Recombinant Lactobacillus johnsonii DNA replication and repair protein recF (recF)

What is the RecF protein in Lactobacillus johnsonii and what is its primary function?

RecF in L. johnsonii is part of the RecF pathway of DNA recombination and repair, which plays a critical role in maintaining genomic integrity. Based on studies of homologous proteins in other bacteria like E. coli, RecF is primarily involved in the resumption of replication at disrupted DNA replication forks and participates in repairing DNA double-strand breaks .

The protein functions in concert with other members of the RecF pathway, particularly RecO and RecR. Together, these proteins enable the reassembly of replication machinery at sites where replication has been disrupted. This function is particularly important for bacterial survival under conditions of DNA damage or stress .

How is the recF gene organized in the L. johnsonii genome?

The recF gene in L. johnsonii is part of the core genome shared among different L. johnsonii strains. Based on genomic analysis of multiple L. johnsonii strains, including strain ZLJ010, NCC533, and others, recF is maintained as part of the essential genomic infrastructure .

Genomic studies reveal that L. johnsonii has a genome size of approximately 1.8-2.0 Mb, with recF being part of the approximately 1,300 gene clusters that constitute the core genome across different L. johnsonii strains . Unlike some strain-specific genes that may be associated with prophages or mobile genetic elements, recF is conserved, reflecting its essential function in DNA metabolism.

What are the most effective methods for creating recombinant L. johnsonii expressing modified RecF protein?

Several genetic modification approaches have been successfully applied to L. johnsonii, with varying efficiencies:

Table 1. Comparison of Genetic Modification Methods for L. johnsonii

For recF modification specifically, the Cre/loxP system has shown excellent results in Lactobacilli, allowing for precise modifications with high efficiency. This approach is particularly valuable for creating functional modifications to the RecF protein while minimizing unwanted genomic alterations .

When expressing recombinant proteins on the cell surface (as might be desired for certain RecF applications), approaches using cell wall anchored proteins like PrtB from Lactobacillus delbrueckii have proven effective. This method has been successfully used to express fusion proteins on the surface of L. johnsonii .

What expression vectors are most suitable for recombinant RecF production in L. johnsonii?

The choice of expression vector depends on the specific research objectives. For therapeutic applications, plasmid stability and food-grade selection markers are essential considerations:

• pPG612-based vectors: Successfully used for expressing bovine GM-CSF in L. johnsonii, showing good stability over 40 generations . This vector system is particularly valuable for therapeutic applications.

• Food-grade vector systems: For applications where antibiotic resistance markers are undesirable, vectors using the conditionally lethal gene pheS* as a negative selection marker combined with temperature-sensitive replicons (such as pGhost9) have shown excellent results in Lactobacillus species .

For RecF protein expression specifically, vector systems should incorporate:

• Strong, preferably inducible promoters

• A secretion signal if extracellular or surface expression is desired

• Appropriate tag sequences for purification or detection

• Stable replicons for maintenance in L. johnsonii

The design should consider the relatively high A-T content of the L. johnsonii genome (~65-70%) for optimal codon usage .

How can one effectively verify the functionality of recombinant RecF protein in L. johnsonii?

Verifying RecF functionality requires a multi-faceted approach:

• UV sensitivity assay: RecF-deficient bacteria show hypersensitivity to UV radiation. Complementation with functional recombinant RecF should restore normal UV resistance . This can be measured by comparing survival rates of wild-type, RecF-deficient, and complemented strains after UV exposure.

• DNA replication fork recovery assay: Since RecF is essential for the resumption of replication at stalled forks, DNA fiber analysis or pulse-field gel electrophoresis can be used to monitor replication restart after induced replication fork stalling (e.g., with hydroxyurea treatment) .

• In vitro reconstitution assay: A biochemical approach involving purified RecF, RecO, RecR, RecA, RecQ, RecJ, and SSB proteins to reconstitute the DNA repair process in vitro. Successful joint molecule formation between linear dsDNA and supercoiled DNA indicates functional RecF .

• Genetic complementation studies: Introduction of recombinant RecF into RecF-deficient strains should complement phenotypic defects if the protein is functional.

• Protein-protein interaction studies: Co-immunoprecipitation or bacterial two-hybrid assays to verify interactions between recombinant RecF and known partners like RecO and RecR.

