Recombinant Lactobacillus johnsonii Bifunctional protein FolD 2 (folD2)

What is the Bifunctional protein FolD 2 (folD2) in Lactobacillus johnsonii and what functions does it serve in bacterial metabolism?

The Bifunctional protein FolD 2 (folD2) in Lactobacillus johnsonii is an enzyme involved in folate metabolism that typically catalyzes two sequential reactions in one-carbon metabolism: the NAD(P)-dependent dehydrogenation of 5,10-methylenetetrahydrofolate to 5,10-methenyltetrahydrofolate and the subsequent hydrolysis to 10-formyltetrahydrofolate. These reactions are critical for purine biosynthesis, amino acid metabolism, and methylation processes within bacterial cells.

The presence of a second folD gene (designated as folD2) suggests possible functional redundancy or specialization in folate metabolism pathways in L. johnsonii. This specialized metabolism may contribute to the bacterium's adaptability to different environmental niches and potentially its probiotic properties. The enzyme's activity influences the availability of one-carbon units essential for DNA synthesis, protein synthesis, and various cellular methylation reactions.

L. johnsonii, as a probiotic bacterium, demonstrates various beneficial metabolic capabilities, including the production of antimicrobial compounds and beneficial biological molecules, which might be influenced by folate-dependent pathways regulated by folD2 .

How does folD2 expression in L. johnsonii differ from other Lactobacillus species?

While the available search results don't provide specific comparative data on folD2 expression across Lactobacillus species, we can analyze differences based on genomic and functional characteristics:

L. johnsonii demonstrates unique metabolic capabilities compared to other Lactobacillus species, including the production of specific bioactive compounds like inulin fructans . The genomic analysis of L. johnsonii reveals its distinct antimicrobial properties, which may be influenced by specialized metabolic pathways that could involve folD2 .

What molecular techniques are commonly used to identify and isolate the folD2 gene from Lactobacillus johnsonii?

The identification and isolation of the folD2 gene from L. johnsonii typically involves a systematic approach using several molecular techniques:

• Genomic analysis and sequence identification:

  • Whole genome sequencing followed by bioinformatic analysis to identify folD2 based on homology to known folD genes

  • Comparative genomic analysis with reference databases

• Gene amplification strategies:

  • PCR amplification using specific primers designed based on the L. johnsonii genome sequence

  • Similar to techniques used for other L. johnsonii genes: "specific primer pairs were utilized to amplify the GM-CSF fragment from the PUC57-GM-CSF"

• Cloning approaches:

  • Restriction enzyme digestion and ligation into suitable vectors

  • Following protocols similar to those demonstrated in L. johnsonii genetic engineering: "The gene was inserted into the expression vector pPG612, resulting in the generation of the recombinant plasmid"

• Verification methods:

  • Restriction enzyme analysis: "Validation of this process was achieved through restriction enzyme digestion and sequencing, confirming the successful construction of recombinant plasmid"

  • DNA sequencing to confirm correct sequence

  • PCR confirmation of correct orientation

These approaches have proven successful for genetic manipulation of L. johnsonii, as demonstrated in the engineering of recombinant L. johnsonii expressing bovine GM-CSF, which could be adapted for folD2 isolation and expression .

What expression vectors are most suitable for recombinant protein production in Lactobacillus johnsonii?

Based on successful genetic engineering experiments with L. johnsonii, several expression vector characteristics are particularly important:

The pPG612 vector has been successfully used for recombinant protein expression in L. johnsonii, making it a tested platform for folD2 expression. The research demonstrates that "the recombinant plasmid pPG612-GM-CSF was transformed into Lactobacillus johnsonii competent cells by electroporation," confirming the functionality of this vector system .

For folD2 expression specifically, this vector could be modified by replacing the insert with the folD2 gene sequence while maintaining the same backbone elements that enable stable expression in L. johnsonii.

How can the electroporation technique be optimized specifically for Lactobacillus johnsonii transformation?

