Recombinant Lactobacillus plantarum Protein RecA (recA)

What is the RecA protein in Lactobacillus plantarum and what are its primary functions?

The RecA protein in Lactobacillus plantarum is a highly conserved protein that plays fundamental roles in DNA metabolism. Similar to its counterpart in other bacteria (including Escherichia coli), the RecA protein is implicated in homologous DNA recombination, SOS induction, and DNA damage-induced mutagenesis . This multifunctional protein exhibits several biochemical activities, including the ability to bind to both double-stranded and single-stranded DNA, facilitate pairing and exchange of homologous DNA sequences, hydrolyze ATP, and perform coproteolytic cleavage of regulatory proteins such as LexA, λcI, and UmuD . The ubiquitous nature of the recA gene across bacterial species reflects its essential role in maintaining genomic integrity and facilitating evolutionary adaptation through recombination processes. In L. plantarum specifically, RecA maintains these conserved functions while exhibiting species-specific sequence characteristics that researchers have leveraged for taxonomic identification purposes.

How is the recA gene utilized as a phylogenetic marker for Lactobacillus species?

The recA gene has proven to be an effective phylogenetic marker for differentiating closely related Lactobacillus species due to its ubiquitous presence and sequence conservation with sufficient variability to distinguish between species. Researchers have successfully used recA gene sequence comparison to differentiate Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, which are genotypically closely related and display highly similar phenotypes . The methodology involves amplifying short homologous regions of approximately 360 bp using PCR with degenerate consensus primers, sequencing these amplicons, and analyzing a 322 bp segment for phylogenetic tree inference . Multiple phylogenetic analysis approaches—including parsimony, maximum likelihood, and neighbor-joining models—have yielded coherent phylograms that clearly separate these three species . This demonstrates that despite the close evolutionary relationship between these Lactobacillus species, the recA gene contains sufficient sequence divergence to serve as a reliable taxonomic marker, establishing its validity for phylogenetic classification where other markers might be insufficient.

What are the characteristic sequence features of the L. plantarum recA gene compared to other Lactobacillus species?

The recA gene in Lactobacillus plantarum contains distinctive sequence features that enable its differentiation from closely related species like L. pentosus and L. paraplantarum. Analysis of the recA gene sequences has revealed species-specific regions that can be targeted for identification purposes. When amplified using multiplex PCR with species-specific primers, the L. plantarum recA gene produces an amplicon of 318 bp, which is distinct from the 218 bp amplicon for L. pentosus and the 107 bp amplicon for L. paraplantarum . These size differences provide a clear visual distinction when PCR products are analyzed by gel electrophoresis. The sequence differences are distributed throughout the gene but concentrate in regions that maintain the protein's functionality while allowing for species-specific variations. These distinguishing features make the recA gene particularly valuable in taxonomic studies and have enabled researchers to develop precise identification protocols for differentiating these otherwise phenotypically similar Lactobacillus species.

How can researchers amplify and analyze the recA gene from L. plantarum?

Researchers can amplify the recA gene from Lactobacillus plantarum using PCR with degenerate consensus primers designed to target conserved regions of the gene. A standard protocol involves adding 300 ng of chromosomal DNA from each Lactobacillus strain to a 50-μl PCR mixture containing 2.5 U of DNA polymerase in appropriate reaction buffer, 50 μM each primer, and 100 μM deoxynucleotide triphosphates . The recommended thermal cycling conditions include an initial denaturation of 1 minute at 94°C followed by 30 cycles of 1 minute at 94°C, 1 minute and 30 seconds at 55°C, and 1 minute at 72°C . The forward primer typically corresponds to amino acid region 92 to 98 of the E. coli RecA protein, with the sequence 5′-TT(C,T) AT(A,T,C) GA(C,T) GC(A,T,C,G) GA(A,G) CA(C,T) GC-3′ .

For analysis, the amplified fragments (approximately 360 bp) should be purified and sequenced. From the sequence data, researchers typically analyze a 322 bp segment for phylogenetic inference using multiple methods, including parsimony, maximum likelihood, and neighbor-joining models to ensure robust classification . The resulting sequences can be used for species identification through comparison with reference sequences or for designing species-specific primers for more targeted analyses.

What multiplex PCR protocol can differentiate L. plantarum from L. pentosus and L. paraplantarum?

A well-established multiplex PCR protocol for simultaneous differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum has been developed based on recA gene sequence analysis. This method utilizes species-specific primers designed from alignments of recA gene sequences that target unique regions within each species .

The multiplex PCR reaction typically contains:

• Template DNA (300 ng of chromosomal DNA)

• PCR buffer with appropriate MgCl₂ concentration

• Species-specific forward primers for each target species

• A common reverse primer

• dNTPs (100 μM)

• Thermostable DNA polymerase

The thermal cycling conditions follow a standard protocol similar to the one used for initial recA amplification. The key distinguishing feature of this approach is that it produces amplicons of different sizes for each species in a single reaction:

• L. plantarum: 318 bp

• L. pentosus: 218 bp

• L. paraplantarum: 107 bp

This size difference allows for immediate visual identification through gel electrophoresis without the need for additional sequencing steps. The method has been validated to permit unambiguous identification of strains belonging to the L. plantarum group, making it particularly valuable for rapid screening of environmental or clinical isolates where species-level identification is required.

