Recombinant Lactobacillus plantarum Ribonuclease 3 (rnc)

What is the function of Ribonuclease III (RNase III) in Lactobacillus plantarum?

RNase III is a global regulator of gene expression that recognizes and cleaves double-stranded RNA (dsRNA) at specific targeted locations. In bacterial systems like L. plantarum, RNase III performs several critical functions:

• Processing of precursor ribosomal RNA (pre-rRNA) for maturation

• Regulating mRNA stability and degradation

• Post-transcriptional regulation of numerous genes, including autoregulation of its own expression

• Participation in rRNA quality control and localization to the nucleoid

In L. plantarum specifically, RNase III is likely analogous to those in B. subtilis, working alongside other ribonucleases such as RNase Y and PNPase to coordinate RNA metabolism .

What are the key components needed to construct a recombinant L. plantarum expressing RNase III?

To construct recombinant L. plantarum expressing RNase III, researchers require:

• Vector selection: Either high copy number (e.g., pCDLbu-1ΔEc-based plasmids) or low copy number (e.g., p256 origin of replication) plasmid backbones

• Promoter selection: Options include:

  • P11: Strong constitutive promoter

  • Ptuf33 and Ptuf34: Alternative constitutive promoters

  • Inducible promoters (e.g., pSIP system) for controlled expression

• Ribosomal binding site (RBS) optimization: The distance between the Shine-Dalgarno sequence and start codon significantly impacts expression, with 8 nucleotides often providing optimal levels

• Cloning strategy: Typically using restriction enzymes (e.g., EcoRI and EcoRV) to insert the gene into expression vectors

• Transformation protocol: Electroporation is commonly used for introducing the construct into L. plantarum

How can I confirm successful expression of recombinant RNase III in L. plantarum?

Multiple complementary techniques should be employed:

• Western blotting: Using anti-RNase III antibodies to detect the expressed protein from cell lysates. This confirms the protein is being produced at the expected molecular weight (typically 25 kDa for RNase III)

• Enzymatic activity assay: Measuring dsRNA cleavage activity using synthetic substrates or known natural substrates like 16S rRNA precursors

• Immunofluorescence microscopy: Using antibodies to visualize localization of the expressed protein (particularly useful for surface-displayed constructs)

• Flow cytometry: Particularly useful for quantifying expression efficiency when the protein is displayed on the bacterial surface (expecting 35-40% positive cells under optimal conditions)

• RNase III complementation test: Introducing the construct into an RNase III-deficient strain (e.g., SK7622) and assessing growth rate recovery (a functional RNase III should restore normal growth)

What strategies can optimize RNase III expression in L. plantarum?

Optimizing RNase III expression requires fine-tuning at multiple levels:

Transcriptional optimization:

• Promoter selection based on desired expression level - P11 provides strong expression while Ptuf promoters offer moderate expression

• Using high copy number plasmids (e.g., pCDLbu-1ΔEc) can increase yield up to 2-fold compared to low copy vectors

Translational optimization:

• Codon optimization for L. plantarum is crucial for heterologous genes

• Optimizing the Shine-Dalgarno sequence to match the consensus sequence (AGGAGG)

• Varying the spacing between SDS and start codon, with 8 nucleotides typically yielding maximum expression

• For surface display, using appropriate secretion signals and anchoring domains (e.g., LPXTG motif)

Induction conditions:

• For inducible systems, optimizing inducer concentration (e.g., 50 ng/mL SppIP)

• Determining optimal induction time (typically 6-10 hours post-induction)

• Cultivation temperature optimization (generally 37°C for L. plantarum)

A systematic approach combining these strategies while monitoring growth parameters is recommended for optimal expression.

How does gene copy number and promoter strength affect RNase III expression in L. plantarum?

The interplay between gene copy number and promoter strength significantly impacts expression levels:

Gene copy number effects:

• High copy number plasmids (e.g., based on the L. buchneri CD034 pCD034-1 origin) can contain >200 copies per chromosome

• Low copy number vectors (e.g., p256 origin) maintain fewer plasmid copies

• High copy number vectors can increase yield approximately 2-fold when combined with the same promoter

Promoter strength effects:

• P11 promoter provides strong constitutive expression

• Ptuf33 and Ptuf34 offer more moderate expression levels

Interaction between variables:

• Strong promoters on high copy vectors may cause excessive metabolic burden

• For RNase III specifically, overexpression can be detrimental as it may disrupt normal RNA processing

• The combination of promoter strength and copy number should be calibrated to the specific application

For RNase III expression, moderate expression levels are often preferable as they prevent disruption of normal cellular RNA processing while providing sufficient enzyme activity .

