Recombinant Lactobacillus plantarum Isoleucine--tRNA ligase (ileS), partial

What is the role of Isoleucine--tRNA ligase (ileS) in Lactobacillus plantarum?

Isoleucine--tRNA ligase (ileS) in Lactobacillus plantarum plays a crucial role in protein synthesis by catalyzing the attachment of isoleucine to its cognate tRNA. This aminoacylation process is essential for accurate translation of genetic information into proteins. The enzyme specifically recognizes isoleucine and attaches it to tRNA^Ile, ensuring proper incorporation of isoleucine residues during protein synthesis. In recombinant L. plantarum systems, ileS function is particularly important for maintaining proper protein expression levels in engineered strains, especially when these strains are designed to express therapeutic proteins or antigens as seen in experimental vaccine development models .

What experimental methods are most effective for cloning and expressing recombinant L. plantarum ileS?

For successful cloning and expression of recombinant L. plantarum ileS, a systematic approach involving several complementary techniques yields optimal results. Begin with PCR amplification of the target ileS gene using high-fidelity DNA polymerase and primers designed with appropriate restriction sites. For expression vector selection, pSIP409-based systems have demonstrated high efficiency in L. plantarum, similar to those used for expressing other recombinant proteins like FomA . The integration can be verified through restriction digestion analysis, PCR, and DNA sequencing to confirm proper insertion and orientation.

For expression, induction conditions should be optimized based on the specific promoter system used. When using inducible promoters similar to those employed in other L. plantarum recombinant systems, expression can be verified through Western blotting, with typical protein yields ranging from 0.1-1 mg/L depending on culture conditions . Surface display systems utilizing anchoring sequences such as PGSA' (from Bacillus subtilis) have shown high display efficiency and may be applicable for partial ileS expression if surface localization is desired .

How does growth media composition affect recombinant L. plantarum ileS expression?

The composition of growth media significantly impacts recombinant L. plantarum ileS expression levels, with several key factors requiring optimization. Based on experimental approaches used for other recombinant L. plantarum proteins, MRS (de Man, Rogosa and Sharpe) medium supplemented with specific amino acids, particularly isoleucine, can enhance expression levels. A typical optimized media formulation includes:

Cultivation temperature (30-37°C) and induction timing (typically at OD₆₀₀ of 0.3-0.5) must be carefully controlled to maximize expression while maintaining proper protein folding . Growth under microaerophilic conditions generally yields better expression results compared to strictly anaerobic or aerobic cultivation.

What are reliable methods for detecting and quantifying ileS expression in recombinant L. plantarum?

For reliable detection and quantification of ileS expression in recombinant L. plantarum, researchers should employ a combination of complementary techniques. Western blotting with antibodies specific to either the ileS protein or to epitope tags incorporated into the recombinant construct provides qualitative confirmation of expression. For quantitative analysis, ELISA offers greater sensitivity, with detection limits typically in the ng/mL range.

RT-qPCR can be used to measure ileS transcript levels, providing insight into transcriptional regulation. For functional assessment, aminoacylation assays measuring the attachment of isoleucine to tRNA can directly quantify ileS activity. When surface display systems like PGSA' are used, flow cytometry provides quantitative data on the percentage of cells expressing the protein and the relative expression levels per cell . Mass spectrometry approaches such as LC-MS/MS offer the most comprehensive characterization, allowing identification of post-translational modifications and verification of the complete amino acid sequence.

How can experimental design be optimized to study the impact of ileS mutations on L. plantarum protein synthesis?

