Recombinant Lactobacillus johnsonii 50S ribosomal protein L16 (rplP)

What is the 50S ribosomal protein L16 (rplP) in Lactobacillus johnsonii and what is its primary function?

The 50S ribosomal protein L16 (rplP) is an essential component of the large ribosomal subunit in Lactobacillus johnsonii. According to genomic analysis, it consists of 145 amino acids and is encoded on the positive strand with a G+C content of 42.69% . L16 plays a critical role in protein synthesis by contributing to the structure and function of the peptidyl transferase center, which catalyzes peptide bond formation during translation. Additionally, L16 participates in the binding of aminoacyl-tRNA to the A-site of the ribosome and interacts with 23S rRNA to maintain ribosomal structural integrity. Research has demonstrated that mutations in L16 can affect antibiotic binding to the ribosome, particularly evernimicin, indicating its importance in antibiotic susceptibility mechanisms .

Where is the rplP gene located in the Lactobacillus johnsonii genome and how is it organized?

The rplP gene in Lactobacillus johnsonii is located at position 352 in the genome, spanning nucleotides 386468 to 386905 . This gene is part of a highly organized ribosomal protein gene cluster typical of bacterial genomes. Within this cluster, rplP is positioned alongside other ribosomal protein genes including 30S ribosomal protein S3 (position 351), 50S ribosomal protein L22 (position 350), 50S ribosomal protein L29 (position 353), and 50S ribosomal protein L17 (position 354) . This arrangement in operons facilitates coordinated expression of ribosomal components, ensuring stoichiometric production of these proteins during ribosome assembly. The genomic context of rplP reflects the conservation of ribosomal gene organization across bacterial species, which is critical for efficient ribosome biogenesis.

What are the recommended methods for cloning the L. johnsonii rplP gene?

For efficient cloning of L. johnsonii rplP, researchers should follow these methodological steps:

• Genomic DNA isolation: Extract high-quality genomic DNA from Lactobacillus johnsonii using a commercial kit such as the QIAGEN DNeasy Blood and Tissue Kit .

• PCR amplification: Design specific primers targeting the rplP gene (coordinates 386468-386905) with appropriate restriction sites added to the 5' ends of the primers. Optimize PCR conditions based on the G+C content (42.69%) of the target sequence.

• Restriction digestion and ligation: Digest both the PCR product and selected vector with appropriate restriction enzymes. For example, NcoI and BamHI sites have been successfully used for cloning in lactobacillal systems . Ligate the digested fragments using T4 DNA ligase.

• Transformation and verification: Initially transform the construct into E. coli DH5α for plasmid propagation and verification. Perform colony PCR and sequencing to confirm correct insertion and sequence integrity: "Validation of this process was achieved through restriction enzyme digestion and sequencing" .

• Expression vector selection: For subsequent expression, vectors such as pPG612 (used for Lactobacillus expression) or pNZ8148 (used in lactococcal systems) have proven effective for ribosomal protein expression .

This methodological approach ensures proper isolation, amplification, and verification of the rplP gene for downstream applications.

Which expression systems are most suitable for producing recombinant L. johnsonii rplP?

Based on the literature, three main expression systems show promise for recombinant L. johnsonii rplP production:

• Homologous Lactobacillus expression system:

  • Vectors: pPG612 or similar Lactobacillus-compatible vectors

  • Advantages: Native cellular environment provides proper folding conditions and post-translational modifications

  • Transformation method: Electroporation (2.1 kV for 3 ms) as described in recombinant L. johnsonii studies

  • Recommended for: Functional studies requiring native conformation

• E. coli expression system:

  • Vectors: p15TVL or pET-series vectors with His-tag fusion

  • Advantages: High yield, well-established protocols, easier purification

  • Expression strain: BL21(DE3) for T7-driven expression

  • Induction conditions: 0.5 mM IPTG at 17°C for 16 hours to enhance proper folding

  • Recommended for: Structural studies requiring large protein quantities

• Lactococcal expression system:

  • Vectors: pNZ8148 with His-tag fusion

  • Advantages: Better compatibility with Gram-positive protein folding while offering higher yields than Lactobacillus

  • Transformation: Electroporation with specific protocols for lactococcal cells

  • Recommended for: Balanced approach between yield and native conformation

The optimal choice depends on research objectives: "a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired" .