What are the optimal conditions for expressing recombinant RecA protein in L. plantarum?

The optimal conditions for expressing recombinant proteins, including RecA, in Lactobacillus plantarum involve several critical parameters that must be carefully controlled. While the search results don't specifically detail RecA expression conditions, they provide insights from the expression of other recombinant proteins in L. plantarum that can be adapted for RecA expression.

Based on research with recombinant SARS-CoV-2 spike protein expression in L. plantarum, the following conditions yielded optimal protein expression:

• Induction with 50 ng/mL of SppIP (induction peptide)

• Temperature of 37°C

• Expression time of 6-10 hours

For RecA expression specifically, researchers should consider:

• Expression vector selection: Choose a vector with appropriate promoters for L. plantarum, such as those used in food-grade expression systems.

• Codon optimization: As observed with other recombinant proteins, codon optimization for L. plantarum significantly improves expression efficiency by addressing codon usage bias .

• Signal peptide selection: For surface display or secretion, appropriate signal peptides native to L. plantarum should be considered.

• Induction systems: Inducible promoters allow controlled expression at desired time points.

• Growth media and conditions: Media composition and growth conditions significantly impact protein expression levels.

• Protein stabilization: The recombinant protein expression system should be optimized to ensure protein stability under various environmental conditions (temperature, pH, salt concentration).

When expressing RecA specifically, additional considerations may include preventing unwanted recombination events by using conditional expression systems or RecA-deficient host strains for initial cloning steps.

How can recombinant RecA from L. plantarum be utilized in genome editing applications?

Recombinant RecA from Lactobacillus plantarum can be utilized in genome editing applications by enhancing homologous recombination efficiency. While traditional genome editing in L. plantarum has relied on heterologous single-stranded DNA recombinases followed by CRISPR-Cas9 selection, research indicates that endogenous recombination machinery, including RecA, can drive efficient recombineering between plasmids harboring homologous sequences .

For RecA-mediated genome editing approaches:

• RecA-dependent homologous recombination: Purified recombinant RecA protein can be introduced alongside donor DNA to enhance recombination rates in transformation procedures.

• Template design optimization: When designing homology templates, researchers should consider:

  • Optimal homology arm length (typically 500-1000 bp for RecA-mediated recombination)

  • Sequence composition to avoid secondary structures that might impede RecA binding

  • Strategic placement of selection markers

• CRISPR-Cas9 integration: Combining RecA-enhanced homologous recombination with CRISPR-Cas9 selection can significantly improve editing efficiency. This approach leverages the native RecA protein while using CRISPR-Cas9 to select for cells that have undergone successful recombination .

• Recombinase-free editing: Research has shown that encoding recombineering templates on replicating plasmids allows efficient genome editing with CRISPR-Cas9 in multiple L. plantarum strains without requiring a recombinase, potentially relying on endogenous RecA activity . This strategy has successfully introduced stop codons, silent mutations, and complete gene deletions.

• Potential challenges: Researchers should be aware that RecA-dependent recombination can sometimes lead to unexpected outcomes, including large-scale genomic rearrangements in some instances .

The integration of recombinant RecA in these genome editing workflows can potentially increase efficiency while reducing the need for heterologous recombinases, streamlining the editing process for L. plantarum and related species.

What role does L. plantarum RecA play in bacterial stress responses and DNA repair mechanisms?

Lactobacillus plantarum RecA plays a crucial role in bacterial stress responses and DNA repair mechanisms, functioning as a central regulator in pathways that maintain genomic integrity under adverse conditions. While the search results don't provide specific details on L. plantarum RecA's stress response role, its functions can be inferred from the highly conserved nature of RecA across bacterial species and the limited information available.

RecA's involvement in stress responses includes:

• SOS response regulation: As in other bacteria, L. plantarum RecA likely mediates the SOS response by facilitating the autocleavage of the LexA repressor when activated by binding to single-stranded DNA at sites of DNA damage . This activates the expression of DNA repair genes.

• DNA damage repair: RecA catalyzes the pairing of homologous DNA sequences and strand exchange reactions essential for homologous recombination-based DNA repair. This function is critical when L. plantarum faces environmental stressors that cause DNA damage.

• Error-prone DNA synthesis: During severe DNA damage, RecA likely activates error-prone polymerases through the cleavage of UmuD, contributing to mutagenesis that, while potentially introducing errors, allows cell survival.

• Acid and bile stress adaptation: As L. plantarum often encounters acidic environments and bile salts in its natural habitats (human gut, fermented foods), RecA-mediated DNA repair likely contributes to its survival under these stress conditions. Research on recombinant L. plantarum has shown stability of expressed proteins under conditions of pH=1.5 and in the presence of bile salts, suggesting adaptation mechanisms that may involve RecA-mediated processes .

• Heat shock response: RecA may participate in repairing DNA damage caused by thermal stress, contributing to the thermotolerance observed in some L. plantarum strains.

Understanding these mechanisms is particularly relevant for applications where L. plantarum is exposed to stressful conditions, such as in probiotic formulations or food fermentation processes.

How can researchers troubleshoot inconsistent results when working with recombinant L. plantarum RecA?

When researchers encounter inconsistent results while working with recombinant Lactobacillus plantarum RecA, several methodological approaches can help identify and resolve the underlying issues:

• Protein expression variability

  • Assessment: Quantify protein expression levels across different experimental batches using Western blot analysis with RecA-specific antibodies.