To optimize experimental design for studying ileS mutations in L. plantarum, researchers should implement a systematic approach combining site-directed mutagenesis with comprehensive phenotypic and molecular analyses. Begin by creating a strategic panel of mutations targeting conserved domains (binding sites, catalytic regions) identified through sequence alignment with other bacterial ileS proteins. A factorial experimental design should be employed to test multiple variables simultaneously, including:

• Growth conditions (varying temperature, pH, and nutrient availability)

• Protein synthesis rates (measured via radioactive amino acid incorporation)

• Cell viability (under stress conditions)

• Complementation experiments (with wild-type ileS)

Statistical power analysis should determine appropriate sample sizes, typically requiring 3-5 biological replicates and 2-3 technical replicates per condition to achieve 80% power with α=0.05 . Controls must include both negative (empty vector) and positive (wild-type ileS) references. To properly analyze the resulting multivariate data, employ ANOVA with post-hoc tests, ensuring models account for interaction effects between variables. This approach resembles the systematic experimental design principles used in other recombinant L. plantarum studies .

What mechanisms explain variability in ileS expression levels across different recombinant L. plantarum strains?

The variability in ileS expression across different recombinant L. plantarum strains stems from a complex interplay of genetic, physiological, and environmental factors. Codon optimization represents a primary determinant, as strain-specific codon usage bias can significantly impact translation efficiency. Analysis of the Codon Adaptation Index (CAI) in various L. plantarum strains reveals up to 30% variation in predicted translation efficiency for heterologous genes .

Promoter context and strength significantly influence expression levels, with inducible systems showing strain-dependent response variations. The genomic integration site affects expression through positional effects, with certain regions permitting higher expression levels due to local chromatin structure. Copy number variations between plasmid-based systems can result in 5-10 fold differences in expression levels .

Additionally, strain-specific differences in proteolytic activities, stress responses, and protein folding machinery contribute to expression variability. Metabolic burden associated with recombinant protein production varies by strain, with faster-growing strains often showing reduced expression of non-essential recombinant proteins. These mechanisms parallel those observed in studies of other recombinant proteins in L. plantarum .

How does recombinant ileS expression affect L. plantarum's interaction with host microbiota?

Recombinant ileS expression in L. plantarum can significantly alter its interactions with host microbiota through multiple mechanisms. Overexpression of ileS may enhance protein synthesis capacity, potentially increasing the production of bacteriocins and other antimicrobial compounds that selectively modulate surrounding microbial populations. This selective pressure can reshape microbial community composition, similar to how L. plantarum AR113 administration enriched beneficial Lactobacillus and Bacteroides while depleting potential pathogens like Flavonifractor and Acetatifactor in experimental models .

Modifications to surface properties resulting from altered protein synthesis patterns can affect L. plantarum's adhesion to intestinal mucosa and interaction with other microbes. Recombinant strains may exhibit altered metabolic profiles, changing the availability of key metabolites in the intestinal environment. Metagenomics analysis of gut microbiota following administration of recombinant L. plantarum reveals significant shifts in diversity indices (Shannon and Simpson indices) and unique taxonomic signatures compared to wild-type strains .

These interactions have functional consequences, potentially altering metabolic networks within the gut microbiome, as evidenced by changes in phospholipid metabolism and amino acid utilization observed in studies with other recombinant L. plantarum strains .

What approaches can resolve contradictory data when characterizing recombinant L. plantarum ileS function?

When faced with contradictory data in recombinant L. plantarum ileS research, a systematic troubleshooting and validation approach is essential. Begin with methodological standardization by implementing rigorous protocol documentation and using identical reagents, equipment, and analytical methods across experiments. Employ orthogonal validation by confirming key findings through multiple independent techniques - for example, combining enzymatic activity assays, mass spectrometry, and in vivo complementation studies to verify ileS function .

Statistical meta-analysis can help identify sources of variability by pooling data across experiments and applying random-effects models to account for inter-study heterogeneity. Control for strain-specific effects by testing identical constructs in multiple L. plantarum backgrounds and including appropriate reference strains. Environmental variables should be systematically investigated using design of experiments (DOE) methodology to identify interaction effects that might explain contradictory results .

Additionally, computational modeling can reconcile seemingly contradictory data by simulating ileS activity under different conditions and predicting outcomes based on mechanistic understanding. This multi-faceted approach has successfully resolved contradictions in other complex recombinant protein systems in L. plantarum .

