Recombinant Lactobacillus plantarum UDP-N-acetylmuramoyl-L-alanyl-D-glutamate--2,6-diaminopimelate ligase (murE)

What is Lactobacillus plantarum and why is it significant for recombinant protein expression?

Lactobacillus plantarum (recently reclassified as Lactiplantibacillus plantarum) is a probiotic bacterium naturally found in the mouth, gut, and fermented foods. As a lactic acid bacterium (LAB), it is internationally recognized as a food-grade microorganism with applications in improving gastrointestinal tract function and immune regulation . Its significance for recombinant protein expression stems from several advantages: it can survive passage through the gastrointestinal tract, possesses GRAS (Generally Recognized As Safe) status, and can efficiently express surface-anchored proteins using appropriate expression vectors. When engineering recombinant L. plantarum, researchers should note the recent taxonomic reclassification from Lactobacillus to Lactiplantibacillus genus in April 2020, although some product labels and publications may still use the former classification .

What is the MurE enzyme and what role does it play in bacterial cell wall synthesis?

The MurE enzyme (UDP-MurNAc-tripeptide ligase) is a cytoplasmic enzyme that catalyzes a critical step in bacterial peptidoglycan biosynthesis. Specifically, it adds meso-diaminopimelic acid (m-DAP) to the nucleotide precursor UDP-N-acetylmuramoyl-L-alanyl-D-glutamate, coupled with ATP hydrolysis . This reaction is essential for building the peptide side chains that will eventually cross-link the glycan strands in bacterial cell walls. The importance of this enzyme is underscored by the fact that peptidoglycan synthesis is a target for many antibiotics, and the cytoplasmic enzymes in this pathway, including MurE, have generated renewed interest as targets for developing new antimicrobials, particularly against resistant bacteria like Mycobacterium tuberculosis .

What expression systems are commonly used for producing recombinant L. plantarum with surface-displayed proteins?

For expressing recombinant proteins on the surface of L. plantarum, researchers typically employ E. coli-Lactobacillus shuttle expression vectors. A common approach involves using polyglutamate synthase A (pgsA) derived from Bacillus subtilis as a surface display element . To address environmental concerns related to antibiotic resistance markers, modern expression systems often utilize antibiotic-free screening markers such as the aspartic acid-β-semialdehyde dehydrogenase (asd) gene and the alanine racemase (alr) gene . The expression strategy may include an asd gene-deficient E. coli strain (e.g., E. coli χ6212) as the plasmid donor and an alr gene deletion L. plantarum strain (e.g., NC8Δ) as the host strain . This system allows for stable expression without introducing antibiotic resistance genes into the environment.

What structural features characterize the MurE ligase and how do they compare to related enzymes in the peptidoglycan synthesis pathway?

The MurE ligase exhibits a complex three-domain structure that contributes to its specific catalytic function. Crystal structure analysis of the Escherichia coli enzyme has revealed that the protein consists of three domains, two of which have a topology similar to equivalent domains found in UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase (MurD) . MurD catalyzes the immediate previous step in peptidoglycan precursor biosynthesis. The refined structural model of MurE has identified the binding site for UDP-MurNAc-L-Ala-gamma-D-Glu-meso-A2pm, providing insights into substrate recognition . Notably, a carbamylated lysine residue has been observed in the active site, similar to MurD. The structural determinant responsible for selecting the specific amino acid to be added to the nucleotide precursor has also been identified . Understanding these structural features is essential for designing mutations to study enzyme function and developing potential inhibitors targeting this enzyme.

What essential residues control the catalytic activity of MurE, and how do mutations affect enzyme function?

Site-directed mutagenesis studies have identified several critical residues essential for MurE catalytic activity. Residues K157, E220, D392, and R451 are crucial for catalysis, as replacing them with alanine significantly impacts enzyme activity . Additionally, the N449 residue plays an important role in substrate binding, as demonstrated by mutations to aspartate . Interestingly, the first 24 amino acid residues at the N-terminus do not appear essential for catalytic activity. Mutations of key residues (K157A, E220A, D392A) result in uncoupled ATP hydrolysis from catalysis, with ATP hydrolysis rates enhanced by at least partial occupation of the uridine nucleotide dipeptide binding site . These findings provide critical insights into the catalytic mechanism and may guide the development of specific inhibitors targeting the MurE enzyme.

How does ATP binding and hydrolysis couple with catalytic activity in MurE, and what experimental approaches can assess this coupling?

