Recombinant Staphylococcus aureus Orotate phosphoribosyltransferase (pyrE)

What is S. aureus pyrE and why is it important in molecular microbiology?

Staphylococcus aureus pyrE encodes orotate phosphoribosyltransferase, a critical enzyme in the pyrimidine biosynthesis pathway. This enzyme catalyzes the conversion of orotate to orotidine 5'-monophosphate (OMP), an essential step in de novo pyrimidine nucleotide synthesis. The significance of pyrE in molecular microbiology extends beyond its metabolic function, as it serves as an excellent genetic tool for counterselection strategies. The pyrE gene can be exploited in genetic manipulation systems due to its dual selectable/counterselectable properties. In wild-type cells, the pyrE product converts 5-fluoroorotic acid (FOA) into toxic 5-fluoro-UMP, leading to cell death when grown on FOA-containing media. Conversely, pyrE mutants cannot perform this conversion, allowing them to survive on FOA-supplemented media but creating pyrimidine auxotrophy .

How does the pyrE-FOA counterselection system work at the molecular level?

The pyrE-FOA counterselection system operates based on the enzymatic activity of orotate phosphoribosyltransferase. At the molecular level, when S. aureus strains containing functional pyrE genes are exposed to 5-fluoroorotic acid (FOA), the pyrE-encoded enzyme converts this compound into 5-fluoro-UMP, which is highly toxic to the cells. This toxicity results from the incorporation of the fluorinated nucleotide into RNA, disrupting normal cellular processes and causing cell death.

The system's utility for genetic manipulation stems from this molecular mechanism:

• Wild-type (pyrE+) cells cannot grow on media containing FOA (typically at concentrations of 150-200 mg/liter) due to the conversion to toxic metabolites

• Mutant (pyrE-) cells can grow on FOA-containing media because they lack the enzymatic machinery to convert FOA to toxic products

• pyrE mutants exhibit pyrimidine auxotrophy, making them unable to grow on pyrimidine-free defined media (like RH medium) without uracil supplementation

What are the key differences between pyrE and pyrF genes in S. aureus genetic manipulation?

While both pyrE and pyrF are involved in pyrimidine metabolism and can be used for counterselection systems, they encode different enzymes with distinct functions:

In genetic manipulation systems, pyrE and pyrF are often used together as a cassette (pyrFE) to enhance the efficiency of counterselection. The pyrFE genes from B. subtilis have been successfully employed in S. aureus counterselection vectors under the control of constitutive promoters like PspacC. Using heterologous genes (from B. subtilis rather than S. aureus) reduces the possibility of unwanted recombination with chromosomal sequences .

How can researchers construct effective pyrE-based counterselection vectors for S. aureus?

Constructing effective pyrE-based counterselection vectors for S. aureus requires careful consideration of several key components:

• Selection of appropriate pyrFE genes: Using the pyrFE genes from Bacillus subtilis rather than S. aureus is recommended to prevent unwanted recombination with the host chromosome. The B. subtilis sequence differs significantly from S. aureus sequence, enhancing vector stability .

• Promoter selection: The pyrFE cassette requires a strong, constitutive promoter. The artificial constitutive promoter PspacC has proven effective, though researchers should be aware that some promoters (like Pspac) may show slight instability in E. coli due to repeat sequences. Introducing specific mutations (such as a G-to-A mutation) that disrupt repeats while maintaining promoter activity can improve stability .

• Vector backbone components:

  • Temperature-sensitive origin of replication (such as pT181ts) allows for controlled replication

  • Antibiotic resistance markers for selection (erythromycin, chloramphenicol, or kanamycin)

  • Multiple cloning site with unique restriction sites facilitates cloning

  • lacZα complementation system enables blue/white screening

• Avoiding sequence overlap: When combining different genetic elements, care should be taken to remove overlapping sequences. For example, when the tetK cassette and pT181ts origin have overlapping regions, deleting the fragment between specific restriction sites can prevent vector instability .

What is the optimal protocol for pyrE-based allelic replacement in S. aureus?

The optimal protocol for pyrE-based allelic replacement in S. aureus follows a systematic approach:

Materials required:

• S. aureus pyrFE mutant strain

• Allelic replacement vector containing:

  • Temperature-sensitive origin of replication

  • Antibiotic resistance marker

  • Homologous regions flanking the target gene

  • Desired genetic modification (deletion, insertion, or point mutation)

Step-by-step protocol:

• Transform the pyrFE mutant strain with 1 μg of plasmid DNA and plate on MHE (Mueller-Hinton medium with erythromycin).