  • Resolution: Standardize induction conditions, including inducer concentration (e.g., 50 ng/mL for SppIP-based systems), induction timing, and culture density at induction .

  • Monitoring: Track growth curves post-induction to ensure consistent cellular responses to recombinant protein expression.

• Protein functionality considerations

  • ATP hydrolysis assay: RecA function depends on ATP hydrolysis; inconsistent ATPase activity may indicate structural issues.

  • DNA binding assay: Electrophoretic mobility shift assays (EMSAs) can assess RecA's ability to bind ssDNA.

  • Resolution: Optimize buffer conditions (particularly Mg²⁺ concentration) and ensure appropriate reducing environments to maintain protein functionality.

• Genomic integration inconsistencies

  • Problem: When using RecA for recombineering applications, recombination efficiencies may vary.

  • Analysis: Sequence the target regions to identify any mutations or variations that might affect homologous recombination.

  • Resolution: Design longer homology arms (>500 bp) to enhance recombination efficiency, and consider using CRISPR-Cas9 selection to enrich for successful recombination events .

• Codon optimization challenges

  • Issue: Sub-optimal codon usage can lead to truncated or misfolded RecA protein.

  • Resolution: Employ L. plantarum-specific codon optimization strategies as demonstrated with other recombinant proteins .

• Protein stability concerns

  • Testing: Assess protein stability under experimental conditions by exposing purified RecA to varying temperatures, pH levels, and salt concentrations.

  • Resolution: Add stabilizing agents such as glycerol or specific divalent cations to buffer systems.

• Strain-specific variations

  • Analysis: Different L. plantarum strains may exhibit varying recombination efficiencies or protein expression capabilities.

  • Resolution: Compare results across multiple validated L. plantarum strains to identify optimal host backgrounds .

By systematically addressing these potential sources of inconsistency, researchers can develop more robust protocols for working with recombinant L. plantarum RecA.

How does L. plantarum RecA compare structurally and functionally to RecA proteins from other bacterial species?

Lactobacillus plantarum RecA shares the core structural and functional features common to RecA proteins across bacterial species while exhibiting species-specific variations that reflect its evolutionary adaptations. Although the search results don't provide specific structural comparisons, several insights can be drawn about its comparative features:

Structural Conservation and Variation:

• The RecA protein's core structure is likely highly conserved in L. plantarum as it is across bacteria, consisting of three domains: a central ATPase core, and N-terminal and C-terminal domains involved in DNA binding.

• The sequence analysis of the recA gene has revealed sufficient variation to distinguish L. plantarum from closely related species like L. pentosus and L. paraplantarum, suggesting species-specific structural adaptations .

• These variations occur primarily in regions that don't affect the core catalytic functions, allowing the protein to maintain its essential activities while acquiring species-specific characteristics.

Functional Comparison:

• Like RecA in other bacteria, L. plantarum RecA likely performs the fundamental functions of homologous DNA recombination, SOS response induction, and DNA damage-induced mutagenesis .

• The RecA protein across all bacterial species exhibits multiple biochemical activities: binding to double and single-stranded DNA, catalyzing pairing and exchange of homologous DNA, ATP hydrolysis, and coproteolytic cleavage of regulatory proteins .

• L. plantarum RecA's functionality in homologous recombination is evidenced by the finding that endogenous recombination machinery can efficiently drive recombineering between plasmids harboring homologous sequences, even without heterologous recombinases .

Evolutionary Implications:

• The recA gene's utility as a phylogenetic marker for Lactobacillus species demonstrates that it evolves at an appropriate rate to reflect speciation events within this genus .

• Phylogenetic analyses using recA gene sequences have yielded coherent phylograms that clearly separate L. plantarum, L. pentosus, and L. paraplantarum, confirming its value as an evolutionary marker .

This comparative perspective is valuable for researchers working with RecA across different bacterial species, helping to predict functional conservation while accounting for potential species-specific variations.

What are the key considerations when designing species-specific primers based on recA for Lactobacillus identification?

Designing effective species-specific primers based on the recA gene for Lactobacillus identification requires careful consideration of several key factors to ensure specificity, sensitivity, and reliability:

• Sequence alignment and polymorphism identification

  • Perform comprehensive multiple sequence alignments of recA gene sequences from all target Lactobacillus species, particularly focusing on L. plantarum, L. pentosus, and L. paraplantarum .

  • Identify regions with species-specific polymorphisms that can serve as targets for primer binding.

  • Analyze the distribution of sequence variations to determine optimal primer placement.

• Primer design parameters

  • Target amplicon size: Design primers that generate distinctly different amplicon sizes for each species to allow visual discrimination on gel electrophoresis (e.g., 318 bp for L. plantarum, 218 bp for L. pentosus, and 107 bp for L. paraplantarum) .

  • Primer length: Typically 18-25 nucleotides to balance specificity and annealing efficiency.

  • GC content: Aim for 40-60% GC content with similar melting temperatures between primer pairs.

  • Terminal nucleotides: Include G or C at the 3' end when possible to promote binding stability.

  • Avoid secondary structures: Check for hairpins, self-dimers, and cross-dimers that could interfere with amplification.

• Multiplex PCR optimization

  • When designing primers for multiplex PCR, ensure all primers have compatible annealing temperatures (within 2-3°C of each other).