How can I assess the activity of recombinant RNase III in L. plantarum?

Several complementary approaches can be used to measure RNase III activity:

In vitro activity assays:

• dsRNA cleavage assay: Using synthetic dsRNA substrates labeled with fluorescent dyes and measuring fluorescence after cleavage

• Gel electrophoresis analysis: Incubating purified recombinant RNase III with known substrates and analyzing cleavage patterns by gel electrophoresis

In vivo functional assessment:

• Growth rate measurement: RNase III activity correlates with bacterial growth; cells lacking functional RNase III grow approximately 25% slower

• rRNA processing analysis: Examining 16S rRNA maturation using Northern blotting or primer extension

• Autoregulation analysis: Quantifying the ability of RNase III to regulate its own mRNA through 5' UTR cleavage

• Nucleoid localization: Assessing the ability of RNase III to localize pre-16S rRNA to the nucleoid using RNA-FISH (fluorescence in situ hybridization)

Complementation studies:

• Transform the recombinant L. plantarum RNase III construct into an RNase III-deficient E. coli strain (e.g., SK7622)

• Measure growth rate recovery compared to wild-type strains

• Analyze rRNA processing patterns to confirm restoration of RNase III activity

What are the key mutations that affect RNase III function and how can they be used in experimental design?

Specific mutations in RNase III can provide valuable experimental tools:

Catalytic domain mutations:

• E117K mutation: Creates a nuclease-null variant that maintains RNA binding ability but eliminates catalytic activity. Useful for distinguishing binding from cleavage effects

• Mutations in the RNase III signature motif: Residues in this 10-residue motif are critical for enzyme function, with most substitutions causing complete activity loss

RNA binding domain mutations:

• Mutations in RBM1 and RBM3: These binding motifs are crucial for dsRNA interaction; mutations here reduce substrate affinity without affecting catalytic capability

• RBM4 and RBM2 mutations: These regions tolerate more substitutions, allowing for tunable binding affinity

Experimental applications:

• Separating binding from cleavage: Use E117K mutant to study RNA binding effects independent of processing

• Conditional activity: Engineer temperature-sensitive RNase III variants for temporal control

• Substrate specificity studies: Mutations at G97, G99, and F188 appear to affect substrate selectivity without eliminating general activity

These mutations provide powerful tools for dissecting RNase III function in L. plantarum gene regulation networks .

How can recombinant L. plantarum expressing RNase III be used for therapeutic applications?

Recombinant L. plantarum expressing RNase III has several potential therapeutic applications:

Autoimmune disease treatment:

• In rheumatoid arthritis, regulatory T-cell development and function is regulated by FOXP3, which is underexpressed in patients. L. plantarum could be engineered to express both RNase III and FOXP3 to restore T-cell functionality and decrease inflammation

• RNase III could help regulate the processing and stability of the FOXP3 mRNA and protein expression

Vaccine delivery platform:

• The RNase III enzyme can be co-expressed with vaccine antigens to modulate immune responses by controlling mRNA stability and antigen expression levels

• This approach has shown promise for vaccines against viral pathogens (e.g., influenza) and parasitic diseases (e.g., trichinellosis)

Cancer immunotherapy:

• L. plantarum expressing tumor antigens like NY-ESO-1 along with RNase III could enhance antigen presentation and immune response

• The regulated expression via RNase III activity enables controlled release of antigens, promoting more effective T-cell responses

For these applications, researchers should consider:

• Designing constructs with appropriate secretion signals or surface-display anchors

• Optimizing RNase III expression to maintain viable bacteria while achieving therapeutic effect

• Validating in appropriate animal models before clinical testing

How does recombinant L. plantarum expressing RNase III affect gut microbiota and immune responses?

