Recombinant Lactobacillus plantarum 30S ribosomal protein S7 (rpsG)

What is the function of 30S ribosomal protein S7 (rpsG) in bacteria?

The 30S ribosomal protein S7 (rpsG) is a crucial component of the bacterial 30S ribosomal subunit. It functions as one of the primary rRNA binding proteins that directly binds to 16S rRNA, where it nucleates the assembly of the ribosomal head domain. This protein is strategically located at the subunit interface close to the decoding center, where it interacts with mRNA and contacts tRNA in both the P and E sites. Functionally, it may block the exit of the E site tRNA and plays an essential role in accurate protein synthesis by ensuring proper codon-anticodon recognition .

How does Lactiplantibacillus plantarum differ from other lactobacilli as a recombinant protein expression system?

Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) stands out among lactobacilli as a robust microbial chassis for synthetic biology applications. It possesses several advantages:

• Cross-genera compatibility: Genetic parts developed in L. plantarum have demonstrated functionality across other lactobacilli genera and species, making it a gateway host for developing widely applicable genetic tools .

• Versatile colonization: L. plantarum can colonize various environments including the gastrointestinal tract of humans and animals, fermented foods, and plant surfaces, providing flexibility in application contexts .

• Enhanced stability: L. plantarum exhibits better stress tolerance compared to other lactobacilli, allowing for improved survival during research applications and therapeutic delivery .

• Established genetic tools: While still limited compared to model organisms like E. coli, L. plantarum has a growing toolkit of promoters, expression vectors (e.g., pSIP series), and surface display systems that facilitate recombinant protein expression .

What are the current methods for genetic transformation of L. plantarum for rpsG expression?

For genetic transformation of L. plantarum to express recombinant rpsG, researchers typically employ electroporation as the primary method. The procedure involves:

• Preparation of competent cells: Growing L. plantarum (e.g., strain WCFS1 or NC8) to mid-log phase, washing cells with decreasing concentrations of sucrose or glycerol buffers to make them electrocompetent .

• Transformation: Mixing purified plasmid DNA (containing the rpsG gene and necessary regulatory elements) with competent cells, followed by electric pulse application (~2.0 kV, 25 μF, 200 Ω) .

• Recovery and selection: Immediate incubation in MRS medium supplemented with specific sugars (often without antibiotics) for 2-3 hours, followed by plating on selective media containing appropriate antibiotics (commonly erythromycin for pSIP vector systems) .

• Confirmation: Verifying successful transformation through colony PCR, restriction digestion of extracted plasmids, and sequencing to confirm the integrity of the rpsG coding sequence .

What promoter systems are most effective for rpsG expression in L. plantarum?

Several promoter systems have been evaluated for recombinant protein expression in L. plantarum, with varying efficacy for potential rpsG expression:

The recently discovered phage-derived promoter system shows exceptional promise, achieving expression levels nearly 9-fold higher than previous systems, with the added advantage of almost complete repression (500-fold reduction) when combined with its cognate repressor .

How can codon optimization improve rpsG expression in L. plantarum?

Codon optimization for rpsG expression in L. plantarum is crucial due to the organism's distinct codon usage bias. Methodologically, this involves:

• Analysis of codon usage patterns in highly expressed L. plantarum genes compared to the native rpsG sequence.

• Substitution of rare codons with those preferred by L. plantarum while maintaining the amino acid sequence. This typically involves increasing the GC content as L. plantarum has a relatively high GC content compared to other lactobacilli.

• Elimination of secondary structures in the mRNA (especially near the ribosome binding site) that might impede translation initiation.

• Removal of internal Shine-Dalgarno-like sequences, potential premature termination signals, and restriction sites that could interfere with cloning procedures.

Researchers have reported 2-5 fold increases in heterologous protein yield following codon optimization in L. plantarum . For rpsG specifically, optimizing the regions encoding the rRNA binding domains can be particularly beneficial for ensuring proper folding and function.

What fusion tags and secretion signals are most appropriate for purification and localization of recombinant rpsG in L. plantarum?

For optimal purification and localization of recombinant rpsG in L. plantarum, the following systems have proven effective:

Fusion Tags for Purification:

Secretion and Localization Signals:

• pgsA' anchoring system: The N-terminal transmembrane region (26-42 amino acid residues) of the pgsA protein has been successfully used to anchor recombinant proteins to the L. plantarum cell membrane and wall. This system has been demonstrated in the NC8-pSIP409-pgsA'-S-DCpep construct, which effectively displayed the S protein on the bacterial surface .

• USP45 signal sequence: Derived from Lactococcus lactis, this secretion signal has been optimized for efficient protein secretion in L. plantarum.