What are the optimal conditions for inducing recombinant L. plantarum ileS expression?

Optimizing induction conditions for recombinant L. plantarum ileS expression requires precise control of multiple parameters to maximize protein yield while maintaining functionality. Based on established protocols for recombinant protein expression in L. plantarum, the following conditions typically yield optimal results:

For nisin-inducible systems similar to those used in other recombinant L. plantarum studies, a dose-response curve should be established for each construct to determine the optimal inducer concentration . Temperature downshift during induction often improves proper protein folding, particularly for complex enzymes like ileS. Supplementation with 0.1-0.2% L-isoleucine during induction can enhance expression by providing abundant substrate for the enzyme, potentially stabilizing the protein structure.

How should researchers design experiments to evaluate the impact of recombinant ileS on L. plantarum metabolic pathways?

When designing experiments to evaluate recombinant ileS impact on L. plantarum metabolic pathways, researchers should implement a systems biology approach integrating multiple analytical techniques. Begin with a factorial experimental design that systematically varies ileS expression levels (using inducible promoters with different induction strengths) and growth conditions (carbon sources, amino acid availability, and stress factors) .

Integrate transcriptomics analysis (RNA-seq) to identify differentially expressed metabolic genes in response to altered ileS levels, with particular attention to amino acid biosynthesis pathways and translation machinery. Metabolomics profiling (LC-MS/MS and GC-MS) should target amino acids (especially branched-chain amino acids), tRNA charging levels, and key metabolic intermediates. Flux analysis using ¹³C-labeled substrates can quantify changes in carbon flow through central metabolic pathways .

Phenotypic characterization must include growth kinetics, stress tolerance, and protein synthesis rates under various conditions. Use statistical design principles to ensure adequate replication (minimum n=3 biological replicates) and include appropriate controls (wild-type, empty vector, and complemented strains). Data integration requires multivariate statistical approaches, including principal component analysis and partial least squares discriminant analysis to identify key metabolic shifts .

What control strains are essential when evaluating recombinant L. plantarum ileS functionality?

A comprehensive set of control strains is essential for rigorous evaluation of recombinant L. plantarum ileS functionality. The experimental design should include the following controls:

• Wild-type L. plantarum: Serves as the baseline reference for natural ileS expression levels and function. This strain establishes normal growth parameters, protein synthesis rates, and metabolic profiles.

• Empty vector control: L. plantarum containing the expression vector without the ileS insert. This control accounts for potential metabolic burden and physiological changes due to the presence of the vector and selection markers.

• ileS knockout/knockdown with complementation: If technically feasible, a strain with reduced native ileS expression complemented with the recombinant version provides the most direct assessment of recombinant functionality.

• Point-mutated ileS variants: Strains expressing ileS with mutations in catalytic sites serve as negative controls for enzyme activity, helping to distinguish specific effects from general overexpression consequences.

• Alternative aminoacyl-tRNA synthetase overexpression: Strains overexpressing a different aminoacyl-tRNA synthetase (e.g., valS) help identify effects specific to ileS versus general effects of tRNA synthetase overexpression.

• Heterologous ileS expression: L. plantarum expressing ileS from different bacterial species helps evaluate species-specific aspects of enzyme function and compatibility with host machinery .

Each control should be characterized under identical experimental conditions, with at least three biological replicates per strain to ensure statistical significance .

How can researchers minimize experimental bias when comparing different recombinant L. plantarum ileS constructs?

To minimize experimental bias when comparing different recombinant L. plantarum ileS constructs, researchers should implement a systematic approach addressing potential sources of variability at each experimental stage. Begin with construct design standardization by maintaining identical vector backbones, regulatory elements, and fusion tags across all constructs, differing only in the specific ileS sequence variants being tested .

Implement blind experimental protocols where researchers performing experiments and data analysis are unaware of construct identities until after data collection and initial processing. Randomize the order of sample processing and analysis to minimize systematic biases related to processing order or equipment drift. All constructs should be transformed into the same parental strain background prepared from a single clone to eliminate strain-to-strain variation .

