Recombinant Lactobacillus plantarum S-ribosylhomocysteine lyase (luxS)

What is S-ribosylhomocysteine lyase (LuxS) in Lactobacillus plantarum?

S-ribosylhomocysteine lyase (EC 4.4.1.21), encoded by the luxS gene, is a metalloenzyme that cleaves the thiol ester bond in S-ribosylhomocysteine (SRH), producing homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD) . In Lactobacillus plantarum, LuxS functions as part of the AI-2/LuxS-mediated quorum sensing (QS) system, which regulates various physiological functions including growth characteristics, bacteriostatic ability, stress tolerance, and adhesion to intestinal epithelial cells . The enzyme exists as a homodimer with two identical active sites at the dimer interface, formed by residues from both subunits, and contains a divalent metal ion (Zn²⁺) in its active sites .

How does the AI-2/LuxS-mediated quorum sensing system function in bacteria?

The AI-2/LuxS-mediated quorum sensing system functions through the following mechanism:

• The LuxS enzyme converts S-ribosylhomocysteine (SRH) into homocysteine and DPD

• DPD undergoes spontaneous rearrangements to form AI-2, the signaling molecule

• AI-2 is secreted by bacteria and can be detected by other bacteria in the environment

• Upon reaching a threshold concentration (indicating a critical cell density), AI-2 triggers changes in gene expression

In L. plantarum specifically, this system regulates physiological functions including biofilm formation, bacteriostatic action, and stress tolerance . The system works differently depending on whether the bacteria are in mono-culture or co-culture with other species. Research shows that in mono-cultivation, luxS deletion may not significantly affect bacteriocin production, while in co-cultivation with other species (e.g., L. helveticus), bacteriocin production significantly decreases in luxS mutants .

What is the role of LuxS in bacterial metabolism beyond quorum sensing?

Beyond its role in quorum sensing, LuxS plays crucial roles in bacterial metabolism:

These metabolic functions explain why luxS mutations have pleiotropic effects beyond just disrupting quorum sensing signals.

What techniques are used to create luxS mutants in L. plantarum for functional studies?

The creation of luxS mutants in L. plantarum typically employs the following methodologies:

• Two-Step Homologous Recombination: This is the most commonly reported method. The process involves:

  • Amplification of luxS gene-flanked fragments using PCR

  • Fusion of these fragments by overlap extension PCR

  • Ligation into a temperature-sensitive plasmid (e.g., pFED760)

  • Transformation into competent E. coli for plasmid propagation

  • Extraction of recombinant plasmids and transformation into L. plantarum by electroporation

  • Two-step homologous recombination controlled by temperature to regulate plasmid replication and suicide

• Homologous Recombination With Selectable Markers: This alternative approach has been used for L. plantarum KLDS1.0391:

  • PCR amplification of the upstream and downstream regions of the luxS gene

  • Construction of a recombinant plasmid carrying homologous fragments

  • Selection of successful transformants using appropriate markers

  • Confirmation of gene deletion by PCR and sequencing

For phenotypic verification of successful luxS deletion, researchers typically measure AI-2 activity using reporter strains like Vibrio harveyi BB170 .

How does luxS deletion affect the bacteriostatic ability of L. plantarum?

LuxS deletion has significant effects on the bacteriostatic ability of L. plantarum, with notable differences between mono-cultivation and co-cultivation conditions:

In Mono-cultivation:

• The bacteriostatic ability of L. plantarum SS-128 ΔluxS mutant shows no significant change compared to wild-type when grown alone .

• Metabolic analysis reveals that wild-type strains exhibit higher pyruvate metabolic efficiency, higher LDH levels, and greater metabolite overflow than ΔluxS mutants, potentially contributing to stronger bacteriostatic ability .

In Co-cultivation:

• The 'spot-on-the-lawn' method demonstrates that bacteriocin production by L. plantarum KLDS1.0391 significantly decreases (P < 0.01) when the luxS mutant is co-cultivated with L. helveticus KLDS1.9207 .

• LC-MS/MS analysis shows that proteins involved in several key metabolic pathways are differentially expressed between wild-type and ΔluxS strains during co-cultivation, while no significant differences in proteins related to bacteriocin biosynthesis are found during mono-cultivation .

These findings suggest that the bacteriostatic effects of LuxS are context-dependent and particularly important during interspecies interactions.

What molecular mechanisms underlie luxS-mediated regulation of bacteriocin production?