Recombinant L. plantarum expressing RNase III influences host biology through several mechanisms:

Effects on gut microbial communities:

• Recombinant L. plantarum can significantly increase microbial diversity as measured by Shannon-Wiener index

• It enhances the species richness (Chao1 index) and alters community structure (beta diversity)

• These changes may be partially mediated by RNase III's role in processing bacterial sRNAs that regulate gene expression

Immune response modulation:

• Humoral immunity: Increases levels of serum IgG, IgG1, IgG2a, and mucosal secretory IgA (sIgA)

• Cellular immunity: Enhances CD4+ T cell and CD8+ T cell responses, particularly IFN-γ producing cells

• Dendritic cell activation: Activates dendritic cells in Peyer's patches, crucial for antigen presentation

• B cell response: Increases the number of B220+IgA+ cells in Peyer's patches

Experimental data from recombinant L. plantarum studies:

The mechanism likely involves RNase III's role in regulating bacterial gene expression and antigen processing, which subsequently affects how the recombinant bacteria interact with host immune cells .

What are the key considerations when designing in vivo experiments with recombinant L. plantarum expressing RNase III?

Designing robust in vivo experiments requires careful consideration of multiple factors:

Animal model selection:

• Mice: Most commonly used for initial studies; BALB/c mice are frequently employed for immunological studies

• Disease-specific models: Consider humanized mice, arthritis models, or other specialized models depending on research focus

Administration protocol:

• Route: Oral administration is most common for L. plantarum (gavage or in drinking water)

• Dosage: Typically 10^8-10^10 CFU per administration, with optimization needed for each construct

• Schedule: Multiple doses are usually required; common schedules include:

  • Primary immunization followed by 1-2 boosters at 2-week intervals

  • Daily administration for 5-10 days

Controls and groups:

• PBS control (negative)

• Empty vector L. plantarum (bacterial backbone control)

• Recombinant L. plantarum expressing RNase III

• Recombinant L. plantarum expressing target antigen without RNase III

• Recombinant L. plantarum expressing both RNase III and target antigen

• Commercial vaccine or treatment (positive control, if available)

Assessment timeline and methods:

• Sampling timepoints: Pre-immunization, 10 days, 20 days, 30+ days post-immunization

• Sample types: Serum, feces, intestinal contents, tissue biopsies

• Analyses: Flow cytometry, ELISA, RT-qPCR, histopathology, and challenge studies when applicable

Ethical considerations:

• Follow appropriate institutional guidelines for animal research

• Use power calculations to determine minimum number of animals needed

• Consider humane endpoints appropriate to the model

How should I design an experiment to investigate RNase III's role in regulating specific gene expression in L. plantarum?

To investigate RNase III's regulatory role, a comprehensive experimental approach is required:

Construct design and strains:

• Wild-type L. plantarum: Baseline control

• RNase III knockout/knockdown: Using CRISPR-Cas9 or antisense RNA approaches

• RNase III overexpression strain: Using strong constitutive promoter

• Catalytically inactive RNase III (E117K): To distinguish binding from cleavage effects

Experimental approaches:

1. Transcriptome analysis:

• RNA-seq comparing wild-type, knockout, and overexpression strains

• Focus on changes in mRNA levels and processing patterns

• Analyze differential expression under various growth conditions (exponential phase, stationary phase, stress conditions)

2. Direct target identification:

• CLIP-seq (Cross-linking immunoprecipitation) to identify RNase III-bound RNAs

• RNA-seq with size selection to identify processing intermediates

• Northern blotting to confirm specific cleavage patterns for candidate targets

3. Functional validation:

• Construct reporter systems (e.g., GFP fusions) with candidate target 5' and 3' UTRs

• Site-directed mutagenesis of putative RNase III recognition sites

• In vitro cleavage assays with purified RNase III and candidate RNA substrates

4. Physiological impact assessment:

• Growth curve analysis under various conditions

• Stress response testing (acid, bile, oxidative stress)

• Competition assays between wild-type and RNase III variant strains

Data analysis approach:

• Identify differentially processed/expressed transcripts

• Map RNase III cleavage sites on target RNAs

• Correlate RNA structural features with cleavage efficiency

• Connect RNA processing to physiological outcomes

What are common challenges in expressing recombinant RNase III in L. plantarum and how can they be addressed?