Statistical design should include power analysis to determine appropriate sample sizes, typically requiring at least 5-8 biological replicates per construct for adequate power (80%) to detect differences of 20% or greater in key parameters . Include technical replicates (typically 2-3) for each biological replicate to account for measurement variability. Growth and induction conditions must be precisely controlled, ideally using bioreactors with automated monitoring of temperature, pH, and dissolved oxygen rather than shake flasks.

Data collection should include multiple parameters (growth, expression levels, enzyme activity) measured at several time points to establish comprehensive phenotypic profiles. Apply appropriate statistical methods including ANOVA with post-hoc tests and correction for multiple comparisons (Bonferroni or Benjamini-Hochberg procedures) .

What statistical approaches are most appropriate for analyzing variability in recombinant L. plantarum ileS expression?

When analyzing variability in recombinant L. plantarum ileS expression, researchers should employ a multi-tiered statistical approach appropriate for the complex biological systems involved. For comparing expression levels across different experimental conditions or construct designs, Analysis of Variance (ANOVA) with appropriate post-hoc tests (Tukey's HSD or Dunnett's test) provides robust identification of significant differences. Data should be checked for normality (Shapiro-Wilk test) and homogeneity of variance (Levene's test) before ANOVA application, with non-parametric alternatives (Kruskal-Wallis) implemented when assumptions are violated .

For time-course experiments measuring expression dynamics, repeated measures ANOVA or mixed-effects models should be employed to account for within-subject correlations. When multiple factors are examined simultaneously (e.g., temperature, pH, media composition), factorial design analysis with interaction terms is essential to identify synergistic or antagonistic effects .

Regression models can quantify relationships between expression levels and continuous variables such as inducer concentration, with polynomial terms included when non-linear relationships are observed. Variability analysis should incorporate calculation of coefficients of variation (CV) both within and between experimental batches, with CV values typically ranging from 15-25% for biological replicates of recombinant protein expression in L. plantarum systems .

How can researchers effectively validate the functionality of recombinant L. plantarum ileS?

Validating the functionality of recombinant L. plantarum ileS requires a comprehensive approach combining biochemical, genetic, and phenotypic analyses. Begin with in vitro enzymatic assays measuring aminoacylation activity, where purified recombinant ileS is incubated with ATP, isoleucine, and tRNA^Ile substrates under physiological conditions. Quantify charged tRNA using either radioactive amino acid incorporation or more modern techniques like acid gel electrophoresis coupled with northern blotting .

Complementation studies provide critical in vivo validation, where the recombinant ileS is expressed in an L. plantarum strain with reduced native ileS activity (through conditional knockdown or temperature-sensitive mutations). Successful restoration of growth under restrictive conditions confirms functionality. Specificity can be assessed by measuring mischarging rates with non-cognate amino acids, particularly structurally similar ones like leucine and valine .

Structural validation through circular dichroism spectroscopy confirms proper protein folding by comparing spectral signatures with wild-type ileS. Thermal shift assays assess protein stability, with functional enzymes typically showing cooperative unfolding transitions. Microscopic techniques including fluorescence recovery after photobleaching (FRAP) can track intracellular localization when using fluorescently-tagged constructs .

Finally, metabolomic analysis should reveal normalized amino acid incorporation patterns comparable to wild-type strains, particularly for isoleucine-rich proteins. These complementary approaches collectively provide robust validation of recombinant ileS functionality .

What approaches help identify and resolve unexpected phenotypes in recombinant L. plantarum ileS strains?

When faced with unexpected phenotypes in recombinant L. plantarum ileS strains, researchers should implement a systematic troubleshooting workflow combining reductionist and systems approaches. Begin with construct verification through sequencing and restriction analysis to confirm the absence of mutations or recombination events. Quantify expression levels of both recombinant and native ileS using qRT-PCR and Western blotting to identify potential dosage effects or interference with native enzyme function .