The recommended approach is to employ multiple methods, as each provides different insights into RecF functionality.

What phenotypic changes can be expected in L. johnsonii strains with modified RecF proteins?

Modifications to the RecF protein can lead to several observable phenotypic changes:

• Altered DNA damage response: Strains with defective RecF show increased sensitivity to DNA-damaging agents, particularly UV radiation . Conversely, enhanced RecF function might confer greater resistance.

• Changes in mutation rates: RecF is involved in maintaining genomic stability; modifications may alter spontaneous mutation frequencies.

• Growth characteristics: Significant RecF modifications may affect growth rates, particularly under stressful conditions. This can be measured through growth curve analysis under various conditions.

• Prophage induction: In L. johnsonii strains containing prophages (like the NCC533 strain with prophages Lj928 and Lj965), RecF function may influence prophage stability and induction rates .

• Recombination efficiency: Changes in homologous recombination rates may be observed, which can be measured using appropriate recombination reporter systems.

When designing experiments to assess these phenotypes, it is essential to include appropriate controls, including wild-type strains and complete RecF deletion mutants for comparison.

How does RecF in L. johnsonii interact with other DNA repair pathway components, and how can these interactions be studied?

RecF functions within a network of protein interactions comprising the RecF pathway. In E. coli, the RecFOR complex (RecF, RecO, RecR) facilitates RecA loading onto SSB-coated ssDNA at processed ssDNA-dsDNA junctions . In L. johnsonii, similar interactions are expected but remain less characterized.

Methods to study these interactions include:

• Bacterial two-hybrid assays: To identify direct protein-protein interactions between RecF and other repair proteins.

• Co-immunoprecipitation followed by mass spectrometry: To identify the interactome of RecF in vivo.

• Fluorescence microscopy using fluorescently tagged proteins: To visualize co-localization of RecF with other repair proteins during DNA damage response.

• In vitro reconstitution assays: Using purified components to reconstruct the DNA repair pathway and study the biochemical requirements for each step.

• ChIP-seq analysis: To map genome-wide binding sites of RecF and associated proteins during normal growth and after DNA damage.

Recent studies using reconstituted systems have shown that RecF, RecO, and RecR have distinct but complementary roles in facilitating RecA loading onto DNA. RecO and RecR mediate exchange of RecA for SSB, while RecF acts with RecO and RecR to load RecA at ssDNA-dsDNA junctions . Similar approaches could be applied specifically to L. johnsonii RecF to elucidate potential species-specific interaction patterns.

How can recombinant L. johnsonii expressing modified RecF be utilized for therapeutic applications?

L. johnsonii has shown significant promise as a delivery vehicle for therapeutic molecules, including vaccines and immunomodulatory proteins. Recombinant L. johnsonii expressing modified RecF could have several therapeutic applications:

• Enhanced DNA damage resistance: Modified RecF that improves DNA repair efficiency could enhance L. johnsonii survival in harsh environments like the gastrointestinal tract, improving probiotic efficacy.

• Vector stability: Optimized RecF function could enhance the genetic stability of therapeutic vectors in L. johnsonii, ensuring consistent expression of therapeutic proteins over time.

• Adjuvant effects: RecF or RecF-derived peptides could potentially serve as immune-stimulating adjuvants when co-expressed with vaccine antigens, enhancing immune responses.

Research by Scheppler et al. demonstrated that L. johnsonii can be effectively used as a mucosal vaccine delivery vehicle, partly due to its ability to survive gastric conditions . By engineering RecF to enhance DNA damage resistance, the survival and efficacy of such vaccine vehicles could potentially be improved.

What are the significant differences between RecF in L. johnsonii and other well-characterized bacterial species?

While RecF is functionally conserved across bacterial species, important structural and regulatory differences exist:

Table 2. Comparative Analysis of RecF Across Bacterial Species

One key research question is whether RecF in L. johnsonii has adapted to the specific genomic architecture and repair needs of this organism, which contains multiple prophage regions and other mobile genetic elements .

What are the optimal culture conditions for maximizing recombinant protein expression in L. johnsonii?

Optimizing culture conditions is critical for efficient recombinant protein production in L. johnsonii. Based on studies with various Lactobacillus strains, the following conditions have shown positive results:

Table 3. Optimized Culture Conditions for L. johnsonii

For specific RecF expression, additional considerations include:

• Protein solubility: RecF is typically soluble when expressed at moderate levels. Overexpression may lead to inclusion body formation.

