Recombinant Lactobacillus plantarum DNA mismatch repair protein MutS (mutS), partial

What is Lactobacillus plantarum and why is it significant in microbiological research?

Lactobacillus plantarum is a versatile gram-positive bacterium widely studied for its probiotic properties and potential therapeutic applications. It has gained significant attention for its ability to disrupt pathogenic biofilms, particularly those formed by cariogenic microorganisms like Streptococcus mutans and Candida albicans . L. plantarum demonstrates superior inhibition of these pathogens by disrupting virulent biofilm formation, reducing bacterial components, and interfering with exopolysaccharide (EPS) production .

Moreover, L. plantarum strains have shown remarkable survival capabilities at low pH (3.0) and in the presence of bile salts (0.3%), making them suitable candidates for oral administration and intestinal colonization . Their ability to adhere to intestinal epithelial cells further enhances their potential as probiotic agents and delivery vehicles for therapeutic proteins and vaccines .

How does the MutS protein function in DNA mismatch repair, and what is its role in bacterial adaptation?

The MutS protein serves as a critical DNA mismatch repair (MMR) enzyme that identifies and initiates repair of mismatched base pairs during DNA replication. Methodologically, MutS functions by:

• Scanning newly synthesized DNA for mismatches

• Binding to mismatched or unpaired bases

• Recruiting additional repair proteins to correct the error

• Preventing the accumulation of spontaneous mutations

Experimentally, transient inactivation of MutS has been demonstrated to decrease DNA replication fidelity, thereby increasing mutation rates and accelerating adaptive evolution in Lactobacillus species . This approach has proven effective for developing strains with enhanced resistance to environmental stressors such as acidic conditions .

What genetic modifications are possible with the MutS system in L. plantarum?

The MutS system can be manipulated in several ways to achieve specific research objectives:

When designing MutS modification experiments, researchers should carefully consider the balance between generating beneficial mutations and maintaining sufficient genetic stability for the intended application .

What are the optimal protocols for creating recombinant L. plantarum strains with modified MutS expression?

Creating recombinant L. plantarum strains with modified MutS expression requires a methodical approach:

• Vector Selection: The pSIP expression system has proven effective for L. plantarum modification, as demonstrated in studies with heterologous protein expression . This inducible system allows for controlled expression using peptide pheromone inducers.

• Transformation Protocol:

  • Prepare electrocompetent L. plantarum cells by growing cultures to OD600 of 0.5-0.7

  • Wash cells in ice-cold electroporation buffer (0.5 M sucrose, 7 mM potassium phosphate, pH 7.4)

  • Mix 100 μL of cells with 0.5-1 μg of plasmid DNA

  • Pulse at 2.5 kV, 25 μF, 200 Ω

  • Immediately add 900 μL of recovery medium (MRS with 0.5 M sucrose)

  • Incubate at 30°C for 2-3 hours before plating on selective media

• Confirmation of Modification:

  • PCR verification of insertion

  • Western blot analysis to confirm protein expression

  • Functional assays to assess MutS activity

  • Genome sequencing to ensure the integrity of the modification

For transient MutS inactivation systems, the experimental design should include a mechanism for restoring normal MutS function after the desired adaptation period, such as inducible promoters or temperature-sensitive alleles .

How can researchers effectively measure mutation rates in MutS-modified L. plantarum strains?

Accurate measurement of mutation rates is critical for evaluating MutS-modified strains:

• Fluctuation Analysis (Luria-Delbrück Method):

  • Grow multiple parallel cultures of both wild-type and MutS-modified strains

  • Plate cultures on selective media to identify mutants (e.g., rifampicin resistance)

  • Calculate mutation rates using appropriate statistical methods (e.g., Ma-Sandri-Sarkar maximum likelihood estimator)

• Genome Sequencing Approach:

  • Perform whole-genome sequencing of parent and adapted strains

  • Identify accumulated mutations through bioinformatic analysis

  • Quantify mutation types and frequencies

  • This approach revealed genetic changes in adapted L. casei strains, including mutations in NADH dehydrogenase (ndh), phosphate transport ATP-binding protein (pstB), and histidine protein kinase (hpk) genes

• Reporter Systems:

  • Integrate specific reporter constructs that detect particular mutation types

  • Measure reporter signal (e.g., fluorescence, colorimetric change) to quantify mutation events

A comprehensive mutation rate analysis should include controls for selection bias and account for growth rate differences between strains.