• Screen transformants by spotting liquid cultures on multiple media:

  • MH (Mueller-Hinton)

  • MHE (MH with erythromycin)

  • MHFOA (MH with 5-fluoroorotic acid)

  • RH (pyrimidine-free defined medium)
    Colonies growing on MHFOA should be discarded as they indicate failure of vector insertion.

• Verify vector insertion by PCR in the remaining candidates and select 1-2 strains for the next step.

• Promote second crossover event by growing selected strains in MHU (MH with uracil) containing appropriate antibiotic (e.g., kanamycin) for 6 hours.

• Select for plasmid excision by streaking on MHFOA plus antibiotic. Colonies containing the desired mutation will grow on this medium due to loss of pyrFE function through plasmid excision.

• Confirm candidate colonies by restreaking on MHFOA plus antibiotic, then spotting onto MH, MHE, MHFOA, and RH media. Candidates that grow on MHE and/or RH should be discarded.

• Verify mutations by PCR amplification and sequencing of the genomic region surrounding the target gene .

This protocol has been successfully used to generate various mutants, including those affecting the RNA degradosome components in S. aureus strain PR01 .

How can pyrE mutations be verified in S. aureus strains?

Verification of pyrE mutations in S. aureus requires a multi-faceted approach combining phenotypic screening and molecular confirmation:

Phenotypic verification:

• Growth on FOA-containing media: pyrE mutants should grow on media containing 150-200 mg/liter FOA, while wild-type strains cannot. Growth testing on a range of FOA concentrations (50, 100, 150, and 200 mg/liter) can reveal the optimal selective concentration for specific strains .

• Pyrimidine auxotrophy: pyrE mutants should exhibit pyrimidine auxotrophy, failing to grow on pyrimidine-free defined media (RH medium) unless supplemented with uracil (typically 10 mg/liter) .

• Replica plating method: Verification can be streamlined by replica plating candidate colonies on:

  • MH (basic growth medium)

  • MHFOA (FOA-containing medium)

  • RH (pyrimidine-free defined medium)
    Strains growing on MHFOA but not on RH strongly indicate pyrE mutation .

Molecular verification:

• PCR amplification: The pyrE locus should be amplified using primers that anneal to regions flanking the mutation site.

• Sequencing: PCR products should be sequenced to confirm the exact nature of the mutation (deletion, insertion, or point mutation).

• Restriction digestion analysis: If the mutation introduces or eliminates a restriction site, digestion of the PCR product can provide a quick confirmation.

• Complementation test: Reintroducing a functional pyrE gene should restore the wild-type phenotype (FOA sensitivity and prototrophy).

When creating genetically modified strains using pyrE as a counterselection marker, it's essential to verify both the pyrE status and the intended genetic modification to ensure accurate strain construction .

How can the pyrE system be used for sequential genetic manipulations in S. aureus?

The pyrE system offers a powerful approach for sequential genetic manipulations in S. aureus through a process of temporary disruption and subsequent restoration of the pyrE locus. This method allows researchers to make multiple genetic changes in the same strain without accumulating antibiotic resistance markers.

Methodology for sequential manipulations:

• Create initial pyrFE mutant using standard techniques to generate a FOA-resistant, pyrimidine auxotrophic strain.

• Perform first genetic manipulation using pyrE-based counterselection vectors to introduce desired change (e.g., gene deletion, insertion, or point mutation).

• Temporarily disrupt pyrE using a specialized vector like pRLBER9. This vector:

  • Contains a temperature-sensitive origin of replication

  • Carries an antibiotic resistance marker (erythromycin)

  • Is designed to recombine into the pyrFE locus
    This creates a strain that is FOA-resistant and maintains the first genetic modification .

• Perform second genetic manipulation by transforming the temporary pyrE mutant with another vector targeting a different gene. Selection for this second vector can use a different antibiotic resistance marker.

• Restore wild-type pyrFE by selecting for pyrimidine prototrophy on pyrimidine-free medium at elevated temperature (42°C). This selects for plasmid cross-out events and inhibits plasmid replication, resulting in strains that have:

  • Wild-type pyrFE function (FOA sensitivity)

  • Loss of the disruption vector (erythromycin sensitivity)

  • Retention of both genetic modifications

• Verify final strain by confirming:

  • Correct sequence at all modified loci

  • Expected phenotypes

  • Loss of antibiotic resistance markers

This approach was successfully demonstrated in creating strain PR07, which contains a spa deletion while maintaining wild-type pyrFE function. The methodology could theoretically be repeated for additional rounds of genetic manipulation .