  • Verify that primers won't cross-react or produce non-specific products when combined in a single reaction.

  • Consider primer concentration adjustments to balance amplification efficiency across all targets.

• Validation strategy

  • Test primers against a panel of reference strains representing target and non-target species.

  • Include closely related non-target species to confirm specificity.

  • Verify results through sequencing of amplicons to confirm target identity.

  • Assess sensitivity by determining the minimum detectable DNA concentration.

• Practical considerations

  • Design primers to work under standard PCR conditions to facilitate adoption in different laboratories.

  • Consider accessibility of target regions (avoid regions that might be difficult to amplify).

  • Include positive controls for each species in the testing protocol.

Following these guidelines will help researchers design robust, species-specific primers based on recA gene sequences for reliable identification of Lactobacillus species, particularly within the L. plantarum group.

How has the understanding of L. plantarum RecA evolved with advances in genomic and proteomic technologies?

The understanding of Lactobacillus plantarum RecA has evolved significantly with advances in genomic and proteomic technologies, transitioning from basic characterization to sophisticated applications in species identification and genetic engineering:

Historical Progression:

• Early characterization (pre-2000s)

  • Initial studies focused on basic identification and preliminary functional characterization of RecA in lactic acid bacteria.

  • Techniques were limited to traditional protein purification and basic genetic analyses.

• Genomic era advancements (2000s)

  • The development of PCR-based techniques and DNA sequencing enabled more detailed analysis of the recA gene.

  • By 2001, researchers had successfully used recA gene sequence comparison to differentiate closely related species like L. plantarum, L. pentosus, and L. paraplantarum .

  • The design of species-specific primers enabled multiplex PCR protocols for simultaneous identification of these species in a single reaction .

• Functional genomics integration (2010s)

  • Whole genome sequencing of multiple L. plantarum strains provided context for recA gene function within the broader genomic landscape.

  • Transcriptomic analyses began to reveal recA expression patterns under various stress conditions.

• Advanced genetic engineering applications (Recent)

  • CRISPR-Cas9 genome editing systems in L. plantarum have highlighted the role of endogenous recombination machinery, potentially including RecA, in facilitating homologous recombination without heterologous recombinases .

  • Studies showing efficient plasmid-based recombineering suggest that RecA may play a significant role in these processes .

Technological Impacts:

• High-throughput sequencing

  • Enabled comparative genomics approaches to study recA variation across numerous strains and species.

  • Facilitated more accurate phylogenetic analyses based on recA and other marker genes.

• Protein structure prediction

  • Advanced bioinformatic tools have allowed better prediction of RecA protein structure and function based on sequence data.

  • Homology modeling has provided insights into species-specific structural variations.

• Genome editing tools

  • CRISPR-Cas9 systems have revolutionized genetic manipulation in L. plantarum, revealing new aspects of RecA function in recombination .

  • The observation that recombinase-free methods can achieve efficient editing highlights the potential role of endogenous RecA .

• Heterologous expression systems

  • Advanced expression systems have enabled the production of recombinant proteins in L. plantarum with optimized conditions, providing templates for RecA expression studies .

This evolution in understanding continues to expand the applications of L. plantarum RecA beyond its original context as a phylogenetic marker to potential roles in sophisticated genetic engineering and biotechnological applications.

How is recombinant L. plantarum being developed for vaccine delivery applications?

Recombinant Lactobacillus plantarum is being developed as a promising vehicle for vaccine delivery, leveraging its natural probiotic properties and genetic tractability. While specific applications of RecA in this context aren't detailed in the search results, the development of L. plantarum as a vaccine delivery platform provides important context for researchers working with recombinant proteins in this organism:

• SARS-CoV-2 vaccine development

  • Researchers have successfully constructed recombinant L. plantarum strains expressing the SARS-CoV-2 spike protein on their surface .

  • The spike gene with optimized codons was efficiently expressed on the bacterial surface and exhibited high antigenicity .

  • Optimal expression conditions were identified as induction with 50 ng/mL SppIP at 37°C for 6-10 hours .

• Stability advantages for oral vaccine delivery

  • Recombinant proteins expressed in L. plantarum have demonstrated remarkable stability under challenging conditions:

    • Temperature stability at up to 50°C

    • Acid resistance at pH as low as 1.5

    • Stability in high salt concentrations

    • Enhanced expression in the presence of 0.2% bile salt

  • These properties make L. plantarum particularly suitable for oral vaccine delivery, as it can withstand gastrointestinal conditions.

• Expression system optimizations

  • Codon optimization has proven critical for efficient heterologous protein expression in L. plantarum .

  • Surface display systems utilizing endogenous signal peptides and anchoring domains have been developed for presenting antigens on the bacterial surface .

  • Inducible expression systems allow controlled production of recombinant proteins.

• Immunological advantages

  • L. plantarum strains have demonstrated high adhesion to intestinal cells and possess strong anti-inflammatory and immunoregulatory functions .

  • As a food-grade bacterium widely recognized as a probiotic, L. plantarum offers a safe platform for vaccine delivery .

  • The bacteria can potentially stimulate both mucosal and systemic immune responses.

• Technical challenges

  • Expression efficiency may be affected by codon usage bias, requiring optimization for each target protein .

  • Protein folding and post-translational modifications may differ from native contexts.