The MurE ligase couples ATP hydrolysis with the formation of an amide bond between meso-diaminopimelic acid and UDP-N-acetylmuramoyl-L-alanyl-D-glutamate. Research has shown that certain mutations (K157A, E220A, D392A) can disrupt this coupling, resulting in ATP hydrolysis without productive catalysis . To experimentally assess this coupling, researchers can employ assays that simultaneously measure ATP hydrolysis rates and product formation. This typically involves quantification of inorganic phosphate release as an indicator of ATP hydrolysis, alongside HPLC or mass spectrometry-based detection of the reaction products. Varying substrate concentrations and determining kinetic parameters (Km and Vmax) for both wild-type and mutant enzymes provides insights into how specific residues contribute to this coupling mechanism. Additionally, structural analysis through X-ray crystallography with ATP analogs can reveal conformational changes associated with nucleotide binding and hydrolysis .

What strategies can be employed for developing antibiotic-free selection systems when creating recombinant L. plantarum?

Developing antibiotic-free selection systems for recombinant L. plantarum requires careful consideration of both the vector design and host strain selection. One effective approach utilizes complementation of essential gene deletions. For example, researchers have successfully employed the aspartic acid-β-semialdehyde dehydrogenase (asd) gene and the alanine racemase (alr) gene as antibiotic-free screening markers . This approach requires:

• Creating a host strain with a deletion in an essential gene (e.g., alr gene deletion L. plantarum strain NC8Δ)

• Using an expression vector containing the complementing gene

• Employing an intermediate host with a different gene deletion (e.g., asd gene-deficient E. coli χ6212)

This strategy ensures plasmid maintenance without selective pressure from antibiotics, as only cells retaining the plasmid can synthesize essential cellular components. When designing such systems, researchers should consider the metabolic burden of complementation, potential growth rate reductions, and the stability of recombinant protein expression over multiple generations .

What techniques are most effective for verifying and quantifying surface display of recombinant proteins on L. plantarum?

Verification and quantification of surface-displayed proteins on L. plantarum requires a multi-faceted approach. Based on established protocols, the following techniques have proven effective:

• Immunoblotting: After collecting recombinant L. plantarum cells, researchers can use either ultrasonic fragmentation or freeze-thaw cycles (typically 5 cycles at -80°C) to prepare samples. SDS-PAGE followed by transfer to membranes and probing with specific primary antibodies (e.g., anti-H7N9 hemagglutinin antibodies for HA1-expressing strains) and appropriate HRP-labeled secondary antibodies allows detection of the expressed protein .

• Flow Cytometry: This technique enables quantitative assessment of surface-displayed proteins on intact cells. Cells are incubated with specific primary antibodies followed by fluorophore-conjugated secondary antibodies (e.g., PE-conjugated anti-mouse IgG). The proportion of positive cells and fluorescence intensity provides information about expression efficiency and uniformity .

• Indirect Immunofluorescence: This microscopy-based approach provides visual confirmation of surface localization. After antibody treatment (similar to flow cytometry preparation), fluorescence microscopy reveals the distribution pattern of the recombinant protein on the bacterial surface .

These complementary approaches provide both qualitative and quantitative data on recombinant protein expression.

How should induction conditions be optimized for maximum expression of recombinant proteins in L. plantarum?

Optimizing induction conditions for maximal expression of recombinant proteins in L. plantarum requires systematic evaluation of several parameters:

• Growth Phase: Induction timing is critical. Research indicates that adding inducing peptides when cultures reach an OD600 of approximately 0.3 (early logarithmic phase) often yields optimal results .

• Induction Agent: The specific inducer depends on the promoter system used. For SppIp-based systems, adding SppIp-inducing peptide at a concentration of 50 ng/mL has proven effective .

• Temperature: While standard growth occurs at 37°C, reducing the temperature to 30°C during induction can enhance protein folding and stability. Maintaining anaerobic conditions during both growth and induction phases is essential for L. plantarum .

• Induction Duration: The optimal duration should be determined empirically, typically ranging from 3-24 hours depending on the specific recombinant protein and expression system.

• Media Composition: Modified MRS media may improve expression yields for specific recombinant proteins.

Researchers should develop a matrix of conditions and assess expression levels using the verification techniques described above to identify optimal parameters for their specific recombinant construct.

How should variations in enzyme kinetic parameters be interpreted when comparing wild-type and mutant MurE enzymes?

• Km Values: Changes in Km reflect altered binding affinity for substrates. Increased Km values for one or more substrates (as observed with mutations K157A, E220A, D392A, R451A, and N449D) indicate reduced binding affinity, suggesting these residues participate in substrate recognition or binding pocket formation . The specific substrate affected by a particular mutation provides insight into which domain of the enzyme interacts with that substrate.

• Vmax and kcat: Reductions in these parameters without changes in Km suggest the mutation affects catalysis rather than substrate binding. Complete loss of catalytic activity (as seen with certain alanine substitutions) indicates an essential role in the reaction mechanism .

• Coupling Efficiency: For ATP-utilizing enzymes like MurE, the ratio of product formed to ATP hydrolyzed is a critical parameter. Mutations that result in ATP hydrolysis uncoupled from product formation (as seen with K157A, E220A, and D392A mutations) suggest roles in coordinating ATP utilization with the addition of meso-diaminopimelic acid .