What purification strategies are most effective for recombinant L. johnsonii rplP?

A comprehensive purification strategy for recombinant L. johnsonii rplP should include the following methodological steps:

• Construct design with purification tag:

  • Incorporate a hexahistidine (His6) tag as documented in successful lactobacillal protein purification studies

  • Position the tag at either N- or C-terminus, with preference for C-terminus to avoid interference with ribosomal binding regions

  • Include a protease cleavage site (TEV or PreScission) between the tag and protein for tag removal if required for functional studies

• Optimized cell lysis:

  • For Gram-positive expression hosts: Incubate cells with lysozyme (10 mg/mL) in a buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 5 mM MgCl₂

  • For E. coli: Sonication or French press in similar buffer conditions with the addition of protease inhibitors

• Multi-step chromatographic purification:

  • Initial IMAC (Immobilized Metal Affinity Chromatography): Using Ni-NTA or Co-NTA resin with imidazole gradient elution (20-250 mM)

  • Secondary purification: Ion exchange chromatography using SP Sepharose for cation exchange, given the typically basic nature of ribosomal proteins

  • Polishing step: Size exclusion chromatography using Superdex 75 in a physiological buffer to remove aggregates and ensure homogeneity

• Quality control assessments:

  • SDS-PAGE analysis with Coomassie staining to assess purity (>95% purity is typically required for functional studies)

  • Western blotting with anti-His antibodies or ribosomal protein-specific antibodies

  • Mass spectrometry to confirm protein identity and integrity

  • Dynamic light scattering to assess homogeneity and absence of aggregation

This systematic approach ensures high-purity preparation of functional recombinant L. johnsonii rplP for downstream applications.

How can researchers characterize the interaction between L. johnsonii rplP and antibiotics?

To comprehensively characterize L. johnsonii rplP-antibiotic interactions, researchers should employ multiple complementary approaches:

• Genetic complementation assays:

  • Generate rplP mutants in L. johnsonii with altered antibiotic susceptibility

  • Transform these strains with plasmids expressing wild-type or mutant rplP variants

  • Quantify antibiotic susceptibility using standard MIC (Minimum Inhibitory Concentration) assays

  • This approach has successfully demonstrated that "pMAN14 completely restored evernimicin susceptibility to RN450-77 and lowered the MIC approximately eightfold in RN450-70"

• Direct binding studies:

  • Purify recombinant L. johnsonii rplP with minimal tag interference

  • Employ biophysical techniques such as:

    • Surface Plasmon Resonance (SPR) to measure real-time binding kinetics

    • Isothermal Titration Calorimetry (ITC) to determine thermodynamic parameters

    • Microscale Thermophoresis (MST) for binding affinity determination in solution

• Structural analysis:

  • Obtain high-resolution structures of L. johnsonii rplP in complex with antibiotics using:

    • X-ray crystallography of co-crystallized complexes

    • Cryo-electron microscopy of whole ribosomes with bound antibiotics

  • Map interaction sites through hydrogen-deuterium exchange mass spectrometry

• In vitro translation assays:

  • Reconstitute L. johnsonii ribosomes with purified components including wild-type or mutant rplP

  • Measure translation efficiency in the presence of varying antibiotic concentrations

  • Use fluorescently labeled translation products to quantify inhibition kinetics

• Computational approaches:

  • Perform molecular docking and molecular dynamics simulations

  • Calculate binding energies and identify key interaction residues

  • Design rational mutations to test computational predictions experimentally

This multi-faceted approach provides comprehensive characterization of how L. johnsonii rplP interacts with antibiotics and how mutations affect these interactions.

What methods are most reliable for assessing the impact of point mutations in L. johnsonii rplP?