Conduct comparative phenotypic profiling using Biolog or similar methods to generate metabolic fingerprints that can direct further investigation toward specific affected pathways. Perform global transcriptome analysis (RNA-seq) comparing the recombinant strain to appropriate controls under identical conditions, focusing on stress response pathways and protein synthesis machinery .

For growth-related phenotypes, detailed growth curve analysis with varying media compositions can identify specific nutritional requirements or sensitivities. Competition assays with wild-type strains under various conditions can reveal subtle fitness effects. Examine protein synthesis rates using pulse-chase experiments with labeled amino acids to identify potential translational impacts .

When membrane or cell wall phenotypes are observed, analyze lipid profiles and cell wall composition using mass spectrometry. If immunological phenotypes appear in animal models, characterize cytokine profiles and immune cell populations to identify specific altered pathways . This systematic approach resembles successful troubleshooting strategies employed in other recombinant L. plantarum systems .

How should researchers interpret changes in host response when administering recombinant L. plantarum expressing modified ileS?

Interpreting changes in host response to recombinant L. plantarum expressing modified ileS requires careful discrimination between ileS-specific effects and general responses to the bacterial vector. Begin by establishing comprehensive baseline measurements using appropriate controls, including wild-type L. plantarum and strains expressing non-functional ileS variants, to isolate effects specifically attributable to the modified enzyme .

Host immunological parameters should be evaluated at multiple levels, including innate responses (cytokine profiles, dendritic cell activation patterns) and adaptive immunity (antibody production, T-cell responses). In experimental animal models, these measurements should span multiple time points (acute: 24-48 hours; intermediate: 7 days; long-term: 21-28 days) to capture the complete response dynamics .

Changes in host microbiome composition should be assessed using 16S rRNA sequencing or metagenomic approaches, with particular attention to shifts in microbial diversity and functional capacity. Correlational analysis between microbiome changes and host parameters can identify potential mechanistic links, similar to approaches used in other L. plantarum studies showing significant correlations between gut microbiota composition and metabolomic profiles .

Metabolomic analysis of host biological fluids (serum, urine, fecal extracts) can detect systemic effects on host metabolism, with particular focus on amino acid profiles and protein turnover markers. Tissue-specific responses should be evaluated in organs with high protein synthesis rates (liver, intestinal mucosa) through histological examination and transcriptomic analysis . Multi-omics data integration using network analysis approaches can then identify key regulatory nodes and pathways mediating the observed host responses.

How can recombinant L. plantarum ileS be utilized in therapeutic applications?

Recombinant L. plantarum ileS offers multiple therapeutic applications by leveraging the probiotic properties of L. plantarum combined with the specific activities of engineered ileS variants. Modified ileS with enhanced substrate specificity could serve as a targeted intervention for protein synthesis disorders by delivering precisely charged tRNAs to tissues with specific isoleucine incorporation defects. This approach parallels the targeted delivery mechanisms demonstrated in recombinant L. plantarum expressing other therapeutic proteins .

For modulating gut microbiome composition, recombinant L. plantarum expressing modified ileS can selectively influence the growth of specific bacterial populations, similar to how L. plantarum AR113 administration enriched beneficial Lactobacillus and Bacteroides populations . This selective microbiome modulation could be particularly valuable in conditions like inflammatory bowel disease where microbiome dysbiosis plays a central role.

Additionally, recombinant L. plantarum ileS can be engineered as a novel adjuvant system for mucosal vaccines, where coordinated expression of ileS variants optimized for antigen production alongside surface-displayed antigens enhances immune response, similar to approaches using L. plantarum to express FomA protein for protection against Fusobacterium-associated diseases . The efficacy of such systems could be enhanced by coupling ileS expression to environmental sensing promoters that activate in specific host compartments.

What long-term effects might recombinant L. plantarum ileS have on gut microbiome stability?