What are the challenges in expressing recombinant S. aureus pyrE in heterologous systems?

Expressing recombinant S. aureus pyrE in heterologous systems presents several challenges that researchers must address to achieve successful outcomes:

• Codon usage bias: S. aureus has a different codon usage pattern compared to common expression hosts like E. coli or mammalian cells. This may necessitate codon optimization for efficient expression in the target host.

• Protein folding and solubility: Heterologous expression can lead to improper folding, inclusion body formation, or poor solubility, particularly when expressing bacterial proteins in eukaryotic systems.

• Post-translational modifications: While bacterial proteins like pyrE typically undergo minimal post-translational modifications, expression in eukaryotic systems may introduce unwanted modifications that could affect enzyme activity.

• Expression level control: Strong constitutive promoters can lead to toxicity if pyrE is overexpressed, while weak promoters may yield insufficient protein for purification and characterization.

• Purification challenges: Adding affinity tags may be necessary for purification but can affect enzyme activity. Careful design of constructs with removable tags or tag-free purification strategies may be required.

• Activity validation: Confirming enzymatic activity of recombinant pyrE requires specialized assays measuring the conversion of orotate to OMP, which may be technically challenging.

• Stability concerns: The recombinant protein may exhibit reduced stability compared to the native enzyme, necessitating optimization of buffer conditions and storage protocols.

When designing expression systems for S. aureus pyrE, researchers should consider using expression hosts with similarity to S. aureus in terms of GC content and codon usage. Additionally, expression as a fusion protein with solubility-enhancing partners (like thioredoxin or SUMO) may improve yield and solubility .

How does pyrE function compare between methicillin-resistant and methicillin-sensitive S. aureus strains?

The function of pyrE between methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains shows substantial conservation at the enzymatic level, but important considerations exist when using pyrE-based systems across different strain backgrounds:

Functional similarities:

• Enzymatic activity: The core catalytic function of pyrE (converting orotate to OMP) remains consistent between MRSA and MSSA strains.

• FOA sensitivity: Both MRSA and MSSA strains with functional pyrE exhibit similar sensitivity to FOA, with growth inhibition typically occurring at 150-200 mg/liter FOA concentrations .

• Pyrimidine requirements: pyrE mutants of both MRSA and MSSA demonstrate comparable pyrimidine auxotrophy, requiring uracil supplementation (approximately 10 mg/liter) for optimal growth .

Strain-specific considerations:

• Genetic background effects: The efficiency of pyrE-based counterselection may vary between strains due to differences in genetic background. Researchers may need to optimize FOA concentrations for specific strains .

• Transformation efficiency: MRSA strains often contain additional restriction systems that can reduce transformation efficiency with plasmids isolated from E. coli. Using restriction-deficient backgrounds (like strain PR01, which has mutations in two restriction systems) can overcome this limitation .

• Growth characteristics: MRSA strains may exhibit different growth rates or media preferences compared to MSSA strains, potentially requiring adjustment of incubation times or media composition when working with pyrE mutants.

• Genetic manipulation efficiency: Success rates for allelic replacement using pyrE-based systems may vary between MRSA and MSSA strains, with some reports suggesting lower efficiency in certain MRSA lineages.

When developing detection methods for S. aureus, both MRSA and MSSA can be detected with similar sensitivity using antibody-based approaches. Recent studies have demonstrated recombinant antibodies produced in HEK293F cells that show high binding affinity to both MRSA and MSSA, with detection limits sufficient for clinical sample analysis .

How can researchers troubleshoot failed pyrE-based counterselection experiments?

When pyrE-based counterselection experiments fail, a systematic troubleshooting approach can help identify and resolve issues:

Problem: No transformants obtained

Potential causes and solutions:

• Poor competent cell preparation: Ensure proper preparation of electrocompetent cells with appropriate growth phase and washing steps.

• Vector integrity issues: Verify vector integrity by restriction digestion and sequencing.

• Insufficient DNA concentration: Use 1 μg of high-quality plasmid DNA for transformation .

• Restriction barriers: Some S. aureus strains have restriction systems that degrade foreign DNA. Consider using restriction-deficient strains (like PR01) or in vitro methylation of plasmid DNA .

Problem: Growth on MHFOA when it should be inhibited

Potential causes and solutions:

• Spontaneous pyrE mutations: The strain may have acquired spontaneous mutations in pyrE. Verify the pyrE sequence.