• Lp_0373 signal peptide: A native L. plantarum signal that can achieve secretion yields of up to 10 mg/L of recombinant protein.

For rpsG specifically, cell surface display using the pgsA' system might be preferable when studying interactions with host immune cells, while cytoplasmic expression with a simple purification tag would be more suitable for structural studies .

How can recombinant L. plantarum expressing rpsG be used to study ribosomal assembly mechanisms?

Recombinant L. plantarum expressing modified or tagged rpsG variants provides a valuable model system for studying ribosomal assembly mechanisms through several methodological approaches:

• In vivo assembly monitoring: By expressing fluorescently-tagged rpsG in L. plantarum, researchers can track the spatiotemporal dynamics of 30S subunit assembly in real-time using advanced microscopy techniques.

• Assembly defect analysis: Systematic mutagenesis of rpsG residues followed by expression in L. plantarum can identify critical amino acids required for proper 30S subunit assembly. This can be quantified by measuring growth rates, translation efficiency, and ribosome profiling of the recombinant strains.

• Interaction partner identification: Tagged rpsG variants can be used in pull-down assays or crosslinking studies to identify novel interaction partners within the ribosomal assembly pathway specific to lactobacilli.

• Comparative assembly studies: Expression of heterologous rpsG proteins (e.g., from E. coli) in L. plantarum can reveal species-specific differences in ribosome assembly pathways, providing insights into evolutionary conservation and divergence of this fundamental process .

The experimental advantage of using L. plantarum lies in its genetic tractability combined with its distinct phylogenetic position compared to model organisms like E. coli, offering new perspectives on ribosomal biology.

What immunomodulatory effects can be expected when using recombinant L. plantarum expressing rpsG as a vaccine delivery system?

Recombinant L. plantarum expressing rpsG as part of a vaccine delivery system can elicit several specific immunomodulatory effects:

Adaptive Immune Responses:

• Enhanced B cell responses: Recombinant L. plantarum strains have been shown to increase the number of IgA+B220+ B cells, suggesting that rpsG-expressing strains could promote mucosal antibody production .

• Elevated antibody production: Similar recombinant L. plantarum constructs (e.g., NC8-pSIP409-pgsA'-S-DCpep) have demonstrated significant increases in both systemic IgG and mucosal sIgA antibody levels in mouse models .

• T cell activation: Expression of specific cytokines including IFN-γ (Th1), IL-4 (Th2), and IL-17A (Th17) has been observed to increase in mesenteric lymph nodes of mice treated with recombinant L. plantarum, indicating balanced T cell responses that could similarly occur with rpsG-expressing strains .

Innate Immune Effects:

• Dendritic cell activation: Recombinant L. plantarum targeting DC-specific receptors (e.g., through DCpep fusion) has been demonstrated to promote dendritic cell activation in the lamina propria, which could enhance antigen presentation of rpsG to adaptive immune cells .

• Altered microbiota composition: Recombinant L. plantarum can significantly influence gut microbiota diversity, potentially creating a more favorable environment for immune responses.

These immunomodulatory effects position rpsG-expressing L. plantarum as a promising candidate for mucosal vaccines, particularly for targeting infections at mucosal surfaces .

What are the key considerations for maintaining genetic stability of recombinant L. plantarum expressing rpsG?

Maintaining genetic stability of recombinant L. plantarum expressing rpsG requires addressing several critical factors:

• Plasmid segregational stability:

  • Challenge: Plasmid loss during continuous culture, especially without selective pressure.

  • Solution: Implement post-segregational killing systems (e.g., toxin-antitoxin pairs) or auxotrophy complementation on expression vectors. Monitor stability by plating on selective vs. non-selective media at regular intervals.

• Structural stability of the rpsG expression cassette:

  • Challenge: Recombination events or mutations due to metabolic burden.

  • Solution: Minimize direct repeats in constructs, use low-copy plasmids or integrate the expression cassette into chromosomal loci using CRISPR-Cas9 systems adapted for L. plantarum.

• Codon optimization considerations:

  • Challenge: Codon optimization might create sequences with unintended secondary structures or regulatory elements.

  • Solution: Perform in silico analysis for potential regulatory elements and RNA secondary structures before synthesis. Test multiple variants if resources permit.

• Metabolic burden management:

  • Challenge: Overexpression of ribosomal proteins can disrupt normal ribosome biogenesis.

  • Solution: Use inducible systems like the phage-derived promoter/repressor system or the pSIP system to control expression levels. The phage system's 500-fold repression capability is particularly valuable for tight regulation .

• Storage and revival protocols:

  • Challenge: Loss of expression during freeze-thaw cycles.

