Recombinant Lactobacillus plantarum Probable endonuclease 4 (nfo)

What is Lactobacillus plantarum endonuclease 4 (nfo) and what is its primary function in bacterial cells?

Lactobacillus plantarum endonuclease 4 (nfo, lp_1976) is an endodeoxyribonuclease (EC 3.1.21.2) that plays a crucial role in DNA repair mechanisms. This enzyme is specifically involved in the base excision repair (BER) pathway, recognizing and cleaving the phosphodiester bond at abasic sites (AP sites) in DNA that result from oxidative damage. In L. plantarum strain ATCC BAA-793/NCIMB 8826/WCFS1, the protein consists of 298 amino acids .

The enzyme functions by:

• Recognizing AP sites in damaged DNA

• Cleaving the phosphodiester backbone at the 5' side of the AP site

• Creating a nick with 3'-OH and 5'-deoxyribose phosphate termini

• Facilitating subsequent repair by DNA polymerase and ligase enzymes

Unlike many other DNA repair enzymes, endonuclease IV does not require metal cofactors for its activity, making it especially valuable in oxidative stress conditions when metals may be sequestered.

What expression systems are most effective for producing recombinant L. plantarum nfo?

Multiple expression systems have been evaluated for recombinant L. plantarum nfo production, each with distinct advantages:

What are the optimal methods for purifying recombinant L. plantarum nfo?

Purification of recombinant L. plantarum nfo typically involves a multi-step process:

• Initial clarification: Cells are lysed using sonication or freeze-thaw cycles as demonstrated effective in the characterization of recombinant L. plantarum proteins

• Affinity chromatography: Using appropriate tag systems (His-tag is common, but alternatives include Avi-tag for biotinylated versions)

• Ion exchange chromatography: To remove contaminants based on charge differences

• Size exclusion chromatography: For final polishing and buffer exchange

The purification protocol should be optimized to maintain enzymatic activity. Studies have shown that protein purity of >85% (as assessed by SDS-PAGE) is achievable and sufficient for most research applications .

How can researchers confirm the structural integrity and activity of purified recombinant L. plantarum nfo?

Verification of recombinant L. plantarum nfo structural integrity and activity requires multiple analytical approaches:

Structural verification:

• SDS-PAGE to confirm molecular weight (~34 kDa)

• Western blotting using specific antibodies

• Mass spectrometry for precise mass determination and peptide mapping

• Circular dichroism spectroscopy to assess secondary structure

Activity assays:

• Substrate cleavage assays using synthetic abasic site-containing oligonucleotides

• Fluorescence-based real-time assays to monitor kinetics

• Complementation assays in nfo-deficient bacterial strains

A typical activity assay involves incubating the purified enzyme with DNA containing abasic sites, followed by gel electrophoresis to visualize the cleaved products. Specific activity should be reported as units per mg protein, where one unit represents the amount of enzyme required to cleave 1 nmol of substrate in 1 minute under standard conditions.

What are the most effective strategies for optimizing the expression of recombinant L. plantarum nfo using the CRISPR/Cas9 system?

CRISPR/Cas9-mediated genomic insertion provides a powerful approach for stable expression of nfo in L. plantarum. Recent advances in CRISPR/Cas9 applications for L. plantarum have demonstrated successful genomic integration with efficiency rates of 40-60% for various gene cassettes .

Recommended methodology:

• Design of the two-plasmid system:

  • One plasmid encoding the recombinase operon (lp0640-42) under an inducible promoter like P* or PsppA*

  • Second plasmid containing the Cas9 protein, sgRNA targeting the insertion site, and homology arms

• Optimization of homology arms:

  • Aim for 500-1000 bp homology arms flanking the insertion site

  • Ensure GC content is balanced (30-60%) for efficient homologous recombination

• Selection of genomic insertion site:

  • Target regions showing high transcriptional activity

  • Avoid disrupting essential genes or regulatory elements

  • Consider chromosomal locations near the origin of replication for potential gene dosage effects

• Induction protocols:

  • Optimize induction timing of recombinase expression (typically early-log phase)

  • Control expression levels using titratable inducible promoters

The success rate of genomic insertion varies significantly depending on the expression cassette size, with observed efficiencies declining for constructs exceeding 1,300 bp . Researchers should note that chromosomal integration typically results in lower expression levels compared to plasmid-based systems, with approximately 6-fold reduction in protein production observed when comparing integrated versus plasmid-expressed genes .

