expression in L. plantarum?

  For accurate RT-qPCR analysis of rplM expression in L. plantarum, appropriate reference genes must be carefully selected. Research has identified the following stable reference genes during exponential growth phase:

  • gmk (guanylate kinase)

  • gyrA (DNA gyrase subunit A)

  • gapB (glyceraldehyde-3-phosphate dehydrogenase)

  The methodology for reference gene validation involves:

  1. Testing multiple candidate reference genes under experimental conditions

  2. Evaluating expression stability using software tools such as GeNorm, BestKeeper, and NormFinder

  3. Selecting a combination of at least three stable reference genes

  This approach ensures accuracy in quantitative expression studies, as housekeeping gene expression can vary with environmental conditions or experimental treatments .

• How can transcriptome analysis be used to understand rplM regulation in L. plantarum?

  Transcriptome analysis provides valuable insights into rplM regulation in L. plantarum under various conditions. The methodological approach includes:

  1. Design and production of a full genome amplicon-based microarray

  2. RNA extraction from cells grown under different conditions

  3. cDNA synthesis, labeling, and hybridization

  4. Data analysis and identification of differentially expressed genes

  5. Validation of key findings using RT-qPCR

  This approach has revealed that sigma factors play a crucial role in transcriptional regulation in L. plantarum. While rplM is likely regulated by the housekeeping sigma factor σA, its expression may also be influenced by global regulators like CcpA under different carbon source conditions .

• What is the impact of oxidative stress on ribosomal protein expression in L. plantarum?

  Oxidative stress significantly impacts ribosomal protein expression in L. plantarum. Research comparing aerobic versus anaerobic growth revealed:

  1. Aerobic growth causes oxidative stress that alters global gene expression patterns

  2. Growth stagnation observed under aerobic conditions is linked to specific transcriptional responses

  3. Metabolic adjustments occur to cope with oxidative stress

  While specific data for rplM is limited, the methodological approach to study this involves:

  1. Culturing L. plantarum under aerobic and anaerobic conditions

  2. RNA extraction at various growth phases

  3. Transcriptome analysis using microarrays or RNA-seq

  4. Validation of expression changes using RT-qPCR

  5. Protein level confirmation using Western blotting

• How can recombinant L. plantarum expressing heterologous proteins be evaluated for immune responses?

  Evaluation of immune responses to recombinant L. plantarum expressing heterologous proteins requires a comprehensive methodological approach:

  1. In vitro assessment:

     • Dendritic cell activation assays

     • Cytokine production measurement

     • T cell proliferation assays

  2. In vivo assessment:

     • Oral administration to mice

     • Analysis of cellular responses:

       • CD4+IFN-γ+ and CD8+IFN-γ+ T cells in spleen and mesenteric lymph nodes

       • B220+IgA+ cells in Peyer's patches

     • Measurement of humoral responses:

       • Serum IgG, IgG1, and IgG2a antibodies

       • Mucosal IgA in intestine and lungs

       • Fecal IgA

  3. Functional assays:

     • Hemagglutination inhibition tests

     • Neutralization assays

     • Challenge studies

  These methodologies have demonstrated that recombinant L. plantarum can effectively induce both systemic and mucosal immune responses .

• What methods can be used to verify surface display of recombinant proteins on L. plantarum?

  Verification of surface display of recombinant proteins on L. plantarum requires multiple complementary techniques:

  1. Western blotting:

     • Cell fractionation to separate membrane proteins

     • Detection using specific antibodies

  2. Flow cytometry:

     • Incubation of intact bacteria with specific antibodies

     • Analysis of fluorescence intensity

  3. Immunofluorescence microscopy:

     • Visualization of surface-displayed proteins using fluorescently labeled antibodies

  4. Enzymatic activity assays (for enzymes):

     • Testing activity of whole cells versus cell lysates

     • Comparing accessibility to substrates

  Research has successfully used these methods to confirm surface display of various proteins, including antigens like HA1 from influenza virus, using different anchoring systems such as pgsA from Bacillus subtilis .

• How does the gut microbiota impact the effectiveness of recombinant L. plantarum as a delivery vehicle?

  The interaction between recombinant L. plantarum and the gut microbiota is bidirectional and complex. Research methodologies to study this relationship include:

  1. Microbiome analysis:

     • 16S rRNA gene sequencing before and after administration

     • Analysis of alpha diversity (Shannon-Wiener index)

     • Beta diversity analysis to assess structural changes

  2. Functional analysis:

     • Metagenomic prediction of microbial functions

     • Metabolomic analysis of gut contents

  3. Immunological assessment:

     • Correlation between microbiota changes and immune responses

     • Analysis of immune cells in gut-associated lymphoid tissues

  Studies have shown that recombinant L. plantarum can modulate the gut microbiota, increasing bacterial diversity and enhancing functions related to metabolism and immune regulation. These changes correlate with improved immune responses, including increased levels of IgG and IgA antibodies and enhanced T and B cell responses .

Experimental Design Questions

• What controls should be included when evaluating recombinant L. plantarum expressing rplM?

  Proper experimental design for evaluating recombinant L. plantarum expressing rplM should include the following controls:

  1. Empty vector control: L. plantarum containing the expression vector without the rplM gene

  2. Wild-type strain: Unmodified L. plantarum

  3. Non-recombinant control: L. plantarum expressing a non-relevant protein

  4. Positive control: Purified rplM protein (if available)

  5. Technical controls: For each assay method

  Research has demonstrated the importance of these controls, particularly the empty vector control (e.g., NC8Δ-pWCF), which provides essential background for understanding the specific effects of the recombinant protein versus those of the vector backbone and expression system .

• How can RNA isolation be optimized from L. plantarum for transcriptomic studies of ribosomal proteins?

  Optimization of RNA isolation from L. plantarum for transcriptomic studies requires a specific methodological approach:

  1. Sample collection:

     • Harvest cells at precise growth phases (e.g., OD600 of 0.3 for exponential phase)

     • Immediately stabilize RNA using RNA protect reagent

     • Flash freeze in liquid nitrogen

  2. Cell lysis:

     • Enzymatic treatment with lysozyme

     • Mechanical disruption (e.g., bead beating)

     • Combined approaches for efficient lysis of gram-positive cell wall

  3. RNA purification:

     • Column-based purification with on-column DNase treatment

     • Phenol-chloroform extraction followed by DNase treatment

     • Assessment of RNA integrity using bioanalyzer

  4. Quality control:

     • RNA integrity number (RIN) > 8

     • A260/A280 ratio ≈ 2.0

     • A260/A230 ratio > 1.8

     • Absence of genomic DNA contamination verified by PCR

  This methodology has been successfully applied in transcriptomic studies of L. plantarum under various conditions .