What are the key considerations for experimental design when studying adaptive evolution in MutS-inactivated Lactobacillus strains?

When designing adaptive evolution experiments with MutS-inactivated strains, researchers should consider:

• Selection Pressure Parameters:

  • Define appropriate stress conditions (e.g., pH 4.0 titrated with lactic acid as used in L. casei studies)

  • Determine optimal stress intensity that balances selection stringency with cell viability

  • Consider gradual versus abrupt application of stress

• Control Groups:

  • Include wild-type strains subjected to identical selection conditions

  • Include MutS-inactivated strains not subjected to selection pressure

  • These controls allow for distinguishing adaptation effects from random mutation effects

• Temporal Sampling:

  • Collect samples at regular intervals to track evolutionary trajectories

  • Preserve samples for retrospective analysis (e.g., freezing glycerol stocks)

  • Consider parallel evolution lines to assess reproducibility

• Phenotypic and Genotypic Characterization:

  • Measure relevant phenotypic parameters (growth rate, stress resistance, metabolite production)

  • Perform genomic analysis to identify adaptive mutations

  • Validate the contribution of identified mutations through targeted gene inactivation

• MutS Restoration:

  • Plan for repair of MutS function after adaptation to stabilize beneficial mutations

  • Develop methods to confirm successful restoration of DNA repair capacity

In a study with L. casei, this approach led to adapted strains with significantly enhanced growth at pH 4.0, higher cell densities, and increased lactic acid production compared to wild-type strains .

How can recombinant L. plantarum strains be used as vaccine delivery vehicles?

Recombinant L. plantarum has emerged as a promising mucosal vaccine delivery platform with several methodological considerations:

• Antigen Selection and Expression Design:

  • Select antigens with strong immunogenic properties

  • Design fusion constructs that optimize antigen presentation

  • In swine influenza studies, the head domain of HA1 was successfully expressed in L. plantarum

• Expression System Optimization:

  • The pSIP401 expression vector has proven effective for heterologous protein expression

  • Surface display can be achieved by fusion with anchoring domains (SP1216, 2578)

  • Western blot analysis and immunofluorescence can confirm successful expression and localization

• Immunization Protocol Development:

  • Oral administration: 10^9 CFU/100 μL in a prime-boost strategy

  • Intranasal administration: 10^7 cells in 10 μL applied to anaesthetized animals

  • Both routes have demonstrated efficacy in murine models

• Immune Response Evaluation:

  • Measure mucosal IgA in intestinal and respiratory secretions

  • Assess systemic IgG antibody titers

  • Evaluate cell-mediated immunity through lymphocyte proliferation assays and cytokine profiling

  • Test protective efficacy through pathogen challenge studies

Research has demonstrated that both oral and intranasal immunization with recombinant L. plantarum expressing the HA1 protein induced effective mucosal, cellular, and systemic immune responses and provided protection against lethal influenza virus challenge in mice .

What genomic mechanisms contribute to the anti-biofilm properties of L. plantarum against pathogenic microorganisms?