How does the catalytic mechanism of L. plantarum nfo compare with other bacterial endonucleases, and what implications does this have for experimental design?

L. plantarum nfo belongs to the endonuclease IV family, characterized by a TIM-barrel fold containing three zinc ions in the active site. The catalytic mechanism differs significantly from that of other AP endonucleases like ExoIII:

Experimental design implications:

• Buffer composition: L. plantarum nfo maintains activity in the absence of added divalent cations, which should be considered when designing reaction buffers.

• pH optimum: The enzyme typically shows optimal activity at pH 7.5-8.5, which differs from the acidic pH optimum of ExoIII family enzymes.

• Substrate specificity: In addition to AP sites, L. plantarum nfo can process certain oxidized bases and unusual DNA structures that ExoIII cannot, requiring careful substrate selection for comparative studies.

• Inhibitor studies: Compounds targeting the zinc-binding site would be effective against L. plantarum nfo but not against ExoIII family enzymes.

These mechanistic differences provide opportunities for developing selective inhibition strategies and for utilizing L. plantarum nfo in specific DNA repair applications where ExoIII activity might be compromised.

What are the challenges in adapting L. plantarum as a delivery vector for heterologous proteins, and how might these be overcome when working with nfo?

Adapting L. plantarum as a delivery vector for heterologous proteins including nfo presents several challenges that require specific strategies to overcome:

Major challenges and solutions:

• Protein expression levels:

  • Challenge: Chromosomal integration typically results in lower expression levels compared to plasmid-based systems

  • Solution: Implement strong constitutive promoters (P23) or inducible systems (PsppA*) and optimize ribosome binding sites. Consider tandem promoter arrangements or integration of multiple gene copies at different genomic loci.

• Protein localization and secretion:

  • Challenge: Ensuring proper localization (cytoplasmic, surface-displayed, or secreted)

  • Solution: Utilize appropriate signal peptides and anchoring domains. For nfo, which naturally functions intracellularly, fusion to lipoprotein anchors has been shown to facilitate surface display while maintaining detectable activity .

• Genetic stability:

  • Challenge: Maintaining stable expression over multiple generations

  • Solution: CRISPR/Cas9-mediated chromosomal integration has demonstrated higher stability than plasmid systems, with retention rates exceeding 95% after 30 generations without selective pressure .

• Immunogenicity control:

  • Challenge: Balancing immune stimulation versus tolerance

  • Solution: Co-expression with immunomodulatory molecules like dendritic cell-targeting peptide (DCpep) can enhance desired immune responses, as demonstrated with other recombinant L. plantarum systems .

Experimental data on recombinant L. plantarum delivery systems:

The choice of delivery strategy should be guided by the intended application. For nfo specifically, maintaining enzymatic activity after recombinant expression is critical, requiring careful design of fusion constructs and expression systems.

How can researchers assess the impact of recombinant L. plantarum nfo on DNA repair mechanisms in diverse cellular environments?

Evaluating the impact of recombinant L. plantarum nfo on DNA repair mechanisms requires multi-level assessment approaches:

In vitro assessment:

• Enzyme kinetics analysis:

  • Determine Km and kcat values using synthetic AP site-containing substrates

  • Compare activity on various DNA damage types (oxidative, alkylation, etc.)

  • Assess processivity and strand specificity

• Competitive inhibition studies:

  • Evaluate interference with endogenous repair pathways

  • Measure displacement of host repair proteins from damage sites

Cellular assessment:

• Complementation assays:

  • Transform nfo-deficient bacterial strains with L. plantarum nfo

  • Measure survival rates after exposure to DNA-damaging agents

  • Compare with wild-type and endonuclease-deficient controls

• DNA damage markers:

  • Monitor γ-H2AX foci formation and resolution

  • Track 8-oxoguanine and AP site levels over time

  • Assess single-strand break accumulation using comet assay

Host-microbe interaction assessment:

• Micronuclei formation:

  • Quantify genomic instability markers in host cells

  • Compare effects of wild-type versus recombinant L. plantarum

• Transcriptional responses:

  • RNA-seq to identify alterations in host DNA repair gene expression

  • Pathway analysis focusing on BER, NER, and oxidative stress response

The relative impact of nfo expression can be quantified using a DNA repair capacity index, calculated as the ratio of repair rate in treated versus control samples, normalized to expression levels. This approach allows for standardized comparison across different experimental conditions and cell types.