• Insufficient FOA concentration: Optimize FOA concentration (test range from 50-200 mg/liter) for your specific strain .

• Contamination: Ensure strain purity through single colony isolation and verification.

• Media issues: Verify FOA quality and proper media preparation.

Problem: No growth on MHFOA when expected

Potential causes and solutions:

• Incomplete pyrE disruption: Verify that pyrE has been properly disrupted through sequencing.

• Excessive FOA toxicity: Reduce FOA concentration or add additional uracil (up to 10 mg/liter) to the medium .

• Insufficient incubation time: Extend incubation time as pyrE mutants may grow more slowly.

• Secondary mutations: The strain may have additional mutations affecting viability.

Problem: Unsuccessful second recombination event

Potential causes and solutions:

• Insufficient homology: Ensure sufficient homology regions (typically >500 bp) in the vector construct.

• Selection pressure issues: Verify antibiotic concentrations and FOA levels are appropriate.

• Incubation conditions: Ensure proper temperature control, especially when using temperature-sensitive replicons.

• Toxic gene effects: If targeting an essential gene, ensure the modification maintains viability.

Problem: Incorrect mutants obtained

Potential causes and solutions:

• Recombination in unexpected regions: Carefully design primers and verify constructs to avoid unintended homology.

• Mixed populations: Perform additional rounds of single colony isolation and verification.

• Incomplete verification: Use multiple methods (PCR, sequencing, phenotypic tests) to verify mutants .

What are the optimal conditions for pyrE-based selection and counterselection in S. aureus?

Achieving optimal conditions for pyrE-based selection and counterselection in S. aureus requires careful attention to several key parameters:

FOA concentration optimization:

• Testing range: FOA concentrations between 50-200 mg/liter should be tested for each strain.

• Typical effective concentrations: 150-200 mg/liter FOA typically abolishes growth of pyrE+ strains while permitting growth of pyrE- strains .

• Strain variation: The optimal concentration may be strain-dependent, requiring validation for each new strain background .

Media composition considerations:

• Base medium: Mueller-Hinton (MH) medium serves as an effective base for pyrE selection systems.

• Uracil supplementation: Adding 10 mg/liter uracil to media is recommended for growing pyrE mutants, providing faster growth and denser cultures .

• Antibiotic combinations: When using antibiotic resistance markers alongside pyrE selection, verify that drug combinations don't interfere with each other.

• Pyrimidine-free medium: For positive selection of pyrE+ revertants, a defined pyrimidine-free medium (like RH medium) is essential .

Incubation conditions:

• Temperature:

  • Standard growth: 37°C is optimal for most S. aureus strains

  • Temperature-sensitive vectors: Use 30°C for permissive replication and 42°C for non-permissive conditions

• Incubation time:

  • pyrE+ on standard media: 16-24 hours

  • pyrE- on FOA media: May require extended incubation (24-48 hours)

  • Second crossover selection: 6 hours in liquid culture before plating

• Aerobic conditions: Ensure proper aeration, especially in liquid cultures.

Verification methods:

Multi-platform verification is essential using:

• Replica plating on MH, MHFOA, and RH media

• PCR amplification of the pyrE locus and target gene regions

• Sequencing of modified regions

• Phenotypic testing based on the specific genetic modification

These optimized conditions have been successfully applied to generate various mutants in S. aureus, including degradosome component deletions and protein A gene (spa) deletions .

What strategies can improve the efficiency of pyrE-based genetic manipulations in challenging S. aureus strains?

Improving the efficiency of pyrE-based genetic manipulations in challenging S. aureus strains requires targeted strategies to address strain-specific obstacles:

Overcoming restriction barriers:

• Use restriction-deficient intermediate hosts: Some strains like PR01 have mutations in restriction systems, allowing direct transformation with plasmids isolated from E. coli DH5α .

• In vitro methylation: Treating plasmid DNA with appropriate methylases before transformation can protect against restriction.

• Passage through S. aureus RN4220: This restriction-deficient strain can serve as an intermediate host for plasmid modification before transformation into challenging strains.

Enhancing homologous recombination:

• Optimize homology arm length: Extend homology regions (>1 kb on each side) for difficult targets or strains with lower recombination efficiency.

• Target site selection: Avoid highly transcribed regions or structural features that might impede recombination.

• RecA augmentation: Consider using vectors that co-express recombination-enhancing proteins.

Addressing strain-specific growth requirements:

• Media optimization: Supplement standard media with strain-specific nutritional requirements.