  • Scale-up for clinical applications requires standardization of production processes.

These developments in recombinant L. plantarum technology provide valuable insights for researchers interested in expressing RecA or utilizing RecA-based systems in vaccine development contexts.

What are the current limitations in working with recombinant L. plantarum RecA and how might they be overcome?

Working with recombinant Lactobacillus plantarum RecA presents several limitations that researchers should be aware of, along with potential strategies to overcome these challenges:

• Expression and purification challenges

  • Limitation: Achieving high-level expression of functional RecA protein can be difficult due to codon usage bias in L. plantarum.

  • Solution: Implement codon optimization strategies specific to L. plantarum, as demonstrated successfully with other recombinant proteins . Consider using controlled induction systems with optimized parameters (50 ng/mL inducer at 37°C) .

• Protein stability concerns

  • Limitation: RecA proteins can be prone to aggregation or inactivation during purification and storage.

  • Solution: Optimize buffer conditions based on stability studies. L. plantarum proteins have shown stability at various temperatures (up to 50°C), pH conditions (down to pH 1.5), and salt concentrations , suggesting that appropriate stabilization conditions can be identified.

• Functional assay limitations

  • Limitation: Verifying proper RecA function (ATP hydrolysis, DNA binding, strand exchange) requires specialized assays.

  • Solution: Develop streamlined activity assays specific for L. plantarum RecA, and benchmark against well-characterized RecA proteins from model organisms.

• Recombination efficiency variability

  • Limitation: When using RecA for recombineering applications, efficiency can be variable or unpredictable.

  • Solution: Encode recombineering templates on replicating plasmids to enhance efficiency . Combine with CRISPR-Cas9 selection to eliminate cells with unedited sequences .

• Strain-specific effects

  • Limitation: Different L. plantarum strains may show varying levels of RecA activity or expression.

  • Solution: Test multiple L. plantarum strains for recombination efficiency to identify optimal host backgrounds . Consider creating a RecA-deficient strain for controlled complementation studies.

• Unexpected recombination outcomes

  • Limitation: RecA-mediated recombination can sometimes lead to unexpected outcomes or genomic rearrangements .

  • Solution: Thoroughly characterize recombination products through whole genome sequencing. Design recombineering templates with unique identifiers or markers to track intended versus unintended recombination events.

• Limited structural information

  • Limitation: Detailed structural information specific to L. plantarum RecA is lacking.

  • Solution: Apply structural biology approaches (X-ray crystallography, cryo-EM) to determine the three-dimensional structure of L. plantarum RecA. In the interim, develop homology models based on well-characterized RecA structures from other species.

Addressing these limitations through targeted methodological improvements will expand the utility of L. plantarum RecA in both basic research and biotechnological applications.

What emerging applications might leverage L. plantarum RecA for synthetic biology and metabolic engineering?

Emerging applications leveraging Lactobacillus plantarum RecA for synthetic biology and metabolic engineering represent a frontier in biotechnology research. While the search results don't directly address these applications, several promising directions can be inferred based on RecA's properties and recent advances in L. plantarum engineering:

• RecA-mediated genome integration systems

  • Application: Development of precise, marker-free genome integration tools that leverage endogenous RecA for targeted incorporation of synthetic pathways.

  • Implementation: Design integration cassettes with optimized homology arms that work efficiently with native RecA, potentially eliminating the need for heterologous recombinases .

  • Advantage: This approach could reduce the genetic burden and potential off-target effects associated with heterologous recombinases.

• Controlled recombination circuits

  • Application: Engineered genetic circuits where RecA activity is inducibly controlled to enable programmed genomic rearrangements.

  • Implementation: Place RecA under the control of synthetic promoters that respond to specific environmental signals, allowing dynamic genome reorganization.

  • Potential: This could enable adaptive strain evolution or conditional pathway activation in response to industrial process conditions.

• RecA-based biosensors

  • Application: Utilize RecA's DNA binding properties to develop whole-cell biosensors for detecting DNA-damaging agents or environmental toxins.

  • Implementation: Couple RecA activation to reporter gene expression through the SOS response pathway.

  • Applications: Environmental monitoring or quality control in food and pharmaceutical industries.

• Enhanced homologous recombination for pathway optimization

  • Application: Overexpress or engineer RecA variants to increase the efficiency of homologous recombination for rapid pathway prototyping.

  • Implementation: Combine with CRISPR-Cas9 for iterative genome editing to optimize metabolic flux in engineered pathways .

  • Benefit: Accelerated strain development cycles for industrial applications.

• RecA-facilitated directed evolution

  • Application: Leverage RecA's role in homologous recombination to develop in vivo directed evolution platforms.

  • Implementation: Create systems where RecA-mediated recombination shuffles genetic variants, followed by selection for improved functions.

  • Target: Evolution of enzymes or pathways for improved activity or novel substrate utilization.

• Probiotics with programmable adaptive capabilities

  • Application: Engineer L. plantarum probiotic strains with RecA-dependent adaptive mechanisms that respond to gut environmental conditions.

  • Implementation: Design genetic systems where specific gut signals trigger RecA-mediated genomic rearrangements, activating beneficial functions.

  • Potential: Next-generation smart probiotics that adapt to individual host conditions.

• Cell-free RecA systems for DNA assembly

  • Application: Develop in vitro DNA assembly methods using purified L. plantarum RecA.