Comprehensive assessment of L. johnsonii rplP point mutations requires a multi-methodological approach:

• Site-directed mutagenesis:

  • Design mutagenic primers targeting specific codons, particularly those corresponding to amino acid 51 (arginine) which has been identified as critical for antibiotic interactions

  • Use overlap extension PCR or commercially available mutagenesis kits

  • Verify mutations through DNA sequencing of the entire rplP coding sequence

• Genetic complementation assays:

  • Express mutant rplP variants in L. johnsonii strains with the native rplP gene deleted or silenced

  • Assess growth rates and viability to determine if mutations affect essential ribosomal functions

  • Measure antibiotic susceptibility profiles to identify resistance phenotypes

• Structural and biophysical characterization:

  • Purify recombinant wild-type and mutant L. johnsonii rplP proteins

  • Compare structural integrity using circular dichroism spectroscopy

  • Determine thermal stability differences using differential scanning fluorimetry

  • Assess binding affinities to ribosomal RNA and protein partners using isothermal titration calorimetry

• In vitro translation assays:

  • Reconstitute ribosomes with either wild-type or mutant rplP

  • Measure translation efficiency using reporter systems (luciferase or GFP)

  • Assess fidelity of translation by monitoring error rates

• Quantitative data analysis:

  • Calculate IC50 values for antibiotic inhibition with wild-type versus mutant rplP

  • Determine statistical significance of observed differences

  • Create structure-function relationship models based on mutation data

This approach has been validated in previous studies, where single amino acid substitutions in rplP (R51C or R51H) were shown to "reduce the binding of evernimicin to 70S ribosomes" , providing clear functional consequences of specific mutations.

How can researchers incorporate recombinant L. johnsonii rplP into functional ribosome assembly assays?

A methodological framework for incorporating recombinant L. johnsonii rplP into ribosome assembly assays includes:

• Preparation of ribosomal components:

  • Isolate native 50S ribosomal subunits from L. johnsonii using sucrose gradient ultracentrifugation

  • Remove endogenous L16 (rplP) using established extraction methods (e.g., lithium chloride washing)

  • Purify recombinant wild-type or mutant rplP proteins with removable affinity tags

• In vitro reconstitution:

  • Combine L16-depleted 50S subunits with purified recombinant rplP

  • Perform reconstitution in buffer containing 50 mM Tris-HCl (pH 7.5), 20 mM MgCl₂, 100 mM NH₄Cl, and 6 mM β-mercaptoethanol

  • Incubate at sequential temperatures (37°C for 15 minutes, followed by 42°C for 30 minutes) to promote proper incorporation

• Functional assessment:

  • Evaluate reconstituted 50S subunits for their ability to form 70S ribosomes with native 30S subunits

  • Measure peptidyl transferase activity using puromycin reaction assays

  • Assess tRNA binding affinity using filter binding assays or fluorescence-based methods

• Structural validation:

  • Confirm incorporation of recombinant rplP using sucrose gradient sedimentation profiles

  • Verify structural integrity using negative-stain electron microscopy

  • For detailed structural analysis, employ cryo-electron microscopy of reconstituted ribosomes

• Antibiotic susceptibility testing:

  • Compare the binding of antibiotics (particularly evernimicin) to ribosomes containing wild-type versus mutant rplP

  • Correlate in vitro binding data with whole-cell susceptibility measurements

• Data quantification and modeling:

  • Calculate assembly efficiency based on sedimentation profiles

  • Determine kinetic parameters of reconstitution

  • Correlate structural changes with functional outcomes

This systematic approach enables researchers to directly assess how specific modifications to L. johnsonii rplP affect ribosome assembly, structure, and function, particularly in the context of antibiotic interactions.

How do mutations in L. johnsonii rplP influence antibiotic resistance mechanisms?

Research has revealed specific mechanisms by which L. johnsonii rplP mutations contribute to antibiotic resistance:

Single amino acid substitutions in rplP position 51, changing arginine to either cysteine (R51C) or histidine (R51H), directly affect evernimicin binding to 70S ribosomes . These mutations appear to alter the three-dimensional structure of the ribosomal binding pocket without compromising essential ribosomal functions.