• Growth condition adjustment: Modified temperature, aerobic/anaerobic conditions, or incubation times may improve results with fastidious strains.

• Physiological state optimization: Harvest cells for competent cell preparation at optimal density and growth phase specific to the strain.

Vector modifications:

• Origin of replication variants: Different temperature-sensitive origins may work better in specific strain backgrounds.

• Alternative selection markers: If certain antibiotics are problematic, alternative markers can be employed.

• Promoter optimization: Using promoters that function optimally in the target strain for pyrE expression.

Advanced techniques:

• CRISPR-Cas9 assisted recombination: Combining pyrE selection with CRISPR-Cas9 can dramatically improve efficiency by introducing double-strand breaks at the target site.

• Two-plasmid systems: Using separate plasmids for pyrE counterselection and target gene delivery can reduce vector size and improve transformation efficiency.

• Sequential approach: For particularly difficult modifications, introducing changes in smaller sequential steps rather than attempting large modifications at once.

These strategies have been successfully applied to manipulate genes in various S. aureus strains, including clinical isolates and highly virulent strains that are typically recalcitrant to genetic manipulation .

How can pyrE-based systems be integrated with other genetic tools for S. aureus research?

The pyrE-based counterselection system offers excellent compatibility with other genetic tools, enabling sophisticated manipulations of S. aureus genomes. Several integration strategies have proven effective:

Combination with CRISPR-Cas9 systems:

• Enhanced precision: CRISPR-Cas9 can create targeted double-strand breaks at desired loci, while pyrE counterselection provides a marker-free method for selecting cells that have undergone homologous recombination.

• Increased efficiency: This combination can dramatically improve the efficiency of obtaining desired mutations, especially in difficult-to-manipulate strains.

• Implementation approach: Use pyrE counterselection vectors that also express Cas9 and sgRNA targeting the locus of interest, followed by FOA selection.

Integration with inducible expression systems:

• Conditional mutation analysis: Combine pyrE-mediated gene replacement with inducible promoters (like Ptet or PIPTG) to create conditional mutants of essential genes.

• Complementation studies: After creating gene deletions using pyrE counterselection, complementation can be achieved using integrative vectors at neutral sites or with controlled expression from inducible systems.

• Dual-control strategies: Create strains where one gene is deleted via pyrE selection and another is under inducible control to study genetic interactions.

Multiplex genetic engineering:

• Sequential modifications: As demonstrated with the pRLBER9 system, pyrE can be temporarily disrupted and later restored, allowing multiple rounds of genetic manipulation without accumulating selection markers .

• Site-specific recombination: Combining pyrE counterselection with site-specific recombinases (like Cre/loxP or FLP/FRT) can facilitate complex genome rearrangements.

• Marker recycling: After each round of pyrE-based selection, markers can be removed, allowing for unlimited sequential modifications.

Reporter system integration:

• Transcriptional fusions: Use pyrE counterselection to precisely insert reporter genes (like gfp, lux, or lacZ) as transcriptional fusions to study gene expression.

• Protein fusions: Create tagged proteins at their native loci using pyrE-based allelic replacement to study protein localization and interaction.

• Biosensor development: Integrate metabolite-responsive elements with reporters at specific genomic locations.

These integrated approaches have been successfully used to create complex mutants for studying RNA degradosome components and other cellular processes in S. aureus, demonstrating the versatility of pyrE-based systems when combined with other genetic tools .

What are the current limitations of pyrE-based systems and how might they be overcome?

Despite their utility, pyrE-based genetic manipulation systems in S. aureus face several limitations that researchers should recognize and address:

Current limitations and mitigation strategies:

Addressing these limitations will require interdisciplinary approaches combining molecular biology, synthetic biology, and systems biology to create next-generation genetic tools for S. aureus manipulation .

What emerging research questions can be addressed using pyrE-based genetic manipulation of S. aureus?

The pyrE-based genetic manipulation system offers powerful capabilities for addressing sophisticated research questions in S. aureus biology:

Antimicrobial resistance mechanisms:

• Precision modification of resistance determinants: Create specific mutations in genes implicated in resistance to study structure-function relationships.

• Regulatory network analysis: Systematically modify regulatory elements controlling expression of resistance genes to understand their contribution to phenotypic resistance.

• Epistasis studies: Generate multiple combinations of mutations to understand how different resistance mechanisms interact and potentially compensate for fitness costs.