  • Implementation: Optimize reaction conditions for RecA-mediated joining of DNA fragments with homologous regions.

  • Advantage: Potentially higher efficiency for assembling GC-rich sequences relevant to Lactobacillus and related species.

These emerging applications highlight the potential for L. plantarum RecA to become a valuable tool in the synthetic biology toolkit, particularly for applications involving lactic acid bacteria in food, health, and industrial contexts.

What are the most significant recent advances in L. plantarum RecA research?

The most significant recent advances in Lactobacillus plantarum RecA research span multiple domains, from fundamental understanding to practical applications in biotechnology. While RecA-specific advances are not extensively detailed in the search results, several related developments provide context for the evolution of this research area:

• Recombinase-free genome editing

  • The discovery that endogenous homologous recombination machinery in L. plantarum, potentially including RecA, can efficiently facilitate genome editing without heterologous recombinases represents a major advancement .

  • This approach has successfully introduced stop codons, silent mutations, and complete gene deletions when combined with CRISPR-Cas9 selection .

  • The finding that encoding recombineering templates on replicating plasmids enhances efficiency suggests new insights into how RecA and related proteins interact with DNA substrates .

• Taxonomic application refinements

  • The recA gene sequence has been established as a reliable phylogenetic marker for differentiating closely related Lactobacillus species, particularly L. plantarum, L. pentosus, and L. paraplantarum .

  • The development of multiplex PCR protocols using species-specific primers designed from recA sequences has enabled rapid and accurate identification of these species in a single reaction .

• Expression system optimizations

  • While not specific to RecA, advances in optimizing recombinant protein expression in L. plantarum provide valuable methodological foundations for RecA production .

  • Identification of optimal induction conditions (50 ng/mL inducer at 37°C for 6-10 hours) and the importance of codon optimization offer templates for RecA expression strategies .

• Stability characterization

  • Research demonstrating the stability of recombinant proteins in L. plantarum under various stress conditions (high temperature, low pH, high salt concentration) provides insights into potential RecA stability properties and experimental considerations .

• CRISPR-Cas9 integration

  • The integration of CRISPR-Cas9 technology with L. plantarum genetic manipulation has opened new avenues for studying RecA function by enabling precise genetic modifications .

  • This combination allows researchers to create defined RecA variants or study its function in various genetic backgrounds.

These advances collectively point toward an expanding toolkit for working with L. plantarum RecA, with implications for both fundamental research and biotechnological applications. The trend toward simplified, more efficient genetic manipulation systems suggests that RecA's role in these processes will continue to be an important area of investigation.

How might further characterization of L. plantarum RecA contribute to improved biotechnological applications?

Further characterization of Lactobacillus plantarum RecA has significant potential to enhance biotechnological applications across multiple domains. Deepening our understanding of this protein could lead to several transformative developments:

• Enhanced genome editing technologies

  • Detailed mechanistic understanding of how L. plantarum RecA facilitates homologous recombination could lead to optimized recombineering protocols with higher efficiency .

  • Structure-function analysis may reveal specific domains or residues that could be engineered to enhance recombination activity or specificity.

  • This knowledge could reduce the need for heterologous recombinases, simplifying genetic modification workflows in L. plantarum and related species .

• Improved strain development for industrial applications

  • Understanding RecA's role in stress responses could enable the development of strains with enhanced robustness for industrial fermentation processes.

  • Engineered RecA variants might facilitate accelerated adaptive laboratory evolution protocols for strain improvement.

  • RecA-optimized homologous recombination could enable more efficient pathway engineering for the production of high-value compounds in L. plantarum.

• Advanced vaccine and therapeutic delivery platforms

  • Building on current developments in recombinant L. plantarum for vaccine delivery , RecA characterization could enhance genetic stability of vaccine strains.

  • Understanding RecA's role in adaptation to gastrointestinal conditions could improve the persistence and efficacy of therapeutic L. plantarum strains.

  • RecA-mediated site-specific integration could enable precise incorporation of immunomodulatory genes for enhanced vaccine efficiency.

• Synthetic biology tools

  • Characterization of RecA's DNA binding specificity could lead to the development of novel inducible recombination systems for synthetic circuit design.

  • RecA variants with altered activity could serve as molecular switches in engineered genetic circuits.

  • Better understanding of RecA regulation might enable the design of genetic systems with programmable genomic plasticity.

• Diagnostic applications

  • Building on RecA's established role in species identification , further characterization could lead to more sensitive diagnostic tools for detecting specific Lactobacillus strains.

  • RecA-based biosensors could be developed for detecting DNA-damaging compounds in food or environmental samples.

• Structural biology advances

  • Solving the three-dimensional structure of L. plantarum RecA would provide valuable insights for protein engineering efforts.

  • Comparative structural analysis with RecA proteins from other species could reveal unique features that might be exploited for biotechnological applications.

By advancing our understanding of L. plantarum RecA's structure, function, and regulation, researchers can unlock new capabilities in precision genome engineering, strain development, and biotechnological applications involving this important probiotic species.

What reagents and equipment are required for RecA purification from L. plantarum?