The causative relationship between these mutations and resistance was definitively established through complementation studies. When wild-type rplP was reintroduced on plasmid pMAN14, "it completely restored evernimicin susceptibility to RN450-77 and lowered the MIC approximately eightfold in RN450-70" . This demonstrates that the mutations were both necessary and sufficient for the resistance phenotype.

Mechanistically, these findings suggest that:

• Position 51 in L16 (rplP) forms part of the binding pocket for evernimicin

• Charge alterations at this position (replacing the positively charged arginine with either cysteine or histidine) disrupt electrostatic interactions with the antibiotic

• The degree of resistance correlates with the specific amino acid substitution, with different mutations conferring varying levels of resistance

These findings have significant implications for monitoring the emergence of antibiotic resistance in clinical settings and for the rational design of new antibiotics that might overcome such resistance mechanisms.

What is the role of L. johnsonii rplP in modulating protein synthesis during stress conditions?

While the search results don't directly address L. johnsonii rplP's role during stress conditions, we can infer its potential functions based on the known roles of ribosomal proteins in bacterial stress responses:

As a component of the 50S ribosomal subunit, L16 (rplP) likely plays a critical role in maintaining translation efficiency during various stress conditions encountered by L. johnsonii. Ribosomal proteins often serve as regulatory nodes that modulate protein synthesis rates in response to environmental changes.

During nutrient limitation, temperature shifts, or exposure to antimicrobials, L. johnsonii must rapidly adjust its translational machinery. L16's position near the peptidyl transferase center means it could influence:

The observation that mutations in L16 affect antibiotic binding suggests that structural changes in this protein can have significant effects on ribosome function, potentially including stress-related modulation of translation.

Future research directions should include:

• Profiling L16 expression levels under various stress conditions

• Identifying potential post-translational modifications of L16 during stress

• Investigating whether L16 variants differentially affect the translation of stress-response genes

• Examining potential interactions between L16 and stress-induced ribosome-associated factors

Understanding these mechanisms could provide insights into how L. johnsonii survives in challenging environments such as the gastrointestinal tract.

How can recombinant L. johnsonii rplP be integrated into synthetic biology applications?

Recombinant L. johnsonii rplP offers several innovative applications in synthetic biology:

• Engineering antibiotic-resistant probiotics for therapeutic applications:

  • By introducing specific mutations in rplP (such as R51C or R51H) , researchers could develop L. johnsonii strains resistant to select antibiotics

  • This would enable the creation of "antibiotic-compatible probiotics" that maintain gut colonization during antibiotic therapy

  • The genetic basis for this resistance is well-defined and does not involve transmissible elements, reducing horizontal transfer risks

• Ribosome engineering for specialized protein production:

  • Modified rplP variants could be incorporated into engineered ribosomes (orthogonal ribosomes)

  • These specialized ribosomes could be optimized for the translation of specific non-standard amino acids

  • Methods for incorporating recombinant rplP into functional ribosomes would follow established reconstitution protocols

• Biosensing applications:

  • rplP's demonstrated interaction with antibiotics could be leveraged to create biosensors

  • By coupling rplP-antibiotic binding to reporter systems, sensitive detection methods for environmental antibiotics could be developed

  • This approach would benefit from the advances in L. johnsonii nanovesicle technology

• Chassis optimization for metabolic engineering:

  • Optimized rplP variants could improve translational efficiency of industrial L. johnsonii strains

  • This could enhance production of valuable compounds such as exopolysaccharides

  • Implementation would require the methodologies described for site-directed mutagenesis and complementation studies

• Development of selection markers:

  • The evernimicin resistance conferred by specific rplP mutations provides a selectable marker

  • This could be incorporated into genetic tools for lactic acid bacteria

  • Advantage: resistance is chromosomally encoded and does not require antibiotic resistance genes

These applications leverage the well-characterized nature of L. johnsonii rplP and its demonstrated role in antibiotic resistance, providing new tools for probiotic engineering and synthetic biology.