  • Solution: Store working aliquots at 4°C for up to one week and avoid repeated freeze-thaw cycles as recommended for similar recombinant proteins.

How can researchers troubleshoot low expression levels of recombinant rpsG in L. plantarum?

When facing low expression levels of recombinant rpsG in L. plantarum, researchers can implement a systematic troubleshooting approach:

Molecular-Level Troubleshooting:

Protocol Optimization:

• Culture conditions: Optimize growth temperature (usually 30°C rather than 37°C for L. plantarum), media composition (MRS media supplemented with specific carbon sources), and induction timing for inducible systems .

• Induction parameters: For the pSIP system, carefully titrate inducer peptide concentrations. Previous work achieved 1800 Miller Unit equivalents of reporter enzyme using optimized induction conditions .

• Extraction methods: For rpsG, which may associate with the ribosome, use specialized extraction buffers containing high salt concentrations (500 mM NaCl) to dissociate the protein from rRNA.

• Detection optimization: Implement sensitive detection methods like immunoblotting with specific antibodies or MS/MS proteomic analysis when expression levels are below standard detection limits.

• Alternative fusion strategies: Test different fusion partners known to enhance solubility and expression, such as thioredoxin or SUMO tags.

How can structural modifications of rpsG in L. plantarum be leveraged to create improved ribosomal targeting antimicrobials?

Leveraging structural modifications of rpsG in L. plantarum to develop improved ribosomal-targeting antimicrobials involves sophisticated protein engineering approaches:

• Structure-guided modification strategy:

  • Map the binding interfaces between rpsG and 16S rRNA in L. plantarum using crosslinking studies similar to those performed in E. coli .

  • Identify residues that differ between pathogenic bacteria and beneficial lactobacilli like L. plantarum.

  • Engineer modified rpsG variants with selective binding capabilities that could compete with native rpsG in pathogens but not in probiotics.

• Chimeric protein development:

  • Create fusion proteins between rpsG and known antimicrobial peptides, positioning the antimicrobial domain to interact with pathogen-specific ribosomal features.

  • Express these chimeric constructs in L. plantarum as a delivery vehicle that can release the modified rpsG upon reaching infection sites.

• Validation methodology:

  • Test the selectivity of engineered rpsG variants using in vitro translation systems derived from both pathogenic bacteria and beneficial microbes.

  • Measure minimum inhibitory concentrations (MICs) against pathogen panels while confirming lack of activity against commensal bacteria.

  • Evaluate in vivo efficacy in infection models using recombinant L. plantarum expressing the engineered rpsG as the delivery system.

This approach could potentially overcome antibiotic resistance by targeting highly conserved ribosomal components while maintaining selectivity through structure-based design .

What are the methodological considerations for using rpsG as a reporter protein in L. plantarum synthetic biology circuits?

Using rpsG as a reporter protein in L. plantarum synthetic biology circuits requires careful methodological considerations:

Design Considerations:

• Promoter-reporter pairing: The newly identified phage-derived promoter system showing 9-fold higher expression than previous systems would provide excellent sensitivity for rpsG reporter constructs .

• Dynamic range calibration: The repressible nature of the phage promoter (500-fold repression capability) enables creation of tunable circuits with wide dynamic ranges .

• Readout mechanism design: Since rpsG lacks intrinsic fluorescence or enzymatic activity, it requires fusion with detection domains or innovative readout mechanisms:

  • Fusion with split fluorescent proteins where functional complementation depends on rpsG expression levels

  • Development of ribosome assembly-dependent reporters where rpsG incorporation into ribosomes triggers a detectable signal

Experimental Implementation:

Performance Assessment:

• Characterize circuit transfer functions by measuring rpsG reporter output across a range of input concentrations.

• Evaluate circuit robustness through perturbation analysis, testing performance across various growth conditions.

• Assess orthogonality by testing for crosstalk with endogenous L. plantarum regulatory networks, particularly those involved in ribosome biogenesis.

This approach could leverage the fundamental role of rpsG in ribosome assembly to create novel synthetic biology applications in L. plantarum .

What statistical approaches are most appropriate for analyzing immunological responses to recombinant L. plantarum expressing rpsG?

When analyzing immunological responses to recombinant L. plantarum expressing rpsG, several statistical approaches are recommended based on experimental design and data characteristics:

For Antibody Titer Analysis:

For Cellular Immune Responses:

• Multivariate analysis: Use principal component analysis (PCA) or partial least squares discriminant analysis (PLS-DA) to identify patterns across multiple cytokines (IFN-γ, IL-4, IL-17A) simultaneously .