The purification of RecA protein from Lactobacillus plantarum requires specific reagents and equipment to ensure isolation of functional protein. While the search results don't provide a direct protocol for L. plantarum RecA purification, a comprehensive list can be compiled based on standard protein purification techniques and insights from similar recombinant protein work:

Biological Materials and Reagents:

• Bacterial strain and expression system

  • L. plantarum strain optimized for protein expression

  • Expression vector containing the recA gene with appropriate promoter

  • Induction system (e.g., SppIP at 50 ng/mL for inducible promoters)

• Growth media and buffers

  • MRS broth or appropriate defined media for L. plantarum cultivation

  • Phosphate-buffered saline (PBS) for washing cells

  • Lysis buffer components:

    • 50 mM Tris-HCl (pH 7.5-8.0)

    • 100-300 mM NaCl

    • 1-5 mM EDTA

    • 1-10 mM DTT or β-mercaptoethanol

    • 5-10% glycerol

    • Protease inhibitor cocktail

• Chromatography materials

  • Affinity chromatography resins (if using tagged RecA):

    • Ni-NTA agarose (for His-tagged RecA)

    • Amylose resin (for MBP-tagged RecA)

    • Glutathione-Sepharose (for GST-tagged RecA)

  • Ion exchange resins (e.g., Q-Sepharose, SP-Sepharose)

  • Size exclusion chromatography media (e.g., Superdex 200)

• Elution and storage components

  • Imidazole (for His-tag elution)

  • Reduced glutathione (for GST-tag elution)

  • Maltose (for MBP-tag elution)

  • Storage buffer components:

    • 20-50 mM Tris-HCl (pH 7.5)

    • 50-200 mM NaCl

    • 0.1-1 mM EDTA

    • 1-10 mM DTT

    • 30-50% glycerol

Equipment:

• Cell culture and harvesting equipment

  • Incubator with temperature control (37°C optimal)

  • Shaking platform

  • Centrifuge for cell harvesting (capacity for 1-10 L cultures)

  • Sterile culture vessels

• Cell disruption equipment

  • French press or high-pressure homogenizer

  • Sonicator with temperature control

  • Bead beater (often effective for Gram-positive bacteria)

• Chromatography systems

  • FPLC (Fast Protein Liquid Chromatography) system

  • Appropriate columns for chosen resins

  • Fraction collector

  • UV detector for protein monitoring

• Analysis equipment

  • SDS-PAGE apparatus

  • Western blot equipment

  • Spectrophotometer for protein quantification

  • Plate reader for activity assays

• Storage equipment

  • -80°C freezer for long-term storage

  • Controlled temperature refrigerator (4°C)

  • Liquid nitrogen container (optional)

Advanced equipment for functional characterization:

• Activity assay equipment

  • Thermocycler for DNA strand exchange assays

  • Fluorescence plate reader for ATP hydrolysis assays

  • EMSA (Electrophoretic Mobility Shift Assay) equipment for DNA binding studies

This comprehensive list provides researchers with the necessary resources to plan RecA purification from L. plantarum, with the understanding that protocol optimization will be required for this specific protein.

What are the detailed steps for designing and validating recA-based species identification primers?

Designing and validating recA-based species identification primers for Lactobacillus requires a systematic approach to ensure specificity, sensitivity, and reliability. Based on successful methods reported in the literature , here is a detailed workflow:

Sequence Acquisition and Analysis

1.1. Collect recA sequences

• Obtain recA gene sequences from L. plantarum, L. pentosus, L. paraplantarum, and related species from GenBank or other databases

• Include multiple strains of each species to account for intraspecies variation

• Consider sequencing recA from new isolates if existing data is limited

1.2. Multiple sequence alignment

• Use software such as CLUSTAL W, MUSCLE, or MAFFT to align sequences

• Manually inspect and refine alignments if necessary

• Identify regions of conserved and variable sequences

1.3. Polymorphism identification

• Locate species-specific sequence signatures or polymorphisms

• Focus on regions with sufficient variation between species but conservation within species

• Create a variability profile across the alignment to identify optimal regions

Primer Design

2.1. Target amplicon planning

• Design primers to generate distinctly different amplicon sizes for visual discrimination:

  • L. plantarum: target ~318 bp

  • L. pentosus: target ~218 bp

  • L. paraplantarum: target ~107 bp

2.2. Primer parameter optimization

• Length: 18-25 nucleotides

• GC content: 40-60%

• Melting temperature (Tm): 55-65°C with <5°C difference between primers

• Avoid repetitive sequences and runs of identical nucleotides

• Include G or C at the 3' end (GC clamp)

2.3. Multiplex compatibility checks

• Ensure all primers have compatible Tm values for multiplex PCR

• Check for primer-dimer formation using software like Primer3 or OligoAnalyzer

• Verify similar GC content across all primers

2.4. In silico validation

• Perform BLAST analysis against genomic databases to check for specificity

• Use PCR simulation software to predict amplification products

• Assess potential cross-reactivity with non-target species

Experimental Validation

3.1. Initial PCR optimization

• Test each primer pair individually before multiplexing

• Optimize annealing temperature using gradient PCR

• Determine optimal MgCl₂ concentration

• Adjust primer concentrations if needed

3.2. Multiplex PCR development

• Combine all primers in a single reaction

• Typical reaction mixture:

  • 300 ng of template DNA

  • 1× PCR buffer with appropriate MgCl₂

  • 100 μM dNTPs

  • 0.5-1 μM of each primer (may require adjustment)

  • 2-2.5 U of DNA polymerase

  • Water to final volume (usually 50 μL)