Researchers frequently encounter several challenges when expressing RNase III in L. plantarum:

Challenge 1: Poor growth of recombinant strains

• Cause: Overexpression of RNase III disrupting normal RNA processing

• Solutions:

  • Use weaker promoters or low-copy plasmids

  • Employ inducible systems with tight regulation

  • Use the native RNase III 5'-UTR to maintain autoregulation

Challenge 2: Low RNase III expression levels

• Causes: Poor translation efficiency, protein instability, or toxicity

• Solutions:

  • Optimize the ribosome binding site and spacing (8 nucleotides optimal)

  • Codon-optimize the RNase III sequence for L. plantarum

  • Verify plasmid stability with repeated culturing

Challenge 3: Difficulty confirming RNase III activity

• Causes: Limited substrate access or improper assay conditions

• Solutions:

  • Use multiple activity assays (in vivo and in vitro)

  • Include the E117K catalytically inactive mutant as a control

  • Purify the protein and perform controlled enzymatic assays

Challenge 4: Plasmid instability

• Causes: Metabolic burden, recombination events

• Solutions:

  • Use appropriate selective pressure (antibiotics)

  • Verify plasmid integrity by restriction analysis after multiple generations

  • Optimize media composition to reduce metabolic stress

Challenge 5: Heterogeneous expression in bacterial population

• Causes: Plasmid loss, variable gene expression

• Solutions:

  • Use flow cytometry to quantify expressing subpopulations

  • Optimize induction conditions and growth phase

  • Consider chromosomal integration for stable expression

How can I troubleshoot inconsistent immune responses in animal studies using recombinant L. plantarum expressing RNase III?

Inconsistent immune responses are a common challenge in animal studies with recombinant L. plantarum. Here's a systematic approach to troubleshooting:

Variable 1: Bacterial preparation and administration

• Assessment: Verify viable cell count, expression level, and administration technique

• Solutions:

  • Standardize culture conditions and harvest at consistent growth phase

  • Confirm RNase III expression by Western blot before each administration

  • Use consistent dosing techniques (e.g., calibrated gavage needles)

Variable 2: Host factors

• Assessment: Examine variability in animal age, weight, gender, and microbiome

• Solutions:

  • Use age and gender-matched animals

  • Consider pre-treatment microbiome analysis

  • House animals under identical conditions for at least 2 weeks before experiments

Variable 3: Bacterial colonization and persistence

• Assessment: Measure fecal shedding of the recombinant bacteria

• Solutions:

  • Perform preliminary colonization studies to optimize dosing

  • Consider repeated administration to maintain colonization

  • Use strain-specific PCR to track recombinant bacteria in the gut

Variable 4: Immune response assessment

• Assessment: Evaluate timing and methods of immune parameter measurement

• Solutions:

  • Collect samples at multiple timepoints

  • Use multiple readouts (flow cytometry, ELISA, ELISpot)

  • Include appropriate positive controls in each assay

Troubleshooting flowchart:

• Verify bacterial preparation:

  • Expression level consistent? → If no: Standardize culture conditions

  • Viability confirmed? → If no: Adjust preparation protocol

• Confirm administration:

  • Consistent dosing? → If no: Standardize administration

  • Timing appropriate? → If no: Adjust schedule

• Assess colonization:

  • Bacteria detectable in feces? → If no: Increase dose or frequency

  • Persistence adequate? → If no: Consider prebiotic supplementation

• Evaluate immune measurements:

  • Multiple parameters assessed? → If no: Expand analysis

  • Appropriate controls included? → If no: Add necessary controls

Implementing this systematic approach will help identify and address sources of variability in immune responses .

How does RNase III function in L. plantarum compare to other bacterial species, and what are the implications for recombinant expression?

RNase III exhibits both conserved and divergent characteristics across bacterial species, with important implications for recombinant expression:

Structural and functional conservation:

• The RNase III signature motif is highly conserved across bacterial species

• The enzyme architecture consisting of a catalytic domain and dsRNA binding domain is preserved

• Basic catalytic mechanism of dsRNA cleavage is consistent across species

Species-specific differences:

Regulatory differences:

• In E. coli, RNase III autoregulates through cleavage of its own 5'-UTR

• RNase III activity in E. coli can be modulated by phosphorylation during phage infection

• The set of natural targets varies between species, affecting the impact of overexpression

Implications for recombinant expression:

• Expression optimization: Species-specific codon usage and regulatory elements must be considered

• Activity assessment: Target selection for activity assays should consider species-specific substrates

• Functional complementation: E. coli RNase III mutants can be used to test L. plantarum RNase III functionality

• Physiological impact: Overexpression effects may differ between species based on the targetome

• Heterologous applications: Consider using RNase III from different species if specific substrate preferences are desired

Understanding these species-specific differences enables more effective design of recombinant expression systems and accurate interpretation of experimental results across different bacterial models .