Successful transformation of L. johnsonii requires careful optimization of electroporation parameters. Research with L. johnsonii provides specific protocol details that can be adapted for folD2 transformation:

• Cell preparation protocol:

  • "The Lactobacillus johnsonii competent cells were thawed in an ice-water slurry for 10 min"

  • "5 μL of the recombinant plasmid was mixed gently with 100 μL of the Lactobacillus johnsonii competent cells"

  • "After incubating on ice for 5 min, transferred to a pre-chilled electroporation cuvette"

• Electroporation parameters:

  • "Applying a single electric pulse of 2.1 kV for 3 ms"

  • "Immediately after electroporation, the cells were chilled on ice for 10 min"

• Recovery conditions:

  • "The transformed cells were then transferred into 1,000 mL of MRS broth containing 15% sucrose"

  • "Incubated anaerobically at 37°C for 2 h"

• Selection protocol:

  • "The culture was centrifuged at 3,000 rpm 10 min, resuspended in 200 μL of MRS broth"

  • "Spread onto an MRS agar plate containing 10 μg/mL of chloramphenicol"

  • "The plate was incubated anaerobically at 37°C overnight"

This protocol has demonstrated efficacy for L. johnsonii transformation and could serve as an excellent starting point for folD2 transformation experiments, with potential adjustments based on specific construct characteristics.

What strategies can improve the stability of recombinant folD2 expression in Lactobacillus johnsonii?

Ensuring stable expression of recombinant folD2 in L. johnsonii requires attention to several key factors:

• Plasmid stability verification:

  • "The stability of the recombinant L. johnsonii pPG612-GM-CSF over 40 generations is demonstrated... providing evidence of its successful inheritance"

  • Regular testing across multiple generations is essential for confirming stable inheritance

• Selection pressure maintenance:

  • Continuous culture in media containing appropriate antibiotic (e.g., 10 μg/mL chloramphenicol)

  • Balanced selection pressure to maintain plasmid without excessive metabolic burden

• Expression level optimization:

  • Promoter selection for appropriate expression level

  • Codon optimization to enhance translation efficiency

  • Avoiding toxic overexpression that might select for non-expressing mutants

• Media and growth condition optimization:

  • "Growth of L. johnsonii MT4 in MRS, BHI, and biofilm medium displayed different degrees of acidification"

  • Media selection can significantly impact protein expression stability

  • pH control may be particularly important given L. johnsonii's acidification tendencies

• Alternative approaches:

  • Chromosomal integration for maximum stability

  • Balanced-lethal systems linking plasmid maintenance to essential gene function

  • Addiction systems to ensure plasmid retention

These strategies can be tailored based on specific experimental goals and the characteristics of the folD2 construct being expressed.

How does codon optimization affect folD2 expression efficiency in Lactobacillus johnsonii?

Codon optimization is a critical factor for efficient folD2 expression in L. johnsonii, which typically shows distinct codon preferences as a Gram-positive bacterium with moderate GC content:

The synthetic gene approach has been successfully used for expression in L. johnsonii: "The gene encoding bovine GM-CSF (456 bp) was artificially synthesized" . This suggests that gene synthesis with codon optimization is a viable approach for expressing folD2 in this species.

For folD2 expression, codon optimization should be considered essential, particularly if the native sequence comes from a phylogenetically distant organism. Experimental comparison between native and codon-optimized folD2 variants would help quantify the expression improvement.

What analytical methods are most effective for confirming the expression of recombinant folD2 protein in Lactobacillus johnsonii?

Confirming successful expression of recombinant folD2 in L. johnsonii requires a multi-level verification approach:

• Genetic confirmation methods:

  • PCR verification: "PCR results confirmed the successful transfer of the recombinant plasmid into L. johnsonii"

  • Sequencing validation: "Sequencing result confirmed that the inserted sequence corresponds to the target gene"

  • Plasmid stability testing: "The stability of the recombinant L. johnsonii over 40 generations is demonstrated"

• Protein expression verification:

  • Western blotting: "Western blotting revealed a band corresponding to the protein in the cell lysates of recombinant L. johnsonii, confirming the successful construction"

  • For folD2, appropriate antibodies would be required, either commercial or custom-developed

  • SDS-PAGE to visualize protein expression levels

• Functional validation approaches:

  • Enzymatic activity assays specific to folD2's bifunctional activities

  • Complementation studies in folD-deficient strains

  • Metabolic product analysis

The systematic verification process demonstrated in L. johnsonii research provides a template for folD2 expression confirmation: genetic confirmation first, followed by protein expression confirmation, and finally functional validation .

How can the enzymatic activity of recombinant folD2 be measured accurately?