The molecular mechanisms underlying luxS-mediated regulation of bacteriocin production in L. plantarum involve multiple pathways and regulatory systems:

• Two-Component Regulatory System: Proteomic analysis reveals that the sensor histidine kinase AgrC (from the LytTR family) is differentially expressed between luxS mutant and wild-type strains during co-cultivation with other bacterial species .

• Metabolic Pathway Regulation: LuxS affects bacteriocin production through its influence on:

  • Carbohydrate metabolism

  • Amino acid metabolism

  • Fatty acid synthesis and metabolism

• Quorum Sensing Signal Integration: AI-2 produced by LuxS functions as a signaling molecule that can affect bacteriocin gene expression directly or indirectly by modulating global regulatory networks .

• Interspecies Communication: The significant decrease in bacteriocin production observed during co-cultivation but not mono-cultivation suggests that luxS plays a crucial role in interspecies communication that ultimately affects bacteriocin synthesis .

These findings demonstrate that bacteriocin production is not simply controlled by direct gene regulation but involves complex metabolic and signaling networks influenced by luxS.

How does luxS influence stress tolerance and adhesion ability in L. plantarum?

The luxS gene significantly impacts stress tolerance and adhesion ability in L. plantarum through multiple mechanisms:

Stress Tolerance:

• When luxS is deleted in L. plantarum KLDS1.0391, acid and bile salt tolerance significantly decrease (p < 0.05) .

• Survival rates in simulated digestive juice (SDJ) are markedly reduced in luxS deletion strains compared to wild-type strains .

• The exact molecular mechanisms may involve altered membrane composition, stress response proteins, or metabolic adaptations affected by luxS deletion.

Adhesion Ability:

• The adhesion ability of luxS deletion strains to Caco-2 cells is significantly lower than that of wild-type strains (p < 0.05) .

• The ability of luxS mutant strains to adhere (competition, exclusion, and displacement) to Escherichia coli ATCC 25922 is significantly reduced compared to wild-type strains (p < 0.05) .

• For adhesion inhibition against Salmonella typhimurium ATCC 14028, a significant decrease is observed only in the exclusion adhesion mechanism of the luxS mutant strain (p < 0.05) .

These findings suggest that luxS plays a crucial role in L. plantarum's ability to survive gastrointestinal transit and colonize the intestinal environment, which are important characteristics for probiotic applications.

What approaches can be used to study structural dynamics of LuxS enzyme?

The structural dynamics of LuxS enzyme can be studied using various computational and experimental approaches:

Computational Approaches:

• Molecular Dynamics (MD) Simulations: This technique can evaluate structural and dynamical properties of LuxS enzyme, including:

  • Dynamics of the interfacial region between monomeric chains

  • Inter-residual contacts and associated interface area in ligand-free and ligand-bound states

  • Binding patterns of inhibitors to the enzyme

• Free Energy Calculations: Techniques like thermodynamic integration can help correlate dynamical properties with inhibition potential of different ligands .

Experimental Approaches:

• X-ray Crystallography: Determines the three-dimensional structure of LuxS, revealing that it exists as a homodimer with two identical active sites at the dimer interface .

• Site-Directed Mutagenesis: Identifies critical residues involved in substrate binding and catalysis.

• Enzyme Kinetics Assays: Measures the catalytic efficiency of wild-type and mutant LuxS enzymes.

A comprehensive study using these approaches can provide insights into the structural basis of LuxS function, potentially leading to the development of novel inhibitors targeting bacterial quorum sensing systems.

How do transcriptomic and metabolomic profiles differ between wild-type and luxS mutant L. plantarum strains?

Comparative analysis of wild-type and luxS mutant L. plantarum strains reveals significant differences in their transcriptomic and metabolomic profiles:

Transcriptomic Differences:

• In L. plantarum SS-128, combined transcriptomics and metabolomics analysis shows that the wild-type strain exhibits differential gene expression related to pyruvate metabolism and energy production compared to the ΔluxS mutant .

• The absence of luxS induces changes in gene expression associated with the cysteine cycle and central metabolic pathways .

Metabolomic Differences:

• Wild-type L. plantarum shows higher pyruvate metabolic efficiency, leading to higher LDH levels and metabolite overflow .