3.3. Reference strain testing

• Test primers against type strains or well-characterized isolates

• Include multiple strains of each target species

• Include closely related non-target species as negative controls

3.4. Amplicon verification

• Sequence amplicons to confirm target identity

• Compare to reference sequences to verify specificity

Assay Optimization and Validation

4.1. Sensitivity assessment

• Determine limit of detection using serial dilutions of template DNA

• Establish minimal cell concentration required for reliable detection

4.2. Reproducibility testing

• Perform inter-laboratory testing if possible

• Assess batch-to-batch variation

• Test different thermal cyclers and reagent sources

4.3. Mixed culture validation

• Test ability to detect target species in mixed bacterial populations

• Determine detection limits in presence of non-target DNA

4.4. Protocol standardization

• Standard thermal cycling conditions:

  • Initial denaturation: 94°C for 3-5 minutes

  • 30-35 cycles of:

    • Denaturation: 94°C for 30-60 seconds

    • Annealing: 55-58°C for 30-60 seconds

    • Extension: 72°C for 30-60 seconds

  • Final extension: 72°C for 5-10 minutes

• Document all optimization parameters for reproducibility

4.5. Application testing

• Validate method with real-world samples (food, clinical, environmental)

• Compare results with established identification methods

This systematic approach ensures the development of robust, reliable recA-based identification primers for Lactobacillus species, particularly for differentiating members of the L. plantarum group.

What statistical methods are appropriate for analyzing RecA sequence data in phylogenetic studies?

Sequence Quality Assessment and Preparation

1.1. Sequence quality metrics

• Calculate Phred quality scores for base calls

• Establish minimum quality thresholds (typically Q20 or higher)

• Trim low-quality regions from sequence ends

1.2. Sequence alignment statistics

• Determine percentage of identical sites

• Calculate pairwise sequence identities

• Identify conserved versus variable regions

• For recA analysis specifically, focus on the informative 322 bp segment identified in previous studies

1.3. Substitution saturation tests

• Implement the Xia test to assess substitution saturation

• Calculate transition/transversion ratios

• Evaluate if sequences are suitable for phylogenetic analysis

Evolutionary Model Selection

2.1. Nucleotide substitution model testing

• Use programs like ModelTest, jModelTest, or the integrated model testers in MEGA

• Compare models using Akaike Information Criterion (AIC)

• Bayesian Information Criterion (BIC) for more conservative model selection

• For RecA specifically, determine if the Kimura 2-parameter, HKY, or GTR models are most appropriate

2.2. Rate heterogeneity assessment

• Test for gamma-distributed rate variation across sites

• Determine optimal shape parameter (α) for gamma distribution

• Assess proportion of invariable sites (I)

2.3. Molecular clock testing

• Likelihood ratio tests to assess clock-like evolution

• Relative rate tests to identify lineages with accelerated or decelerated evolution

Phylogenetic Tree Construction Methods

3.1. Distance-based methods

• Neighbor-joining (NJ) with appropriate distance metrics

• Calculate bootstrap support values (typically 1000 replicates)

• For recA analysis, this approach has been successfully used in conjunction with other methods

3.2. Maximum parsimony (MP)

• Conduct heuristic tree searches with TBR branch swapping

• Calculate consistency index (CI) and retention index (RI)

• Perform bootstrap analysis (1000+ replicates)

• This method has proven effective for recA-based differentiation of Lactobacillus species

3.3. Maximum likelihood (ML)

• Implement with the best-fit evolutionary model

• Calculate likelihood scores for alternative topologies

• Conduct approximate likelihood ratio tests (aLRT) or bootstrap analysis

• This approach has successfully separated Lactobacillus species based on recA sequences

3.4. Bayesian inference

• Run multiple MCMC chains (typically 4) for convergence assessment

• Calculate posterior probabilities as node support values

• Implement in MrBayes or BEAST software packages

• Particularly useful for more complex evolutionary models

Tree Comparison and Consensus Methods

4.1. Topological congruence assessment

• Calculate Robinson-Foulds distances between trees from different methods

• Implement Shimodaira-Hasegawa (SH) test to compare alternative topologies

• For recA analysis, comparing trees from different methods is recommended as done in previous studies

4.2. Consensus tree building

• Generate majority-rule consensus trees

• Calculate support values across methods

• Identify consistently recovered clades across analytical approaches

Statistical Tests for Phylogenetic Signal and Resolution

5.1. Likelihood mapping

• Assess phylogenetic signal strength in the sequence data

• Identify regions of "tree-likeness" versus star-like regions

5.2. Parametric bootstrapping

• Generate simulated datasets under the best-fit model

• Test specific phylogenetic hypotheses

• Establish confidence intervals for branch lengths

5.3. Species delimitation statistics

• Calculate average nucleotide identity (ANI) between sequences

• Implement ABGD (Automatic Barcode Gap Discovery) or similar methods

• Particularly relevant for recA-based species identification applications

Visualization and Robustness Assessment

6.1. Node support visualization

• Display bootstrap values or posterior probabilities on branches

• Consider only nodes with >70% bootstrap support as well-supported

• For recA phylograms, coherent separation of species should be evident across methods

6.2. Sensitivity analysis

• Test effect of taxon sampling on tree topology

• Assess impact of alignment method on phylogenetic reconstruction

• Evaluate robustness to model misspecification