Accurate measurement of folD2's bifunctional enzymatic activity requires careful assay design and optimization:

• Methylenetetrahydrofolate dehydrogenase (MTHFD) activity assay:

  • Principle: Spectrophotometric measurement of NAD(P)+ reduction to NAD(P)H at 340 nm

  • Substrate: 5,10-methylenetetrahydrofolate

  • Reaction conditions: pH 7.0-7.5, 30-37°C, appropriate buffer system

• Methenyltetrahydrofolate cyclohydrolase (MTHFC) activity assay:

  • Principle: Spectrophotometric monitoring of 5,10-methenyltetrahydrofolate conversion

  • Detection: Absorbance changes at wavelengths specific to substrate/product (typically 350 nm)

  • Controls: Heat-inactivated enzyme and substrate-free reactions

• Sample preparation considerations:

  • Cell lysis optimization: Enzymatic, mechanical, or chemical methods specific to L. johnsonii

  • Protein extraction conditions that preserve enzymatic activity

  • Buffer composition to maintain enzyme stability

• Kinetic parameter determination:

  • Km and Vmax measurement for both enzymatic activities

  • Effects of pH, temperature, and ionic strength

  • Potential inhibitor profiling

While the search results don't provide specific methods for measuring folD2 activity, enzymatic kit-based approaches similar to those used for analyzing lactate production in L. johnsonii might be adaptable: "The DL-lactate kit (Megazyme) was used following the manufacturer's recommendations with minor changes" .

What approaches can be used to study the interactions between recombinant folD2 and other proteins in Lactobacillus johnsonii?

Studying protein-protein interactions involving folD2 in L. johnsonii requires specialized techniques adapted to bacterial systems:

To study potential interactions between folD2 and other folate metabolism enzymes, these approaches could be integrated with metabolic analyses to correlate physical interactions with pathway functionality. The co-aggregation properties of L. johnsonii, as demonstrated in its interactions with other microorganisms, suggest that protein-protein interactions play important roles in this bacterium's physiology .

How can researchers assess the impact of folD2 overexpression on Lactobacillus johnsonii metabolism?

Assessing the metabolic impact of folD2 overexpression requires a multi-omics approach:

These comprehensive analyses would provide insights into how folD2 overexpression affects both direct folate metabolism and broader cellular physiology in L. johnsonii.

How can recombinant Lactobacillus johnsonii expressing folD2 be applied in folate metabolism studies?

Recombinant L. johnsonii expressing folD2 offers several valuable applications for folate metabolism research:

• Enhanced folate production models:

  • Overexpression studies to identify rate-limiting steps in folate biosynthesis

  • Analysis of gene dosage effects on pathway flux

  • Comparative studies with other folate metabolism enzymes

• Metabolic engineering platform:

  • Creation of strains with enhanced folate production capacity

  • Study of pathway regulation and feedback mechanisms

  • Development of bacterial folate biofactories

• Structure-function relationship studies:

  • Expression of folD2 variants with targeted mutations

  • Domain-specific analysis of the bifunctional enzyme

  • Investigation of substrate specificity determinants

• Ecological and evolutionary studies:

  • Competitive fitness assessment of folD2-enhanced strains

  • Coevolution studies with host systems

  • Cross-feeding experiments in microbial communities

• Methodological approach:

  • Similar engineering strategies to those demonstrated for other L. johnsonii recombinant proteins: "recombinant Lactobacillus johnsonii strain was engineered to express bovine GM-CSF and administered to pregnant mice via vaginal perfusion"

  • Animal models for in vivo functional studies

  • Biofilm models for community interactions: "L. johnsonii and C. albicans were suspended in biofilm medium and seeded in multiwell plates or into μ-Slide 8-well chambered slides"

These applications demonstrate how recombinant L. johnsonii expressing folD2 could serve as a valuable research tool for understanding folate metabolism both within bacterial systems and in host-microbe interactions.

What are the potential advantages of using Lactobacillus johnsonii as an expression system for folD2 compared to other bacterial systems?

L. johnsonii offers several distinct advantages as an expression system for folD2 compared to other bacterial platforms:

For folD2 expression specifically, L. johnsonii's natural involvement in folate metabolism creates a physiologically relevant expression environment that may facilitate proper protein folding and function. The successful engineering of L. johnsonii to express other functional proteins demonstrates the system's viability for folD2 expression .