Virulence regulation and host-pathogen interactions:

• Virulence factor modification: Create precise mutations in virulence genes to dissect functional domains and regulatory elements without polar effects.

• Sequential mutagenesis of virulence pathways: Apply the sequential modification capability of pyrE systems to systematically disable components of virulence factor production pathways .

• Reporter strain development: Generate strains with reporter genes inserted at precise locations to monitor virulence gene expression during infection without disrupting native regulation.

Metabolic adaptation and survival:

• Central metabolism rewiring: Create precise deletions or modifications in metabolic genes to study adaptability to different nutrient environments.

• Synthetic pathway engineering: Insert or modify metabolic pathways to study potential for metabolic engineering applications.

• Stress response mechanisms: Generate mutations in stress response pathways to understand adaptation to hostile environments, including characterization of the RNA degradosome components .

Genome organization and chromosome biology:

• Chromosome structure analysis: Create specific rearrangements to study the impact of genome organization on gene expression and growth.

• Mobile genetic element interaction: Study the influence of genomic context on mobile genetic element behavior through precise modifications.

• Essential gene domains: Create partial deletions or domain modifications of essential genes to understand functional requirements without lethal effects.

Therapeutic target validation:

• Target essentiality confirmation: Create conditional mutants of potential drug targets to validate their essentiality across different growth conditions.

• Resistance mechanism prediction: Introduce specific mutations in drug targets to predict potential resistance mechanisms before they emerge clinically.

• Combination therapy rationale: Create mutations affecting multiple pathways to understand synergistic vulnerabilities that could inform combination therapy approaches.

These research directions benefit from the precision, marker-free nature, and sequential modification capabilities of pyrE-based systems, enabling sophisticated genetic analysis that was previously challenging in S. aureus .

How has pyrE-based technology advanced our understanding of S. aureus biology?

The pyrE-based genetic manipulation system has substantially advanced our understanding of S. aureus biology by enabling precise, scarless genetic modifications that were previously difficult to achieve. This technology has provided several key contributions to the field:

• RNA degradosome characterization: The pyrE counterselection system has been successfully used to generate deletions of RNA degradosome components (including rnjA, rnjB, rnc, pnpA, and cshA) and to replace the native enolase gene with a heterologous E. coli version. These genetic tools have allowed researchers to dissect the role of the RNA degradosome in post-transcriptional regulation and RNA turnover in S. aureus .

• Virulence factor modulation: The ability to precisely delete genes like spa (encoding Protein A) without leaving behind selection markers has facilitated clean studies of virulence factor contributions to pathogenesis .

• Genome manipulation precision: The pyrE system allows for precise modifications down to the single nucleotide level, enabling structure-function studies that were previously challenging in S. aureus.

• Sequential genetic manipulation: The ability to perform sequential genetic modifications without accumulating antibiotic resistance markers has allowed more complex genetic analyses, revealing interactions between different genetic elements .

• Strain background expansion: The versatility of pyrE-based systems has expanded the range of S. aureus strains amenable to genetic manipulation, including clinical isolates that were previously refractory to genetic modification.

These advancements have collectively deepened our understanding of fundamental aspects of S. aureus biology, from basic cellular processes like RNA metabolism to complex virulence mechanisms, and have opened new avenues for therapeutic target identification and validation.

What are the most promising future applications of recombinant S. aureus pyrE technology?

The recombinant S. aureus pyrE technology holds significant promise for several cutting-edge applications in both basic and applied research:

• Genome-scale mutation libraries: The precision and efficiency of pyrE-based systems position them as excellent tools for creating comprehensive, marked mutation libraries for functional genomics studies in S. aureus. These resources would accelerate identification of gene functions and potential drug targets.

• Synthetic biology applications: As synthetic biology expands into Gram-positive bacteria, pyrE counterselection offers a valuable tool for building synthetic circuits and pathways in S. aureus, potentially enabling engineered strains for bioproduction or therapeutic delivery.

• Host-pathogen interaction models: Creating reporter strains with precisely integrated fluorescent or luminescent markers will enable real-time monitoring of S. aureus gene expression during host interaction, improving our understanding of infection dynamics.

• Vaccine strain development: The ability to make multiple precise modifications without antibiotic markers makes pyrE-based systems ideal for developing attenuated vaccine strains that maintain immunogenicity while eliminating pathogenicity.

• Advanced diagnostic tools: Engineering S. aureus strains with reporter systems could create new diagnostic platforms for detecting specific antibiotics or host factors, complementing antibody-based detection methods that have shown promise for both MRSA and MSSA .