The long-term effects of recombinant L. plantarum ileS on gut microbiome stability involve complex ecological interactions operating across multiple temporal and spatial scales. Initial colonization dynamics typically show transient dominance of the administered strain, gradually equilibrating to a new steady state over 2-4 weeks, based on patterns observed with other recombinant L. plantarum strains . The modified ileS could confer competitive advantages through enhanced protein synthesis efficiency, potentially extending colonization duration compared to wild-type strains.

Ecological niche displacement may occur through several mechanisms: altered metabolic profiles affecting resource utilization, modified interactions with host epithelium changing spatial distribution within the gut, and potential horizontal gene transfer of the recombinant ileS construct to other microbiome members . The magnitude of these effects depends on dosage, administration frequency, and host factors including diet and immune status.

Longitudinal microbiome analysis in animal models receiving recombinant L. plantarum reveals that taxonomic perturbations typically affect 15-20% of resident genera, with functional redundancy often preserving metabolic pathway representation despite taxonomic shifts . Core metabolic functions show greater resilience than specialized pathways, with the latter showing more sustained alterations. Potential consequences include altered colonization resistance to pathogens, modified metabolite profiles affecting host physiology, and recalibration of immune homeostasis, particularly in mucosal immunity .

How does recombinant ileS expression influence L. plantarum's ability to function as a probiotic?

Recombinant ileS expression can significantly alter L. plantarum's probiotic functionality through several interconnected mechanisms. Enhanced ileS activity potentially increases growth rates and stress tolerance by optimizing protein synthesis capacity, particularly under nutrient-limited conditions found in the gastrointestinal tract. This improved fitness could extend gut transit time and enhance competitive colonization ability against potential pathogens .

Modified protein synthesis machinery may alter the production of key probiotic effectors, including bacteriocins, exopolysaccharides, and immunomodulatory proteins. Studies with other recombinant L. plantarum strains demonstrate that even single gene modifications can significantly alter the secretome profile, affecting host-microbe interactions . Changes in surface protein expression patterns due to altered ileS function could modify adherence to intestinal mucosa, a critical determinant of probiotic efficacy.

Metabolic consequences of recombinant ileS expression include potential shifts in amino acid utilization and production, affecting both bacterial physiology and host-microbe metabolic interactions. These changes may enhance or diminish specific probiotic benefits such as immune modulation, epithelial barrier function support, and pathogen exclusion . Administration of L. plantarum with enhanced ileS activity might therefore require recalibration of dosing regimens and timing to optimize probiotic effects compared to wild-type strains.

What ethical considerations should researchers address when developing recombinant L. plantarum ileS for research or therapeutic purposes?

Researchers developing recombinant L. plantarum ileS must address several critical ethical considerations throughout the research and development process. Biosafety concerns are paramount, requiring robust containment strategies to prevent unintended environmental release. Horizontal gene transfer risk assessment should evaluate the potential for antibiotic resistance markers or recombinant ileS sequences to spread to other microorganisms, with design modifications implemented to minimize these risks .

Informed consent protocols for human studies must clearly communicate the nature of genetically modified organisms and potential unknown long-term effects, particularly for first-in-human applications. Transparency regarding the genetic modifications and their purpose is essential for maintaining public trust in biotechnology applications .

Ecological impact assessments should model potential consequences of environmental exposure, including effects on non-target organisms and ecosystem functions. Sustainability considerations include evaluating manufacturing processes, energy requirements, and waste management associated with large-scale production .

Equitable access concerns arise particularly for therapeutic applications, necessitating strategies to ensure that beneficial technologies are available to diverse populations globally. Regulatory compliance pathways must be navigated early in development, with consideration of varying international standards for genetically modified organisms in different contexts (research, food, pharmaceutical) .

Ongoing post-approval monitoring requirements should be established to detect unexpected consequences, with clear communication channels to rapidly address emerging concerns. These ethical frameworks should be integrated throughout the research process rather than applied retrospectively, ensuring responsible innovation in this promising area .