• Substrate Specificity: Changes in substrate preference or the ability to use alternative substrates can reveal insights about residues involved in substrate discrimination.

These parameters should be analyzed collectively rather than in isolation to develop a comprehensive understanding of how specific residues contribute to enzyme function.

What statistical approaches are most appropriate for analyzing immunological responses to recombinant L. plantarum in experimental models?

When analyzing immunological responses to recombinant L. plantarum in experimental models, researchers should employ appropriate statistical methods based on the specific measurements:

• For Cellular Immune Responses: When quantifying increases in specific cell populations (e.g., CD4+IFN-γ+ and CD8+IFN-γ+ cells in the spleen and mesenteric lymph nodes), repeated measures ANOVA with post-hoc tests can determine significant differences between treatment groups over time .

• For Humoral Immune Responses: Analysis of antibody titers (e.g., IgG, IgG1, IgG2a, and IgA) typically employs non-parametric tests like Mann-Whitney U test or Kruskal-Wallis with Dunn's post-test, as antibody data often does not follow normal distribution .

• For Functional Assays: Hemagglutination inhibition (HI) assays should be analyzed using appropriate dilution-based statistical approaches, considering the semi-quantitative nature of these assays .

• Sample Size Determination: Power analysis should be conducted prior to experiments to ensure sufficient statistical power (typically 0.8 or greater) to detect biologically meaningful differences.

• Multiple Testing Correction: When performing multiple comparisons, researchers should apply appropriate corrections (e.g., Bonferroni, Holm-Sidak, or false discovery rate) to control type I error rates.

What are common challenges in expressing functional MurE in recombinant L. plantarum, and how can they be addressed?

Expressing functional MurE in recombinant L. plantarum presents several challenges that researchers should anticipate and address:

• Protein Folding and Solubility: MurE is a multi-domain protein that may face folding challenges when overexpressed. This can be addressed by optimizing growth temperature (typically lowering to 30°C during induction), including molecular chaperones, or using fusion tags that enhance solubility .

• Codon Usage Discrepancy: Differences in codon preference between the source organism of MurE and L. plantarum may reduce expression efficiency. Codon optimization of the MurE gene for L. plantarum is recommended, particularly for rare codons.

• Protein Toxicity: If MurE overexpression disrupts host cell wall synthesis, it may be toxic to the host. Using tightly regulated inducible promoters and optimizing induction conditions can mitigate this issue .

• Surface Display Challenges: For surface-anchored MurE, proper fusion with anchor proteins (like pgsA) is critical. Verification of surface localization using flow cytometry and immunofluorescence is essential to confirm correct display .

• Enzymatic Activity Retention: Fusion tags or surface anchoring may affect enzyme activity. Including appropriate linker sequences between the enzyme and fusion partners can help maintain proper folding and activity.

• Stability of Recombinant Strains: Without antibiotic selection, plasmid loss may occur. Using complementation-based selection systems (asd/alr) and verifying plasmid retention over multiple generations is recommended .

By anticipating these challenges and implementing the suggested solutions, researchers can improve the likelihood of successfully expressing functional MurE in recombinant L. plantarum.

What protocols are most effective for isolating and purifying MurE enzyme for biochemical and structural studies?

Effective isolation and purification of MurE enzyme for biochemical and structural studies requires a systematic approach:

• Expression System Selection: While L. plantarum is valuable for in vivo studies, E. coli expression systems are often preferred for obtaining large quantities of purified MurE for in vitro studies . BL21(DE3) or similar strains with T7 promoter-based expression vectors typically yield high protein amounts.

• Affinity Tag Selection: N-terminal or C-terminal His6-tags facilitate purification by immobilized metal affinity chromatography (IMAC). Tags should be positioned to minimize interference with enzyme activity, and cleavable tags may be considered for structural studies .

• Cell Lysis Protocol: Sonication in a buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and protease inhibitors typically provides good initial extraction. For membrane-associated variants, inclusion of 0.5-1% non-ionic detergents may improve solubilization.

• Purification Strategy:

  • Initial IMAC using Ni-NTA columns with imidazole gradient elution

  • Ion-exchange chromatography (typically Q-Sepharose) as a secondary purification step

  • Size-exclusion chromatography for final polishing and buffer exchange

• Activity Preservation: Including 10% glycerol, 1 mM DTT, and 1 mM EDTA in storage buffers helps maintain enzyme activity during storage at -80°C.

• Quality Control: Purity assessment by SDS-PAGE, activity verification using coupled enzyme assays, and dynamic light scattering to confirm monodispersity are essential before proceeding to detailed biochemical or structural studies .

This systematic approach has been successful in obtaining highly purified, active MurE enzyme suitable for crystallization and detailed enzymatic characterization.