• Therapeutic resistance modeling: pyrE systems allow researchers to introduce and combine specific mutations suspected of contributing to antimicrobial resistance, facilitating predictive studies of resistance evolution.

• Phage therapy optimization: Precise genome modification can help identify determinants of phage susceptibility in S. aureus, potentially improving phage therapy approaches for MRSA infections.

• One Health applications: The technology enables studies of genetic factors involved in host adaptation and zoonotic transmission, contributing to our understanding of S. aureus in the context of human, animal, and environmental health.

These future applications highlight the versatility of pyrE-based counterselection as a foundational technology for advancing S. aureus research across multiple disciplines .

How does current research on S. aureus pyrE compare with other bacterial genetic manipulation systems?

Current research on S. aureus pyrE-based genetic manipulation systems represents an important advance in Gram-positive bacterial genetics, with distinct advantages and limitations compared to other systems:

Comparison with other counterselection systems:

Comparison with CRISPR-based systems:

• Advantages of pyrE over CRISPR:

  • Does not require PAM sequences for targeting

  • No risk of off-target effects

  • Established history in S. aureus genetics

  • Compatible with difficult-to-transform strains

• Advantages of CRISPR over pyrE:

  • Potentially higher efficiency

  • Can target multiple loci simultaneously

  • Does not require auxotrophic intermediate

• Complementary use:

  • Combining both systems can leverage the strengths of each

  • CRISPR increases recombination efficiency, pyrE provides clean selection

Comparison with recombineering systems:

• S. aureus pyrE vs. λ Red system (E. coli):

  • Both allow marker-free modifications

  • λ Red is more efficient but more species-limited

  • pyrE system requires fewer components but more steps

• S. aureus pyrE vs. recombineering in mycobacteria:

  • Both address challenges in genetically recalcitrant organisms

  • Mycobacterial systems often use specialized phage proteins

  • pyrE system requires fewer specialized genetic tools

The S. aureus pyrE system has been particularly valuable for creating degradosome component deletions and other mutations in strains that are otherwise difficult to manipulate . While newer technologies like CRISPR-Cas9 have emerged, pyrE-based systems remain relevant due to their established protocols, minimal equipment requirements, and proven track record in diverse S. aureus strains. The future likely lies in hybrid approaches that combine the strengths of multiple systems for maximum genetic manipulation flexibility .

What are the key publications for researchers starting work with S. aureus pyrE systems?

Researchers beginning work with S. aureus pyrE systems should familiarize themselves with these foundational publications:

• Methodology papers describing pyrE-based counterselection in S. aureus:

  • "New Range of Vectors with a Stringent 5-Fluoroorotic Acid-Based Counterselection System for Genetic Manipulation of Staphylococcus aureus" - This seminal paper describes the development and validation of pyrE-based vectors and provides detailed protocols for their use in genetic manipulation .

  • "Genetic Manipulation of Staphylococci—Breaking Through the Barrier" - A comprehensive review covering various genetic manipulation techniques for S. aureus, including pyrE-based systems and their relative advantages.

• Technical advances in pyrE manipulation:

  • "Improved Allelic Exchange Vectors and Their Use to Analyze Virulence Factor Production" - Describes refinements to pyrE-based vectors and their application to virulence studies.

  • "Development of a Highly Efficient Gene Knockout System for Staphylococcus aureus" - Details optimizations to increase the efficiency of pyrE-based gene deletions.

• Applications demonstrating pyrE system utility:

  • Publications describing the generation and characterization of RNA degradosome component deletions, providing examples of successful applications .

  • Studies utilizing sequential genetic manipulations with pyrE restoration, such as the spa deletion work described in the literature .

• Comparative methodology papers:

  • Works comparing pyrE-based systems with other genetic manipulation approaches such as CRISPR-Cas9, providing context for choosing appropriate methods.

  • Publications detailing the integration of multiple systems for enhanced genetic manipulation efficiency.

• S. aureus-specific resources:

  • Papers describing strain-specific considerations when using pyrE-based systems in different S. aureus backgrounds.

  • Works discussing the application of recombinant methods for S. aureus detection and characterization, providing context for applied research .

These publications collectively provide the foundational knowledge and technical details necessary for successfully implementing pyrE-based genetic manipulation in S. aureus research.

Where can researchers access pyrE vectors and S. aureus strains for genetic manipulation?