The anti-biofilm properties of L. plantarum involve complex genomic mechanisms that can be studied through the following methodological approaches:

• Transcriptomic Analysis:

  • RNA sequencing of multi-species biofilms with and without L. plantarum

  • Differential expression analysis to identify downregulated virulence genes

  • In studies with S. mutans-C. albicans biofilms, L. plantarum significantly downregulated genes involved in:

    • Exopolysaccharide formation

    • Carbohydrate metabolism

    • Glycan biosynthesis and metabolism

    • C. albicans antifungal resistance (ERG4)

    • Fungal cell wall chitin remodeling (CHT2)

    • Resistance to oxidative stress (CAT1)

• Bacteriocin Characterization:

  • Identification of bacteriocin-encoding genes (plnD, plnG, plnN)

  • Purification and functional analysis of bacteriocins

  • Testing specific bacteriocins against target pathogens

• Biofilm Visualization and Quantification:

  • Confocal laser scanning microscopy to visualize biofilm structure

  • Quantification of bacterial and fungal components

  • Assessment of exopolysaccharide production

This research approach has revealed that L. plantarum can disrupt pathogenic biofilms by interfering with the metabolic cooperation between different species and downregulating key virulence factors .

How does MutS-based hypermutation compare with other adaptive evolution strategies for Lactobacillus strain improvement?

Comparison of different adaptive evolution strategies provides valuable insights for researchers:

MutS-based hypermutation has demonstrated particular effectiveness in developing acid-resistant strains, as evidenced in L. casei studies where adapted MutS-deficient derivatives showed greater increases in growth and lactic acid production compared to other methods .

What are the common challenges in working with recombinant L. plantarum MutS systems and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant L. plantarum MutS systems:

• Plasmid Stability Issues:

  • Challenge: Loss of plasmids carrying MutS modifications during prolonged cultivation

  • Solution: Use integrative vectors for chromosomal incorporation of constructs

  • Methodology: Implement antibiotic selection during propagation and verify plasmid retention by PCR

• Expression Level Control:

  • Challenge: Difficulty in achieving appropriate MutS expression levels

  • Solution: Utilize inducible promoter systems like pSIP with titratable induction

  • Methodology: Determine optimal inducer concentration through dose-response experiments

• Phenotypic Characterization:

  • Challenge: Distinguishing MutS-related phenotypes from adaptation to laboratory conditions

  • Solution: Include appropriate controls and perform adaptation experiments in parallel

  • Methodology: Measure mutation rates using fluctuation tests and compare with theoretical predictions

• Genetic Stability After Adaptation:

  • Challenge: Continued hypermutation after desired adaptations are achieved

  • Solution: Develop systems for restoring MutS function after adaptation

  • Methodology: Design genetic switches or complementation strategies to restore DNA repair

• Strain Degeneration:

  • Challenge: Accumulation of deleterious mutations affecting growth or survival

  • Solution: Optimize duration of hypermutation period and selection intensity

  • Methodology: Monitor growth parameters and perform competitive fitness assays

By anticipating these challenges and implementing appropriate solutions, researchers can maximize the effectiveness of MutS-based adaptive evolution strategies.

How should researchers validate the specific contributions of mutations identified in MutS-based adaptive evolution experiments?

Validating the contributions of specific mutations requires a systematic approach:

This multi-faceted validation approach provides comprehensive evidence for the contributions of specific mutations to the adapted phenotype and distinguishes primary adaptive mutations from passenger mutations.

What bioinformatic tools and pipelines are most appropriate for analyzing genomic data from MutS-modified Lactobacillus strains?

Analysis of genomic data from MutS-modified strains requires specialized bioinformatic approaches:

• Variant Calling Pipelines:

  • Software Tools: GATK, FreeBayes, VarScan2

  • Methodological Considerations:

    • Use variant callers optimized for microbial genomes

    • Adjust parameters for hypermutator strains (higher mutation frequency)

    • Implement filters to distinguish true variants from sequencing errors

• Mutation Spectrum Analysis:

  • Software Tools: MutationalPatterns (R package), SomaticSignatures

  • Methodological Approach:

    • Categorize mutations by type (transitions, transversions)

    • Analyze trinucleotide context of mutations

    • Compare mutation spectra between different conditions

• Evolutionary Analysis:

  • Software Tools: PAML, dN/dS calculators, PopGenome

  • Key Analyses:

    • Calculate selective pressures on coding sequences

    • Identify genes under positive selection

    • Track evolutionary trajectories over time

• Functional Impact Prediction:

  • Software Tools: SIFT, PolyPhen, PROVEAN

  • Application:

    • Predict functional consequences of amino acid substitutions

    • Prioritize mutations for experimental validation

    • Identify potentially adaptive mutations

• Pathway and Network Analysis:

  • Software Tools: KEGG Pathway, STRING, Cytoscape

  • Approach:

    • Map mutations to metabolic and regulatory pathways

    • Identify enriched pathways among mutated genes

    • Construct protein-protein interaction networks of affected genes

When analyzing genomic data from L. plantarum and related Lactobacillus species, researchers should use reference genomes appropriate for their specific strain background and consider the unique genomic features of lactic acid bacteria.

How might MutS-based systems be combined with other genetic engineering approaches to create enhanced L. plantarum strains for therapeutic applications?

Integrating MutS-based systems with other genetic engineering approaches offers promising opportunities:

• CRISPR-Cas + MutS Modulation:

  • Methodological Approach: Use CRISPR-Cas to introduce specific beneficial mutations identified through MutS-based evolution

  • Potential Applications:

    • Enhanced stress resistance

    • Improved therapeutic protein expression

    • Targeted modification of metabolic pathways

• Synthetic Biology + Adaptive Evolution:

  • Methodology: Design synthetic genetic circuits with optimized components, then fine-tune through MutS-mediated adaptation

  • Research Applications:

    • Environmentally responsive therapeutic delivery

    • Conditional colonization systems

    • Programmable interactions with host microbiome

• Multi-Omics Guided Engineering:

  • Approach: Integrate transcriptomic, proteomic, and metabolomic data from adapted strains to inform rational design

  • Implementation Strategy:

    • Identify key regulatory nodes from multi-omics data

    • Engineer these nodes for enhanced function

    • Validate through targeted phenotypic assays

• Heterologous Expression Optimization:

  • Methodology: Use MutS-based adaptation to optimize expression of heterologous proteins like vaccine antigens

  • Example Application: Improved expression of viral antigens such as influenza HA1

  • Potential Improvements: Enhanced protein folding, increased secretion efficiency, optimized codon usage

This integrated approach could significantly advance the development of L. plantarum as a therapeutic delivery platform while overcoming current limitations in expression efficiency and in vivo functionality.

What are the emerging research questions regarding L. plantarum-host interactions in therapeutic applications?

Key emerging research questions include:

• Strain-Specific Immunomodulatory Effects:

  • Research Question: How do genetic differences among L. plantarum strains impact their immunomodulatory properties?

  • Methodological Approach: Comparative transcriptomics of host immune cells exposed to different L. plantarum strains

  • Expected Insights: Identification of bacterial factors that influence T helper cell differentiation and cytokine production

• Epithelial Barrier Function Modulation:

  • Research Question: By what mechanisms does L. plantarum enhance epithelial barrier integrity?

  • Experimental Design: Ex vivo intestinal organoid models with recombinant L. plantarum variants

  • Measurement Approaches: Transepithelial electrical resistance, tight junction protein expression, permeability assays

• Microbiome Interactions:

  • Research Question: How does engineered L. plantarum interact with resident microbiota in different host niches?

  • Methodological Approach: Metagenomic and metatranscriptomic analysis following L. plantarum administration

  • Analytical Framework: Ecological network analysis to identify cooperative and competitive interactions

• Heterologous Antigen Presentation Optimization:

  • Research Question: What factors determine the efficacy of antigen presentation by L. plantarum?

  • Experimental Design: Systematic comparison of different surface display systems and administration routes

  • Key Measurements: Mucosal IgA production, T cell activation, protection in challenge studies

• Long-Term Colonization Determinants:

  • Research Question: What genetic factors influence long-term persistence of L. plantarum in different host niches?

  • Methodological Approach: Longitudinal tracking of marked strains with different genetic backgrounds

  • Analysis Framework: Machine learning to identify genetic predictors of colonization success

Addressing these questions will require interdisciplinary approaches combining microbiology, immunology, and systems biology to fully understand and optimize L. plantarum-host interactions.