• Proteomic analysis using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) reveals that luxS deletion alters the expression level of proteins involved in:

  • Carbohydrate metabolism

  • Amino acid metabolism

  • Fatty acid synthesis and metabolism

  • Two-component regulatory system

Table 1: Key Differential Metabolic Pathways Between Wild-Type and ΔluxS L. plantarum

These differences explain the phenotypic changes observed in luxS mutants, including altered bacteriostatic ability, stress tolerance, and adhesion properties.

What controls should be included when studying luxS function in L. plantarum?

When designing experiments to study luxS function in L. plantarum, the following controls are essential:

• Wild-Type Control: Always include the parent strain as a positive control to establish baseline phenotypes and functions .

• Complemented Strain: A luxS-mutant strain complemented with a functional luxS gene should be included to confirm that observed phenotypic changes are specifically due to luxS deletion rather than polar effects or secondary mutations .

• Chemical Complementation: Addition of synthetic AI-2 to luxS mutant cultures can help distinguish between metabolic effects of luxS deletion and signaling effects mediated by AI-2 .

• Mono-cultivation vs. Co-cultivation: Both conditions should be tested as luxS effects can differ dramatically between these scenarios (e.g., bacteriocin production) .

• Time-Course Studies: Include multiple time points to capture temporal dynamics of luxS-dependent processes, as quorum sensing is inherently dependent on growth phase and cell density .

• Reporter Strains: For AI-2 activity assays, validated reporter strains like Vibrio harveyi BB170 should be used as positive controls .

• Environmental Variables: Control for pH, temperature, and media composition, as these factors can influence quorum sensing dynamics and potentially mask or exaggerate luxS-dependent phenotypes.

How can quantitative methods be applied to measure AI-2 production in L. plantarum studies?

Several quantitative methods can be applied to measure AI-2 production in L. plantarum studies:

• Bioluminescence Assay with Reporter Strains:

  • Vibrio harveyi BB170 reporter strain responds to AI-2 by producing bioluminescence

  • Cell-free supernatants from L. plantarum cultures are added to the reporter strain

  • Bioluminescence is measured using a luminometer

  • Results are typically expressed as relative light units (RLU) or fold-induction compared to negative controls

• High-Performance Liquid Chromatography (HPLC):

  • AI-2 precursor DPD can be quantified using HPLC

  • Samples are derivatized to improve detection

  • Known concentrations of synthetic DPD are used to generate standard curves

  • This method provides absolute quantification of AI-2 precursor molecules

• Mass Spectrometry:

  • Liquid chromatography-mass spectrometry (LC-MS) can be used for sensitive detection of AI-2

  • This technique allows for structural identification and quantification

  • Multiple reaction monitoring (MRM) can be applied for improved sensitivity and specificity

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Antibodies against AI-2 can be used in competitive ELISA formats

  • Provides high-throughput quantification option

  • Less commonly used due to challenges in generating specific antibodies

These methods enable researchers to correlate AI-2 production levels with phenotypic changes observed in wild-type and luxS mutant strains, facilitating mechanistic understanding of quorum sensing in L. plantarum.

What are the challenges in purifying recombinant LuxS from L. plantarum expression systems?

Purification of recombinant LuxS from L. plantarum expression systems presents several challenges:

• Protein Solubility: LuxS is known to form inclusion bodies when overexpressed, requiring optimization of expression conditions (temperature, induction time, and inducer concentration) to enhance solubility.

• Metal Cofactor Requirement: LuxS is a metalloenzyme containing a divalent metal ion (Zn²⁺) in its active site . Ensuring proper metal incorporation during expression and purification is critical for obtaining active enzyme.

• Homodimeric Structure: LuxS exists as a homodimer with two identical active sites at the dimer interface . Purification conditions must preserve this quaternary structure to maintain enzyme activity.

• Expression Host Considerations:

  • When expressing LuxS in L. plantarum, endogenous LuxS activity may interfere with characterization of the recombinant enzyme

  • Using luxS knockout strains as expression hosts can overcome this challenge

• Purification Strategy:

  • Affinity tags (His-tag, GST) can facilitate purification but may affect enzyme activity

  • Tag removal may be necessary for structural and functional studies

  • Multi-step purification protocols involving ion-exchange chromatography, size-exclusion chromatography, and affinity chromatography are often required

• Stability During Purification: LuxS may exhibit reduced stability during purification steps, necessitating the addition of stabilizing agents (glycerol, reducing agents) to buffers.

Understanding and addressing these challenges is essential for obtaining pure, active recombinant LuxS for structural and functional studies.

How can researchers resolve data contradictions in luxS function across different L. plantarum strains?