How might recombinant folD2 expression in Lactobacillus johnsonii impact antimicrobial properties?

The expression of recombinant folD2 in L. johnsonii could potentially influence its antimicrobial properties through several mechanisms:

• Metabolic interaction with antimicrobial pathways:

  • Folate metabolism interconnects with numerous biosynthetic pathways

  • Changes in one-carbon metabolism could affect production of antimicrobial compounds

  • Research shows that "L. johnsonii displays pH-dependent and pH-independent antagonistic interactions against C. albicans, resulting in inhibition of C. albicans planktonic growth and biofilm formation"

• Potential impacts on lactic acid production:

  • Folate-dependent pathways influence central carbon metabolism

  • Could affect organic acid production profiles: "Lactobacilli acidified MRS broth to pH 3.9 (from 6.5), BHI to pH 5.5 (from 7.5), and biofilm medium to pH 6 (from 8.4)"

  • Altered acidification could modify antimicrobial efficiency

• Effects on biofilm formation capacity:

  • "L. johnsonii auto-aggregates and co-aggregates with C. albicans"

  • Folate metabolism may influence surface properties and co-aggregation

  • Potential impact on competitive biofilm formation

• Interaction with host defense mechanisms:

  • Similar to engineered L. johnsonii expressing GM-CSF that "significantly reduced inflammation levels"

  • Potential immunomodulatory effects through folate-dependent pathways

  • Influence on host-microbe-pathogen interactions

• Experimental approaches:

  • Antimicrobial activity assays comparing wild-type and folD2-expressing strains

  • Co-culture experiments with pathogens like C. albicans

  • Biofilm inhibition studies using methods similar to those described: "Lactobacilli were found in physical proximity with Candida cells, particularly along the hyphae"

These potential impacts highlight the interconnected nature of bacterial metabolism and antimicrobial functions, suggesting that folD2 overexpression could have multifaceted effects on L. johnsonii's beneficial properties.

What experimental models are most appropriate for studying recombinant Lactobacillus johnsonii expressing folD2?

Selecting appropriate experimental models for studying folD2-expressing L. johnsonii depends on the specific research questions:

• In vitro models:

  • Pure culture systems for basic characterization

  • Growth in different media: "MRS, BHI, and biofilm medium (80% RPMI, 10% BHI, 10% FBS)"

  • Co-culture models with other microorganisms: "L. johnsonii (red) auto-aggregates and co-aggregates with C. albicans (blue)"

• Biofilm models:

  • Single-species biofilm: "Lactobacilli were seeded in multiwell plates or into μ-Slide 8-well chambered slides"

  • Multi-species biofilm interactions: "Lactobacilli were found in physical proximity with Candida cells, particularly along the hyphae"

  • Physical separation systems: "Millicell® 0.4 μm PCF Cell Culture Insert... inserts were placed into the wells containing C. albicans"

• Cell culture models:

  • Intestinal epithelial cell lines

  • Immune cell interaction studies

  • Host-microbe interface models

• Animal models:

  • Similar approaches to those used for other recombinant L. johnsonii: "administered to pregnant mice via vaginal perfusion"

  • Gnotobiotic animal models

  • Specialized models for studying folate metabolism

• Model selection considerations:

  • Research question specificity

  • Physiological relevance

  • Technical feasibility

  • Alignment with previous successful approaches

The diverse experimental models described in the search results demonstrate the versatility of L. johnsonii as a research organism, with applications ranging from pure culture biochemical studies to complex host-microbe interaction investigations .

What are common challenges in achieving stable expression of recombinant folD2 in Lactobacillus johnsonii?

Researchers working with recombinant folD2 expression in L. johnsonii may encounter several challenges:

• Plasmid stability issues:

  • Challenge: Loss of expression plasmid over multiple generations

  • Evidence: Importance of stability testing - "the stability of the recombinant L. johnsonii pPG612-GM-CSF over 40 generations is demonstrated"

  • Solution: Regular subculturing with antibiotic selection, chromosomal integration strategies

• Expression level variability:

  • Challenge: Inconsistent protein yield between experiments

  • Evidence: Growth condition sensitivity - "Growth of L. johnsonii MT4 in MRS, BHI, and biofilm medium displayed a different degree of acidification"

  • Solution: Standardized culture conditions, internal controls, optimized media composition

• Protein folding issues:

  • Challenge: Incorrectly folded or non-functional folD2

  • Solution: Temperature optimization, co-expression of chaperones, fusion partners

• Metabolic burden:

  • Challenge: Growth inhibition due to overexpression

  • Solution: Inducible promoters, balanced expression levels, adaptation periods

• Media and pH considerations:

  • Challenge: L. johnsonii significantly acidifies growth media

  • Evidence: "Acidified MRS broth to pH 3.9 (from 6.5), BHI to pH 5.5 (from 7.5)"

  • Solution: Buffered media, pH monitoring, fed-batch cultivation

These challenges can be systematically addressed through careful experimental design and optimization, building on the successful expression systems demonstrated for other recombinant proteins in L. johnsonii .

How can the enzymatic activity of recombinant folD2 be preserved during extraction and analysis?

Preserving folD2 enzymatic activity during extraction and analysis requires careful attention to buffer conditions and handling procedures:

• Cell disruption optimization:

  • Gentle lysis methods to prevent protein denaturation

  • Buffer selection based on enzyme stability requirements

  • Addition of protease inhibitors: "Supernatants were deproteinized with ice-cold 1 M hydrochloric acid 1 M NaOH"

• Buffer composition considerations:

  • pH optimization: Important given L. johnsonii's acidification tendencies

  • Inclusion of stabilizing agents (glycerol, reducing agents)

  • Appropriate cofactors: NAD(P)+ for folD2 activity

  • Testing multiple buffer systems to identify optimal conditions

• Temperature management:

  • Sample maintenance at 4°C during processing

  • Avoiding freeze-thaw cycles

  • Controlled storage conditions

• Rapid analysis protocols:

  • Minimizing time between extraction and analysis

  • Development of high-throughput activity assays

  • Parallelization of sample processing

• Activity verification methods:

  • Standard curves with known enzyme concentrations

  • Positive controls with commercial folD enzymes

  • Multiple analytical approaches to confirm activity

While the search results don't specifically address folD2 activity preservation, the enzymatic assay approaches used for L. johnsonii metabolite analysis provide relevant methodological insights: "Standard curves were prepared in the corresponding culture medium. Absorbance was measured at λ = 340 nm, and D−/L-lactate concentrations were calculated" .

What strategies can optimize the yield of recombinant folD2 in large-scale Lactobacillus johnsonii cultures?

Scaling up folD2 production in L. johnsonii requires attention to both genetic and process engineering factors:

The successful expression of recombinant proteins in L. johnsonii demonstrated in the search results provides a foundation for these optimization strategies . Additionally, understanding L. johnsonii's growth characteristics in different media compositions offers valuable insights for process development .

For large-scale production, an integrated approach combining genetic optimization (promoter strength, codon usage) with bioprocess engineering (media design, feeding strategies) would likely yield the best results for recombinant folD2 production.

How can researchers troubleshoot unexpected metabolic effects when expressing recombinant folD2 in Lactobacillus johnsonii?

When unexpected metabolic effects occur in folD2-expressing L. johnsonii, systematic troubleshooting approaches are essential:

• Characterization of growth phenotypes:

  • Compare growth curves between wild-type and recombinant strains

  • Assess media acidification patterns: "Regardless of the starting inoculum size, lactobacilli acidified MRS broth to pH 3.9"

  • Evaluate stress response indicators

• Metabolic profiling:

  • Targeted analysis of folate pathway metabolites

  • Broader metabolomic analysis to identify perturbations

  • Comparison across different growth conditions

• Expression level assessment:

  • Quantify folD2 expression levels

  • Correlation of expression with metabolic effects

  • Adjustment of expression through promoter engineering

• Pathway analysis:

  • Examine related metabolic pathways that may be affected

  • Consider metabolic burden of heterologous protein production

  • Investigate potential regulatory crosstalk

• Mitigation strategies:

  • Media supplementation to address metabolic imbalances

  • Adjustment of culture conditions

  • Genetic modifications to compensate for metabolic changes

• Experimental design:

  • Include appropriate controls (empty vector, inactive mutant)

  • Step-wise expression level increases

  • Time-course analysis to identify onset of metabolic effects

This systematic approach allows researchers to identify the mechanisms behind unexpected metabolic effects and develop strategies to mitigate them, ensuring the successful application of recombinant L. johnsonii expressing folD2 in research contexts.