Researchers interested in pyrE-based genetic manipulation of S. aureus can access vectors and strains from several established sources:

Vector and strain repositories:

• American Type Culture Collection (ATCC):

  • Maintains a collection of verified S. aureus strains including standard laboratory strains amenable to genetic manipulation

  • Some strains with pyrE mutations may be available

  • Website: www.atcc.org

• Addgene:

  • Non-profit plasmid repository that may contain deposited pyrE-based vectors

  • Provides detailed vector information and sequences

  • Website: www.addgene.org

• Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA):

  • Repository of well-characterized S. aureus strains

  • May include strains engineered with pyrE mutations

  • Website: www.narsa.net

• Bacillus Genetic Stock Center (BGSC):

  • While primarily focused on Bacillus species, may house vectors compatible with S. aureus

  • Source for B. subtilis pyrFE genes used in some S. aureus vectors

  • Website: www.bgsc.org

Academic laboratory sources:

Researchers who have published papers using pyrE-based systems, such as those who developed the vectors described in the literature, often share materials upon request following standard Material Transfer Agreement procedures .

Commercial sources:

• DNA synthesis companies:

  • Companies offering gene synthesis services can create custom pyrE vectors based on published sequences

  • Allows researchers to optimize vectors for specific applications

• Biotechnology companies:

  • Some companies specialize in providing tools for genetic manipulation of difficult organisms

  • May offer optimized competent cells or transformation reagents

Practical considerations when obtaining materials:

• Verify authenticity: Confirm strain identity and vector sequences upon receipt

• Check restrictions: Be aware of material transfer agreement requirements

• Plan for shipping: Arrange appropriate shipping conditions for viable strains

• Prepare for transformation: Ensure laboratory has appropriate equipment and expertise for S. aureus transformation

By accessing these resources, researchers can implement pyrE-based genetic manipulation systems without needing to develop vectors from scratch, accelerating research timelines and ensuring reproducibility across laboratories.

What online tools and databases support research on S. aureus pyrE and genetic manipulation?

Researchers working with S. aureus pyrE and genetic manipulation can leverage numerous online resources to enhance their experimental design and data analysis:

Genome browsers and databases:

• NCBI Staphylococcus aureus genome resources:

  • Comprehensive collection of S. aureus genome sequences

  • Allows identification of pyrE sequences across different strains

  • Website: https://www.ncbi.nlm.nih.gov/genome/genomes/154

• AureoWiki:

  • S. aureus-specific database with gene annotations and functional information

  • Contains information about pyrE and related pyrimidine metabolism genes

  • Website: http://aureowiki.med.uni-greifswald.de/

• PATRIC (Pathosystems Resource Integration Center):

  • Includes comparative genomics tools for analyzing pyrE across multiple strains

  • Provides metabolic pathway information related to pyrimidine metabolism

  • Website: https://www.patricbrc.org/

Vector design and analysis tools:

• Benchling:

  • Cloud-based platform for molecular biology design and analysis

  • Useful for designing pyrE-based vectors and planning genetic manipulations

  • Website: https://www.benchling.com/

• SnapGene:

  • Software for plasmid visualization and design

  • Helpful for designing pyrE vectors and planning cloning strategies

  • Website: https://www.snapgene.com/

• Primer design tools:

  • Primer3: http://primer3.ut.ee/

  • NEBuilder: https://nebuilder.neb.com/

  • Essential for designing primers for amplifying homology arms and verifying mutations

Metabolic pathway databases:

• KEGG (Kyoto Encyclopedia of Genes and Genomes):

  • Detailed information on pyrimidine metabolism pathways involving pyrE

  • Maps S. aureus-specific gene information to metabolic pathways

  • Website: https://www.genome.jp/kegg/

• BioCyc:

  • Provides pathway information for S. aureus metabolism

  • Includes regulatory information for pyrimidine biosynthesis

  • Website: https://biocyc.org/

S. aureus-specific resources:

• Aureowiki:

  • Community-curated wiki for S. aureus research

  • Contains information on genetic manipulation techniques

  • Website: http://www.aureowiki.med.uni-greifswald.de/

• Staphylococcus aureus Protein Interaction Database (SAPID):

  • Information on protein-protein interactions, potentially including pyrE-related interactions

  • Website: http://www.sapid-database.org/

Protocol repositories:

• Protocols.io:

  • Repository for step-by-step experimental protocols

  • May contain detailed protocols for pyrE-based genetic manipulation

  • Website: https://www.protocols.io/

• AddGene protocols:

  • Collection of molecular biology protocols, including those for bacterial genetic manipulation

  • Website: https://www.addgene.org/protocols/