Resolving data contradictions in luxS function across different L. plantarum strains requires systematic approaches:

• Standardized Experimental Protocols:

  • Establish uniform methods for luxS mutant construction

  • Use consistent growth conditions and media compositions

  • Adopt standardized assays for phenotypic characterization

  • Implement identical analytical techniques for transcriptomic and metabolomic analyses

• Strain Characterization and Genomic Analysis:

  • Perform whole-genome sequencing to identify potential genetic variations that could influence luxS function

  • Analyze genomic context of luxS gene across strains to identify differences in regulatory elements

  • Compare strain phylogeny to determine evolutionary relationships

• Comparative Studies:

  • Conduct side-by-side comparisons of multiple L. plantarum strains under identical conditions

  • Include reference strains with well-characterized luxS function

  • Analyze multiple isolates of the same strain to account for laboratory adaptation

• Context-Dependent Effects:

  • Explicitly test environmental and growth conditions that might influence luxS function

  • Examine mono-cultivation versus co-cultivation scenarios, as strain-specific differences may only emerge during interspecies interactions

  • Investigate growth phase-dependent effects, as quorum sensing dynamics can vary with cell density

• Meta-Analysis Approach:

  • Compile and systematically analyze published data on luxS function across L. plantarum strains

  • Identify patterns and sources of variation

  • Develop predictive models to explain strain-specific differences

By implementing these approaches, researchers can distinguish between genuine strain-specific differences in luxS function and experimental artifacts, leading to a more cohesive understanding of luxS-mediated quorum sensing in L. plantarum.

What are the potential applications of engineered LuxS variants in probiotic research?

Engineered LuxS variants offer several potential applications in probiotic research:

• Enhanced Stress Resistance: Given that luxS influences stress tolerance , engineered variants with optimized function could improve probiotic survival during gastrointestinal transit and food processing.

• Improved Adhesion Properties: Since luxS affects adhesion to intestinal epithelial cells , variants that enhance this property could increase probiotic colonization efficiency and persistence in the gut.

• Modified Bacteriocin Production: Engineered LuxS variants could potentially modulate bacteriocin production , enabling the development of probiotics with enhanced antimicrobial activity against specific pathogens.

• Controlled Quorum Sensing: LuxS variants with altered catalytic properties could modify AI-2 production rates, allowing fine-tuned control over quorum sensing-dependent functions in probiotic strains.

• Interspecies Communication Modulators: Engineered LuxS could influence how probiotics interact with the host microbiome by altering AI-2 production and recognition patterns.

• Biofilm Formation Control: LuxS affects biofilm formation , and engineered variants could potentially enhance beneficial biofilm formation while reducing pathogenic biofilm development.

• Biosensor Development: LuxS variants could be incorporated into biosensor systems for detecting specific metabolites or environmental conditions in the gut.

These applications could contribute to next-generation probiotic design with improved functionality, stability, and therapeutic efficacy.

How might systems biology approaches advance our understanding of luxS function in L. plantarum?

Systems biology approaches can significantly advance our understanding of luxS function in L. plantarum through integrative analysis:

• Multi-omics Integration:

  • Combining transcriptomics, proteomics, and metabolomics data from wild-type and luxS mutant strains can reveal interconnected networks regulated by LuxS

  • This approach can identify emergent properties not observable through individual omics analyses

• Network Modeling:

  • Construction of regulatory and metabolic networks can map the global impact of luxS mutation

  • Flux balance analysis can predict metabolic rewiring in response to luxS deletion

  • Boolean network models can capture regulatory dynamics of quorum sensing systems

• Genome-Scale Models:

  • Development of genome-scale metabolic models of L. plantarum incorporating luxS-dependent pathways

  • In silico prediction of phenotypic changes under various conditions

  • Identification of essential genes and pathways that interact with luxS

• Host-Microbe Interaction Models:

  • Systems-level analysis of how luxS-mediated quorum sensing affects interactions between L. plantarum and host cells

  • Integration of host response data (transcriptomics, proteomics) with bacterial omics data

• Temporal and Spatial Dynamics:

  • Time-resolved analyses to capture the dynamics of luxS-dependent processes

  • Spatial models to understand how quorum sensing operates in structured environments like biofilms

• Machine Learning Applications:

  • Pattern recognition in large-scale datasets to identify subtle luxS-dependent phenotypes

  • Predictive modeling of quorum sensing responses under various conditions