What potential biotechnological applications exist for recombinant L. plantarum nfo beyond basic research?

Recombinant L. plantarum nfo holds promising potential for various biotechnological applications:

Therapeutic applications:

• DNA damage mitigation:

  • L. plantarum as a probiotic carrier for nfo could potentially help mitigate DNA damage in intestinal epithelial cells

  • Targeted delivery to tissues experiencing oxidative stress

  • Particular relevance in inflammatory bowel disease where oxidative DNA damage is elevated

• Cancer adjuvant therapy:

  • Selective enhancement of chemotherapy effects by interfering with cancer cell DNA repair

  • Modulation of immune responses through engineered L. plantarum

Biotechnological tools:

• DNA manipulation:

  • Site-specific DNA cleavage at abasic sites for directed mutagenesis

  • Generation of defined DNA fragments for cloning applications

  • Removal of damaged DNA from genomic preparations

• Biosensors:

  • Development of whole-cell biosensors for genotoxic compounds

  • Activity-based probes for measuring oxidative DNA damage

• Bioremediation:

  • Engineered L. plantarum expressing nfo for degradation of genotoxic compounds in fermented foods

  • Reduction of mutagenic potential in fermentation processes

Industrial applications:

• Food preservation:

  • Enhanced stability of starter cultures through improved DNA repair capacity

  • Protection against processing-induced DNA damage

  • Extended shelf-life of probiotic preparations

• Fermentation optimization:

  • Improved stress resistance during industrial fermentation

  • Enhanced genetic stability in continuous culture systems

Recent investigations into L. plantarum's adaptability to diverse environmental niches support its potential as a versatile delivery platform for functional proteins like nfo . The combination of L. plantarum's natural stress tolerance and the DNA repair functions of nfo creates unique opportunities for applications in challenging environments where DNA damage is prevalent.

What are the most effective protocols for genetically engineering L. plantarum to express recombinant nfo?

The following protocol outlines the most effective approach for engineering L. plantarum to express recombinant nfo, based on recent methodological advances:

CRISPR/Cas9-based genomic integration protocol:

• Plasmid construction:

  • Construct pSIPSh71_LpRec with inducible overexpression of the recombinase operon (lp0640-42)

  • Amplify the nfo gene using primers with appropriate restriction sites

  • Clone into a vector containing strong promoters like P* or PsppA*

  • Include appropriate selection markers (typically erythromycin resistance)

• Transformation procedure:

  • Prepare electrocompetent L. plantarum cells in exponential growth phase

  • Transform with recombinase-expressing plasmid first

  • Induce recombinase expression with appropriate inducer (sakacin P inducing peptide)

  • Prepare second round of electrocompetent cells from these transformants

  • Transform with the Cas9-sgRNA plasmid containing nfo and homology arms

• Selection and verification:

  • Plate on selective media containing appropriate antibiotics

  • Screen colonies by colony PCR targeting insertion junctions

  • Verify recombinants by sequencing across insertion sites

  • Confirm nfo expression by Western blotting and activity assays

Optimization tips:

• Use codon optimization for the nfo gene based on L. plantarum codon usage

• Consider using the native nfo promoter region if expression timing is critical

• For secreted or surface-displayed versions, fuse with appropriate signal peptides and anchoring domains

This method typically achieves transformation efficiencies of 10²-10³ CFU/μg DNA for the first transformation and 10¹-10² CFU/μg for the second transformation, with knock-in efficiencies ranging from 40-60% for most constructs .

What techniques should be employed to accurately measure enzymatic activity of recombinant L. plantarum nfo?

Accurate measurement of recombinant L. plantarum nfo enzymatic activity requires specialized techniques that account for its specific catalytic properties:

Standard activity assay protocol:

• Substrate preparation:

  • Synthesize duplex oligonucleotides (21-25 bp) containing a single tetrahydrofuran (THF) AP site analog

  • 5'-end label one strand with 32P or fluorescent tag for detection

  • Anneal complementary strands in equimolar ratios

• Reaction conditions:

  • Buffer: 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM DTT, 0.1 mg/ml BSA

  • No additional metal cofactors required

  • Temperature: 37°C

  • Time course: 0-30 minutes

• Activity determination:

  • Resolve reaction products on 20% denaturing polyacrylamide gels

  • Quantify cleaved product using phosphorimager or fluorescence detection

  • Calculate initial velocities from linear portions of time courses

  • Determine specific activity as pmol substrate cleaved/min/pmol enzyme

Advanced kinetic analysis:

• Determine Km and kcat using substrate concentration ranges of 1-100 nM

• Assess product inhibition by including increasing concentrations of cleaved oligonucleotides

• Evaluate temperature stability profile by pre-incubating enzyme at 25-65°C before activity measurement

High-throughput fluorescence-based alternatives:

• Molecular beacon substrates with fluorophore-quencher pairs that separate upon cleavage

• Real-time monitoring in microplate format with excitation/emission appropriate for chosen fluorophore

• Standard curves using defined amounts of cleaved substrate

Typical wild-type L. plantarum nfo exhibits a Km of approximately 10-20 nM for THF-containing oligonucleotides and a kcat of 1-5 min⁻¹ under optimal conditions. Recombinant versions should be compared against these baseline parameters to assess functional integrity.

How should researchers design experiments to investigate the potential immunomodulatory effects of L. plantarum expressing recombinant nfo?

Investigating immunomodulatory effects of L. plantarum expressing recombinant nfo requires careful experimental design across multiple immunological parameters:

In vitro assessment protocol:

• Dendritic cell (DC) activation studies:

  • Isolate murine bone marrow-derived DCs or human monocyte-derived DCs

  • Co-culture with wild-type or recombinant L. plantarum (MOI 1:10)

  • Measure DC maturation markers (CD80, CD86, MHC-II) by flow cytometry

  • Quantify cytokine production (IL-10, IL-12, TNF-α) by ELISA

• T cell response evaluation:

  • Perform DC-T cell co-culture assays

  • Measure CD4+ and CD8+ T cell proliferation using CFSE dilution

  • Determine IFN-γ production in CD4+ and CD8+ T cells by intracellular staining

  • Compare responses to wild-type versus nfo-expressing strains

In vivo assessment protocol:

• Mucosal immune response:

  • Administer wild-type or recombinant L. plantarum orally to mice (10⁹ CFU)

  • Primary and booster administrations 2 weeks apart

  • Collect samples at days 0, 14, and 28

  • Analyze B220+IgA+ cells in Peyer's patches by flow cytometry

  • Measure secretory IgA levels in intestinal lavage by ELISA

• Systemic immune response:

  • Collect serum samples at days 0, 14, and 28

  • Measure specific antibody production (IgG, IgG1, IgG2a)

  • Perform spleen and mesenteric lymph node analysis for T cell responses

  • Compare CD4+IFN-γ+ and CD8+IFN-γ+ populations

Data interpretation framework:

• Calculate stimulation indices for proliferation assays (SI = CPM stimulated/CPM unstimulated)

• Determine statistical significance using appropriate tests (ANOVA for multiple group comparisons)

• Perform correlation analysis between bacterial colonization levels and immune parameters

• Consider confounding factors such as strain variation in adhesion properties

Based on previous studies with recombinant L. plantarum expressing foreign antigens, researchers should expect to see significant differences in T cell populations between recombinant and control groups, with increases of 1.5-3 fold in IFN-γ+ subpopulations being considered biologically significant .

What are the most common challenges in expressing functional recombinant L. plantarum nfo and how can they be addressed?

Researchers frequently encounter several challenges when expressing functional recombinant L. plantarum nfo. Here are the most common issues and their solutions:

Expression level problems:

Functionality issues:

Quality control checkpoints:

• Verify gene sequence before expression (Sanger sequencing)

• Confirm protein production by Western blot with anti-nfo antibodies

• Assess protein purity using SDS-PAGE (target >85%)

• Verify enzymatic activity using standardized AP-site substrates

• Evaluate stability by activity retention after storage at different temperatures

For chromosomal integration using CRISPR/Cas9, transformation efficiency can be increased by optimizing electroporation conditions (field strength 2.0-2.5 kV/cm) and by ensuring proper induction of the recombinase system prior to the second transformation .

How can researchers distinguish between endogenous and recombinant nfo activity in experimental systems?