• Non-parametric tests: For flow cytometry data (e.g., DC activation, B cell responses), use non-parametric tests like Mann-Whitney U when sample sizes are small or distributions are skewed.

• Correction for multiple comparisons: When analyzing multiple immune parameters, implement Benjamini-Hochberg false discovery rate or Bonferroni correction to control for Type I errors.

For Microbiome Interaction Analysis:

This multi-layered statistical approach allows for robust interpretation of the complex immunological data generated in studies of recombinant L. plantarum expressing rpsG .

How can researchers distinguish between immune responses triggered by the rpsG protein versus the L. plantarum vehicle?

Distinguishing between immune responses triggered by the rpsG protein versus the L. plantarum vehicle requires carefully designed experimental controls and analytical methods:

Experimental Design Controls:

Analytical Approaches:

• Epitope mapping: Perform epitope mapping studies using peptide arrays or phage display to identify specific rpsG epitopes recognized by antibodies from immunized subjects. Compare these to known L. plantarum epitopes.

• Adoptive transfer experiments: Transfer T cells from immunized animals to naive recipients and challenge with either purified rpsG or L. plantarum alone to determine specificity of the cellular response.

• In vitro stimulation assays: Isolate immune cells from immunized subjects and measure proliferation and cytokine responses upon re-stimulation with purified rpsG versus L. plantarum antigens.

• Competitive inhibition analysis: Pre-incubate serum from immunized animals with excess purified rpsG before testing reactivity against whole bacteria to determine what proportion of the antibody response is rpsG-specific.

• Cytokine signature analysis: Apply machine learning algorithms to cytokine profiles to identify patterns unique to rpsG-specific responses versus general L. plantarum responses.

This comprehensive approach allows researchers to deconvolute the complex immune responses elicited by recombinant L. plantarum expressing rpsG, enabling more precise vaccine design and development .

What emerging gene editing technologies could enhance the precision of rpsG modifications in L. plantarum?

Several emerging gene editing technologies show promise for enhancing the precision of rpsG modifications in L. plantarum:

CRISPR-Cas Systems Optimized for Lactobacilli:

• CRISPR-Cas9 with enhanced specificity: Newly engineered high-fidelity Cas9 variants (e.g., HiFi Cas9) could reduce off-target effects when modifying rpsG in its native chromosomal context. Implementation would involve:

  • Designing sgRNAs specific to the L. plantarum rpsG gene

  • Delivery via theta-replicating plasmids with temperature-sensitive origins

  • Screening for precise modifications using deep sequencing approaches

• Base editors for L. plantarum: Cytosine or adenine base editors fused to catalytically impaired Cas9 could introduce point mutations in rpsG without double-strand breaks, preserving ribosomal function while altering specific properties:

  • C→T or A→G conversions to modify key functional residues

  • No requirement for homology-directed repair, which is inefficient in L. plantarum

  • Target window optimization for the GC-rich coding sequences typical in L. plantarum

• Prime editing: This technology, which uses a fusion of Cas9 nickase with reverse transcriptase, could enable precise insertion of specific modifications in rpsG:

  • Installation of affinity tags within the rpsG sequence

  • Introduction of mutations affecting ribosome assembly without disrupting essential functions

  • Creation of conditional alleles through insertion of regulated protein degradation domains

Delivery and Selection Innovations:

These emerging technologies could facilitate the creation of rpsG variants with defined modifications to study ribosomal function and develop novel biotechnological applications in L. plantarum .

How might structural comparisons between rpsG proteins from different bacterial species inform the design of species-specific ribosomal targeting drugs?

Structural comparisons between rpsG proteins from different bacterial species can inform the design of species-specific ribosomal targeting drugs through several sophisticated approaches:

Comparative Structural Analysis Methodology:

• High-resolution structure determination:

  • Generate atomic-resolution structures of rpsG from multiple species including L. plantarum, pathogens, and human mitochondrial/cytoplasmic counterparts

  • Utilize cryo-EM to capture rpsG in the context of the assembled ribosome

  • Apply hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map dynamic regions

• Binding pocket analysis:

  • Identify species-specific pockets using computational solvent mapping

  • Calculate electrostatic potential surfaces to reveal charge distribution differences

  • Analyze sequence conservation patterns mapped onto structural elements

Drug Design Applications:

Validation and Development Pipeline:

• Use recombinant L. plantarum expressing various rpsG variants (wild-type, mutant, chimeric) to validate structural predictions.

• Implement fragment-based drug discovery approaches targeting identified species-specific pockets.

• Develop high-throughput screening assays based on ribosome assembly or function that can distinguish between effects on different bacterial species.

• Test candidate compounds against panels of bacteria representing diverse taxonomic groups to confirm selectivity profiles.