What are emerging applications of recombinant L. plantarum expressing RNase III in research and therapeutics?

Several innovative applications are emerging for recombinant L. plantarum expressing RNase III:

Precision RNA therapeutics:

• Using RNase III expression to selectively degrade specific pathogenic RNAs

• Engineering substrate specificity through rational mutation of residues G97, G99, and F188

• Developing strain-specific RNA targeting for microbiome modulation

Synthetic biology applications:

• Creating tunable gene expression circuits using RNase III processing

• Developing post-transcriptional regulatory switches based on conditional RNA cleavage

• Designing artificial RNA processing systems for metabolic engineering of L. plantarum

Novel vaccine delivery platforms:

• Multi-antigen delivery systems with controlled expression via RNase III processing

• SARS-CoV-2 and other respiratory pathogen vaccine candidates utilizing surface-displayed antigens

• Cancer vaccine development using tumor-associated antigens with RNase III-regulated expression

Therapeutic microbiota modulation:

• Targeted alteration of microbiome community structure for disease treatment

• Engineering precise strain-specific RNA interference via RNase III-mediated processing

• Developing probiotics with enhanced immune-modulatory properties for inflammatory conditions

Diagnostic applications:

• Biosensor development using RNase III-dependent reporter systems

• In situ detection of specific bacterial targets in complex microbiomes

• Monitoring gut health through engineered sentinel bacteria expressing RNase III-regulated reporters

Challenges and future directions:

• Improving strain stability and expression consistency

• Developing better methods for in vivo monitoring of RNase III activity

• Establishing appropriate regulatory frameworks for clinical applications

• Optimizing colonization and persistence in the human gut

What are the current knowledge gaps in understanding RNase III function in L. plantarum and how might they be addressed?

Despite significant progress, several important knowledge gaps remain in our understanding of RNase III function in L. plantarum:

Gap 1: Comprehensive targetome characterization

• Current status: Limited knowledge of natural RNA targets in L. plantarum

• Research approaches:

  • Genome-wide RNA structure mapping (SHAPE-seq, DMS-seq)

  • RNase III CLIP-seq to identify binding sites

  • Comparative transcriptomics of wild-type and RNase III-deficient strains

Gap 2: Regulatory networks and environmental responsiveness

• Current status: Poor understanding of how RNase III activity changes under different conditions

• Research approaches:

  • Quantitative proteomics to measure RNase III levels under various stresses

  • Activity assays under different environmental conditions

  • Identification of potential post-translational modifications

Gap 3: Species-specific structural and functional features

• Current status: L. plantarum RNase III structure has not been solved

• Research approaches:

  • X-ray crystallography or cryo-EM structure determination

  • Systematic mutagenesis to identify species-specific functional residues

  • Substrate preference profiling compared to other bacterial RNase III enzymes

Gap 4: In vivo dynamics and localization

• Current status: Limited knowledge of subcellular localization and dynamics

• Research approaches:

  • Fluorescent protein tagging for live-cell imaging

  • Super-resolution microscopy to determine co-localization with RNA substrates

  • Single-molecule tracking to analyze enzyme kinetics in vivo

Gap 5: Role in host-microbe interactions

• Current status: Unknown impact of L. plantarum RNase III on host interactions

• Research approaches:

  • Comparing colonization efficiency of wild-type vs. RNase III variants

  • Host transcriptome analysis after exposure to different L. plantarum strains

  • Investigation of RNase III's role in stress responses relevant to gut survival

Proposed integrated research strategy:

• Generate comprehensive RNase III variant library in L. plantarum

• Perform parallel phenotyping under multiple conditions

• Apply multi-omics approaches (transcriptomics, proteomics, structuromics)

• Develop computational models to predict RNase III activity

• Validate in relevant in vivo models including gut colonization studies