Distinguishing between endogenous and recombinant nfo activity is crucial for accurate data interpretation. The following strategies enable reliable differentiation:

Genetic approaches:

• Epitope tagging:

  • Add unique epitope tags (FLAG, HA, c-myc) to recombinant nfo

  • Perform immunoprecipitation with tag-specific antibodies before activity assays

  • Use Western blotting with tag-specific antibodies to quantify recombinant protein

• Site-directed mutagenesis:

  • Introduce silent mutations that preserve activity but alter restriction sites

  • Create catalytically enhanced variants with higher activity than wild-type

  • Design substrate specificity mutations that allow selective activity measurement

Biochemical approaches:

• Differential inhibition:

  • Identify inhibitors with differential effects on endogenous versus recombinant enzyme

  • Use antibodies specific to the recombinant version to selectively inhibit its activity

  • Exploit differences in metal ion requirements if appropriate mutations are introduced

• Activity signatures:

  • Design substrate panels that reveal different cleavage patterns

  • Measure activity under conditions that selectively favor the recombinant enzyme

  • Analyze reaction kinetics to identify biphasic behavior indicating two enzyme populations

Experimental controls:

When working with L. plantarum as the expression host, initial characterization of endogenous nfo activity is essential, as baseline activity levels will vary between strains. Recent genomic studies of L. plantarum strains indicate that endonuclease activity can vary by as much as 3-fold between isolates from different environmental niches .

What are the emerging opportunities for utilizing engineered L. plantarum nfo in synthetic biology applications?

Emerging synthetic biology applications for engineered L. plantarum nfo represent an exciting frontier with several promising research directions:

DNA circuit components:

• Programmable DNA editors:

  • Engineer nfo variants with altered substrate specificity

  • Create inducible DNA damage response systems

  • Develop conditional genetic switches based on DNA repair mechanisms

• Genetic toggle switches:

  • Use nfo to selectively cleave regulatory elements containing modified bases

  • Create stress-responsive genetic circuits that activate under oxidative conditions

  • Design autoregulatory feedback loops utilizing nfo activity

Biosensing applications:

• Genotoxicity detectors:

  • Engineer whole-cell biosensors with fluorescent reporters coupled to nfo activity

  • Develop portable detection systems for environmental mutagens

  • Create high-throughput screening platforms for DNA-damaging compounds

• Diagnostic tools:

  • Design nfo-based assays for detecting DNA lesions in clinical samples

  • Develop point-of-care tests for oxidative stress biomarkers

  • Create microbiome-based sensors for intestinal inflammation

Therapeutic delivery systems:

• Targeted DNA repair:

  • Engineer L. plantarum for selective colonization of damaged tissues

  • Develop systems for controlled release of nfo at sites of oxidative damage

  • Create hybrid systems combining nfo with other DNA repair enzymes

Recent advancements in CRISPR/Cas9-mediated genome editing of L. plantarum provide powerful tools for these applications, with demonstrated success in integrating functional expression cassettes ranging from 800 to 1,300 bp with 40-60% efficiency . The proven ability to display functional proteins on the L. plantarum surface also opens opportunities for creating cell-based platforms with accessible enzymatic activity .

What are the potential applications of recombinant L. plantarum nfo in addressing DNA damage-related health conditions?

Recombinant L. plantarum nfo shows significant promise for addressing DNA damage-related health conditions through various mechanisms:

Gastrointestinal applications:

• Inflammatory bowel disease:

  • L. plantarum naturally colonizes the GI tract

  • Probiotic delivery of nfo could help mitigate oxidative DNA damage in intestinal epithelial cells

  • Potential for reducing inflammation-associated cancer risk

• Colorectal cancer prevention:

  • Reduce genotoxic burden from dietary carcinogens

  • Enhance DNA repair capacity in at-risk epithelial cells

  • Complement L. plantarum's demonstrated anti-inflammatory properties

Systemic applications:

• Oxidative stress-related conditions:

  • Delivery of nfo to tissues experiencing chronic oxidative stress

  • Potential applications in neurodegenerative disorders where DNA damage accumulates

  • Adjuvant therapy for conditions with impaired DNA repair capacity

• Aging-related DNA damage:

  • Counteract age-associated decline in DNA repair efficiency

  • Address mitochondrial DNA damage through targeted delivery systems

  • Combine with L. plantarum's metabolic benefits for multi-faceted approach to aging

Therapeutic development considerations:

The proven safety profile of L. plantarum as a probiotic combined with its ability to be genetically modified using CRISPR/Cas9 systems provides a strong foundation for developing these therapeutic applications. Pre-clinical research should focus on establishing proof-of-concept in animal models of oxidative stress-related conditions, with particular attention to bioavailability, enzymatic activity in target tissues, and safety profiles.