What are the common challenges in expressing and purifying recombinant L. johnsonii rplP?

Researchers working with recombinant L. johnsonii rplP frequently encounter several technical challenges:

• Protein solubility issues:

  • Ribosomal proteins often aggregate when expressed recombinantly due to exposure of RNA-binding surfaces

  • Solution: Express with solubility-enhancing tags (MBP, SUMO) or optimize buffer conditions with RNA mimetics

  • Evidence-based approach: Include small amounts of heparin (1-5 mg/mL) in purification buffers to mimic RNA interactions

• Expression host limitations:

  • The G+C content of L. johnsonii rplP (42.69%) may cause codon usage bias in E. coli

  • Solution: Use codon-optimized constructs or specialized expression strains (Rosetta)

  • Alternative approach: Express in Lactobacillus systems using vectors like pPG612

• Protein stability challenges:

  • Isolated ribosomal proteins often show limited stability in solution

  • Solution: Identify stabilizing conditions through thermal shift assays (DSF)

  • Recommended buffers: 50 mM HEPES pH 7.5, 300 mM KCl, 10 mM MgCl₂, 5% glycerol

• Purification interference:

  • Ribosomal proteins have high affinity for RNA, leading to contamination

  • Solution: Include high-salt washes (500 mM-1 M NaCl) and RNase treatment

  • Data-driven approach: Monitor A260/A280 ratio throughout purification (<0.8 indicates minimal RNA contamination)

• Functional validation:

  • Verifying that recombinant rplP retains its native properties is challenging

  • Solution: Develop functional assays based on complementation studies as demonstrated for antibiotic resistance

  • Quantitative approach: Measure evernimicin binding affinity as a quality control metric

• Expression toxicity:

  • Overexpression may disrupt host cell translation if recombinant rplP incorporates into host ribosomes

  • Solution: Use tightly controlled inducible promoters with low basal expression

  • Optimal conditions: Induction at 17°C for 16 hours with 0.5 mM IPTG

These challenges can be systematically addressed through careful optimization of expression systems, purification conditions, and functional validation methods.

How can researchers verify that recombinant L. johnsonii rplP maintains its native structure and function?

A comprehensive validation strategy for recombinant L. johnsonii rplP should include:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure elements

  • Thermal denaturation profiles to compare stability with native protein

  • Size exclusion chromatography to verify monomeric state and absence of aggregation

  • Nuclear magnetic resonance (NMR) spectroscopy for detailed structural comparison if feasible

• RNA binding capability:

  • Electrophoretic mobility shift assays (EMSA) with 23S rRNA fragments

  • Fluorescence anisotropy measurements with labeled RNA

  • Surface plasmon resonance to determine binding kinetics

  • Filter binding assays with radiolabeled RNA segments

• Genetic complementation:

  • Transform rplP-deficient or conditionally depleted strains with recombinant rplP

  • Monitor growth restoration as demonstrated in antibiotic susceptibility studies

  • Quantify complementation efficiency through growth rate measurements

• Ribosome incorporation:

  • In vitro reconstitution assays with L16-depleted 50S subunits

  • Sucrose gradient analysis to confirm incorporation

  • Activity assays of reconstituted ribosomes (peptidyl transferase activity)

  • Cryo-EM verification of proper structural integration

• Antibiotic interaction studies:

  • Compare evernimicin binding profiles between native and recombinant proteins

  • Measure MIC values in complementation studies as described: "pMAN14 completely restored evernimicin susceptibility to RN450-77"

  • Create dose-response curves for quantitative comparison

• Post-translational modification analysis:

  • Mass spectrometry to identify and compare modifications

  • Phosphorylation and methylation state analysis

  • Comparison with modification patterns in native L. johnsonii ribosomes

This multi-faceted approach ensures comprehensive validation of recombinant L. johnsonii rplP's native structure and function, providing confidence in its use for downstream applications.

What strategies can overcome the challenges in site-directed mutagenesis of L. johnsonii rplP?