• Induction strategy: For temperature-sensitive or chemical-inducible promoters, gradual induction may yield better results than sudden induction.

• Growth media supplements: Addition of specific fatty acids like erucic acid may modify L. johnsonii membrane composition, potentially affecting protein expression and activity .

A sequential quadratic programming approach, as described by Lee et al., can be used to optimize multiple parameters simultaneously for maximum recombinant protein production .

What are the critical factors to consider when designing experiments to study RecF function in L. johnsonii?

When studying RecF function in L. johnsonii, several critical experimental design factors must be considered:

• Genetic background selection:

  • Use well-characterized L. johnsonii strains with complete genome information (e.g., NCC533, N6.2)

  • Consider prophage content of selected strains, as prophages represent ~50% of strain-specific DNA in some L. johnsonii strains and may influence recombination processes

• Control constructs:

  • Include wild-type RecF expression as positive control

  • Use catalytically inactive RecF mutants (e.g., Walker A motif mutations) as negative controls

  • Consider complementation controls with RecF from well-characterized species like E. coli

• DNA damage induction:

  • Use appropriate DNA damaging agents at optimized concentrations

  • UV irradiation: 10-50 J/m² (calibrated for Lactobacillus sensitivity)

  • Chemical agents: mitomycin C (0.1-1 μg/ml), methyl methanesulfonate (0.01-0.1%)

• Phenotypic assays:

  • Growth curves under normal and stress conditions

  • Survival assays following DNA damage

  • Microscopy to observe cell morphology and nucleoid structure

  • Recombination frequency measurements

• Protein expression verification:

  • Western blotting with specific antibodies

  • Activity assays (e.g., ATP hydrolysis for RecF)

  • Protein localization studies

• Statistical considerations:

  • Minimum of three biological replicates

  • Appropriate statistical tests based on data distribution

  • Power analysis to determine sample size

When designing mutations or variants of RecF, conserved functional domains should be identified through multiple sequence alignment with well-characterized RecF proteins from model organisms.

What are common challenges in generating stable recombinant L. johnsonii strains, and how can they be addressed?

Researchers often encounter several challenges when developing recombinant L. johnsonii strains:

• Low transformation efficiency:

  • Problem: L. johnsonii typically shows lower transformation efficiency compared to model organisms.

  • Solution: Optimize electroporation conditions (field strength 1.5-2.5 kV/cm, buffer composition); use glycine treatment (1-2%) to weaken cell wall; ensure high-quality, unmethylated plasmid DNA .

• Plasmid instability:

  • Problem: Recombinant plasmids may be lost during consecutive passages.

  • Solution: Use selectable markers appropriate for L. johnsonii; verify plasmid stability over at least 40 generations as demonstrated for pPG612-based plasmids ; consider chromosomal integration for long-term stability.

• Heterologous protein expression issues:

  • Problem: Poor expression levels or inactive protein.

  • Solution: Codon optimization for L. johnsonii (high A+T content); use L. johnsonii-derived promoters and signal peptides; consider fusion with well-expressed L. johnsonii proteins.

• Unwanted recombination events:

  • Problem: Homologous recombination between repeated sequences in expression constructs.

  • Solution: Minimize sequence repetition in constructs; use recombination-deficient host strains for plasmid propagation; verify construct integrity after transformation.

• Selective marker limitations:

  • Problem: Limited options for food-grade selectable markers.

  • Solution: Employ food-grade selection systems using auxotrophy complementation or food-grade antibiotics; use counterselectable markers like pheS* for marker removal .

• Phage contamination:

  • Problem: Prophage induction or external phage infection.

  • Solution: Choose L. johnsonii strains with characterized prophage content; implement phage monitoring; consider CRISPR-based approaches for phage resistance.

For RecF-specific expression challenges, careful attention to protein folding and potential toxicity of overexpressed DNA-binding proteins is essential. Expression as fusion proteins with solubility-enhancing partners or under tightly controlled inducible promoters may mitigate these issues.

How can researchers address inconsistent experimental results when working with recombinant L. johnsonii RecF?

Inconsistent results are a common challenge in research with recombinant proteins in non-model organisms like L. johnsonii. Several approaches can help address this issue:

• Standardize growth and induction conditions:

  • Use consistent media preparation methods

  • Control growth parameters precisely (temperature, pH, oxygen level)

  • Standardize cell density at induction (OD600 = 0.6-0.8)

  • Harvest cells at consistent time points post-induction

• Implement rigorous quality control:

  • Verify plasmid sequence before each experiment

  • Confirm protein expression by Western blot

  • Check for potential mutations in expression strains

  • Implement routine phenotypic tests to confirm strain identity

• Control for genetic drift:

  • Make working stocks from master cultures

  • Limit passage number before returning to original stocks

  • Store multiple glycerol stocks at -80°C

• Address physiological variability:

  • Pre-adapt cultures to experimental conditions

  • Use biological triplicates from independent colonies

  • Control for growth phase effects by synchronizing cultures

• Optimize biochemical assays:

  • Determine linear range for each assay

  • Include internal controls in each experiment

  • Validate key results with orthogonal methods

• Systematic troubleshooting approach:

  • Isolate variables one at a time

  • Document all experimental conditions meticulously

  • Implement positive and negative controls for each assay

For RecF specifically, its function in DNA metabolism means that experimental conditions causing DNA stress (suboptimal media, contaminating DNA-damaging compounds, excessive aeration) may significantly impact results by triggering native stress responses that interact with recombinant RecF function.

What are promising new approaches for studying RecF function in L. johnsonii?

Emerging technologies offer new opportunities for understanding RecF function in L. johnsonii:

• CRISPR interference (CRISPRi) for tunable gene repression:

  • Allows titration of RecF expression levels without complete knockout

  • Enables study of essential genes like recF that may be lethal when fully deleted

  • Can be implemented with dCas9 systems adapted for L. johnsonii

• Single-molecule techniques:

  • Single-molecule fluorescence resonance energy transfer (smFRET) to observe RecF-DNA interactions

  • DNA curtain assays to visualize RecF activity on DNA substrates in real-time

  • Optical tweezers to study mechanical aspects of RecF-mediated DNA transactions

• In situ structural studies:

  • Cryo-electron tomography to visualize RecF in cellular context

  • Proximity labeling approaches (BioID, APEX) to map RecF interaction network in vivo

  • In-cell NMR to study RecF conformational changes during DNA damage response

• Systems biology approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics) to understand global effects of RecF modification

  • Network analysis to position RecF within the broader DNA damage response network

  • Mathematical modeling of RecF pathway dynamics

• Synthetic biology tools:

  • Split protein complementation assays for RecF to study domain interactions

  • Optogenetic control of RecF expression or activity

  • Engineering of orthogonal RecF variants with novel functionalities

These approaches, combined with the genetic engineering tools specifically developed for Lactobacillus species , offer powerful ways to advance our understanding of RecF biology in L. johnsonii.

How might engineered RecF variants enhance the utility of L. johnsonii as a probiotic or therapeutic delivery system?

Engineered RecF variants could significantly enhance L. johnsonii applications through several mechanisms:

• Stress resistance enhancement:

  • RecF variants with enhanced DNA repair capacity could improve L. johnsonii survival during:

    • Gastrointestinal transit (acid, bile, oxidative stress)

    • Manufacturing processes (lyophilization, storage)

    • Therapeutic delivery to inflammatory environments

  • This could extend shelf life and in vivo persistence of probiotic preparations

• Genetic stabilization of therapeutic constructs:

  • Optimized RecF could reduce mutation rates in expression cassettes

  • Enhanced homologous recombination capacity could facilitate stable chromosomal integration of therapeutic genes

  • Reduced prophage activation through improved DNA maintenance

• Controlled biocontainment:

  • Engineered conditional RecF variants could enable environmental containment strategies

  • Inducible RecF inactivation could trigger programmed cell death under specific conditions

  • This addresses biosafety concerns with engineered probiotics

• Adjuvant properties:

  • RecF-derived peptides could potentially act as immune-stimulating molecules

  • Co-expression with antigens might enhance immunological memory

  • This could improve L. johnsonii-based vaccine efficacy

• Delivery system optimization:

  • RecF engineering could enhance the efficiency of DNA delivery to mammalian cells

  • Modified DNA transfer mechanisms might improve therapeutic nucleic acid delivery

  • This could create new applications in gene therapy