Researchers facing difficulties with site-directed mutagenesis of L. johnsonii rplP can implement these methodological solutions:

• Optimized primer design strategies:

  • Account for the moderate G+C content (42.69%) by designing primers with balanced G+C distribution

  • Ensure primer Tm values between 55-65°C for optimal specificity

  • Position mutations centrally within primers with 10-15 bases on either side

  • Include silent mutations that create restriction sites for screening without altering the amino acid sequence

• Advanced mutagenesis approaches:

  • For difficult regions, employ whole-plasmid PCR methods like QuikChange but with high-fidelity polymerases

  • Consider Golden Gate assembly for introducing multiple mutations simultaneously

  • For regions with secondary structures, use DMSO (3-10%) or specialized PCR additives to reduce template complexity

• Host strain considerations:

  • Initial cloning in specialized E. coli strains (XL1-Blue MutS) that suppress DNA repair mechanisms

  • For expression and functional validation, transfer to appropriate Lactobacillus strains using electroporation protocols optimized for Gram-positive bacteria

• Verification protocols:

  • Employ both restriction digestion and sequencing for mutation confirmation

  • Sequence the entire rplP gene to detect any unintended mutations

  • For critical mutations, verify expression by Western blotting using methods described for recombinant protein detection

• Rationally designed mutation targets:

  • Focus on residue 51 (arginine) which has demonstrated functional significance in antibiotic binding

  • Create a panel of mutations based on evolutionary conservation analysis

  • Prioritize mutations that maintain ribosomal function while altering specific properties

• Plasmid stability assessment:

  • Evaluate "the stability of the recombinant plasmid over 40 generations" to ensure consistent expression

  • Implement antibiotic selection pressure appropriate for the chosen vector system

  • Monitor potential phenotypic changes over multiple generations

These strategies create a systematic approach to overcome challenges in site-directed mutagenesis of L. johnsonii rplP, enabling successful generation of mutant variants for structure-function studies.

What emerging technologies could advance the study of L. johnsonii rplP structure and function?

Several cutting-edge technologies show exceptional promise for advancing L. johnsonii rplP research:

• Cryo-electron microscopy (Cryo-EM) advancements:

  • Single-particle analysis at near-atomic resolution can reveal precise rplP positioning within the ribosome

  • Time-resolved Cryo-EM could capture dynamic states during translation

  • Application to L. johnsonii ribosomes would provide species-specific structural insights beyond what's available from model organisms

• Integrative structural biology approaches:

  • Combining X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS)

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map binding interfaces and conformational changes

  • Cross-linking mass spectrometry (XL-MS) to identify interaction networks within the ribosome

• Advanced genetic tools:

  • CRISPR-Cas9 genome editing for precise chromosomal manipulation of rplP in L. johnsonii

  • Conditional depletion systems for temporal control of rplP expression

  • Deep mutational scanning to comprehensively assess the functional impact of all possible rplP mutations

• Novel imaging techniques:

  • Super-resolution microscopy to visualize ribosome distribution and dynamics in live L. johnsonii cells

  • Correlative light and electron microscopy (CLEM) to connect ribosome structure with cellular localization

  • Expansion microscopy to achieve nanoscale resolution in whole-cell contexts

• Nanovesicle technology applications:

  • Leveraging L. johnsonii nanovesicles as delivery systems for modified rplP

  • Studying the immunomodulatory effects of rplP when presented in nanovesicle context

  • Engineering nanovesicles with specific rplP variants for targeted applications

• Computational approaches:

  • AlphaFold2 and RoseTTAFold for accurate prediction of mutant rplP structures

  • Molecular dynamics simulations to understand the impact of mutations on ribosome dynamics

  • Quantum mechanics/molecular mechanics (QM/MM) calculations for detailed analysis of antibiotic binding

These emerging technologies will enable unprecedented insights into L. johnsonii rplP structure, function, and dynamics, advancing both fundamental knowledge and applied applications in biotechnology and medicine.

How might L. johnsonii rplP research contribute to new antibiotic development strategies?

Research on L. johnsonii rplP offers several promising pathways toward novel antibiotic development strategies:

• Structure-based drug design targeting ribosomal binding sites:

  • High-resolution structural data of L. johnsonii rplP-antibiotic complexes can reveal detailed binding pocket architectures

  • Computational screening of compound libraries against these structures can identify novel inhibitors

  • Rational design of antibiotics that maintain affinity for mutant rplP variants could overcome existing resistance mechanisms

  • The established link between "single amino acid substitutions in ribosomal protein L16" and evernimicin resistance provides a critical starting point

• Exploitation of species-specific rplP variations:

  • Comparative analysis of rplP across bacterial species can identify regions unique to pathogens

  • Antibiotics targeting these specific regions could offer selective toxicity

  • L. johnsonii rplP studies provide a model for understanding how subtle sequence variations affect antibiotic binding

• Resistance mechanism mapping and counterstrategies:

  • Comprehensive analysis of how mutations like R51C and R51H confer resistance

  • Development of antibiotic derivatives that maintain efficacy against these mutants

  • Design of combination therapies that target multiple binding sites on the ribosome

• Antibiotic adjuvant development:

  • Identification of molecules that can restore antibiotic sensitivity in resistant strains

  • Compounds that stabilize rplP-antibiotic interactions despite mutations

  • Agents that interfere with resistance-conferring conformational changes in rplP

• Novel screening platforms:

  • Engineered L. johnsonii strains with modified rplP for high-throughput screening

  • Reporter systems based on ribosome function for rapid identification of inhibitors

  • Differential screening against wild-type and mutant rplP to identify resistance-overcoming compounds

This research direction is particularly valuable as ribosomal targets remain among the most successful sites for antibiotic action, and understanding resistance mechanisms at the molecular level is essential for developing next-generation antimicrobials.

What potential applications exist for engineered L. johnsonii strains with modified rplP in medical and industrial settings?

Engineered L. johnsonii strains with modified rplP offer diverse applications in both medical and industrial contexts:

• Therapeutic probiotic development:

  • Creation of antibiotic-resistant probiotics through specific rplP mutations

  • These modified strains could maintain gut colonization during antibiotic treatment

  • Potential applications in prevention of Clostridioides difficile infection following antibiotic therapy

  • Enhanced delivery of therapeutic proteins through engineered L. johnsonii, building on established expression systems

• Immunomodulatory applications:

  • L. johnsonii strains producing modified rplP could stimulate specific immune responses

  • Research has shown that L. johnsonii can "promote systemic effects on the circulating leukocytes and metabolites"

  • Potential application in inflammatory disorders, leveraging the observation that "L. johnsonii expressing GM-CSF demonstrated protective effects against postpartum endometritis in bovines by reducing inflammatory cytokines"

• Industrial fermentation optimization:

  • Strains with optimized rplP could exhibit enhanced protein synthesis capacity

  • Improved production of valuable metabolites and enzymes

  • Greater stress tolerance in industrial fermentation conditions

  • Enhanced production of exopolysaccharides, which are "key components of the surfaces of their bacterial producers"

• Biocontainment strategies:

  • L. johnsonii strains with synthetic rplP variants requiring non-standard amino acids

  • These could function as genetically contained probiotics for environmental applications

  • Reduced risk of horizontal gene transfer of engineered traits

• Diagnostics and biosensing:

  • L. johnsonii nanovesicles containing modified rplP as biomarkers

  • Development of diagnostic assays based on specific antibody responses to modified rplP

  • Building on the finding that "plasma from individuals administered L. johnsonii N6.2 showed that IgA and IgG antibodies were generated against NV and Sdp domains in vivo"

• Biocatalysis applications:

  • Strains with modified translation machinery for incorporation of non-canonical amino acids

  • Production of novel peptides and proteins with enhanced functions

  • Expanded genetic code applications for industrial enzyme production

The development of these applications requires careful consideration of regulatory pathways and safety assessments, but the foundation established by current L. johnsonii rplP research provides a solid platform for these innovative directions.