Recombinant Chromobacterium violaceum Orotate phosphoribosyltransferase (pyrE)

What is Orotate phosphoribosyltransferase (OPRTase) and what is its biological role in C. violaceum?

Orotate phosphoribosyltransferase (OPRTase), encoded by the pyrE gene, is a key enzyme in de novo pyrimidine nucleotide biosynthesis. In C. violaceum and other bacteria, OPRTase catalyzes the reaction between α-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate (OA) in the presence of Mg²⁺ to produce pyrophosphate and orotidine 5'-monophosphate (OMP), which is a critical precursor in pyrimidine nucleotide synthesis . This enzyme is essential for uracil synthesis, and pyrE gene mutations typically result in uracil auxotrophy, meaning the organism requires external uracil to grow . The pyrE gene is particularly significant in C. violaceum as it can be leveraged for genetic manipulation systems, similar to those established in other bacteria like Clostridium difficile .

How does the catalytic mechanism of C. violaceum OPRTase function?

The catalytic mechanism of OPRTase involves several key steps that have been elucidated through structural and computational studies in related enzymes. Based on research with Escherichia coli OPRTase (EcOPRTase), which shares significant homology with C. violaceum OPRTase, the reaction proceeds as follows:

• Binding of substrates: PRPP binds to the active site along with Mg²⁺, followed by orotate (OA)

• Proton transfer: The most feasible mechanism involves a proton transfer from the N1 atom of orotate to a water molecule, and from that water molecule to the α-phosphate O2A atom of PRPP

• Nucleophilic attack: The N1 atom of orotate attacks the C1 atom of PRPP

• Formation of products: This reaction yields OMP and pyrophosphate

Several conserved residues, including Lys73, Asp125, Lys103*, Arg99*, and the Mg²⁺ ion, play crucial roles in electrostatically stabilizing the transition state and maintaining a closed conformation of the flexible catalytic loop during the enzymatic reaction .

What are the structural characteristics of C. violaceum OPRTase?

C. violaceum OPRTase exists as a dimeric enzyme with a flexible catalytic loop that establishes hydrogen bond interactions with the pyrophosphoryl group of PRPP. Molecular dynamics (MD) simulations and X-ray crystallography studies of related OPRTases reveal significant conformational changes in this loop during catalysis . The active site contains several highly conserved residues that coordinate substrate binding and catalysis, including those that interact with the Mg²⁺ ion required for activity. The enzyme's structure includes domains for PRPP binding and orotate recognition, which work together to position the substrates optimally for the reaction to occur.

How can I generate and select for pyrE deletion mutants in C. violaceum?

Creating pyrE deletion mutants in C. violaceum can be accomplished using homologous recombination approaches similar to those described for other bacteria. The process involves:

• Design and construction of a deletion plasmid:

  • Amplify upstream and downstream homology arms (~1000 bp each) of the pyrE gene

  • Include a selectable marker (e.g., kanamycin resistance gene) between the homology arms

  • Clone these fragments into a suitable suicide vector (e.g., pMAD-like vectors)

• Introduction into C. violaceum:

  • Transform or conjugate the construct into C. violaceum

  • Select for integration of the plasmid using the appropriate antibiotic

• Selection of deletion mutants:

  • Screen for resistance to 5-fluoroorotic acid (5-FOA), as pyrE mutants are resistant to this compound

  • Confirm uracil auxotrophy by testing growth with and without uracil supplementation

• Verification:

  • Confirm the deletion using PCR with primers flanking the pyrE locus

  • Verify by sequencing to ensure no unwanted mutations have occurred

The resulting pyrE mutant will be auxotrophic for uracil and resistant to 5-FOA, making it an excellent base strain for subsequent genetic manipulations .

What considerations are important when designing pyrE complementation experiments?

When designing pyrE complementation experiments, several factors must be considered:

• Gene dosage effects:

  • Avoid using multicopy plasmids that may lead to overexpression

  • Consider chromosomal integration at the native locus for physiological expression levels

• Complementation strategy options:

  • Direct restoration of the native locus: Using allele-coupled exchange (ACE) to restore the wild-type pyrE gene

  • Complementation with heterologous pyrE: Using a pyrE gene from a related species (e.g., C. sporogenes pyrE has been used in C. difficile systems)

• Selection considerations:

  • The restoration of pyrE function allows selection on minimal media without uracil

  • This provides a clean selection method without requiring additional antibiotic markers

• Verification protocols:

  • PCR verification using primers that anneal to chromosomal genes residing up- and downstream of the pyrE gene

  • Nucleotide sequencing of the amplified fragment to confirm the wild-type sequence

  • Growth tests on media with and without uracil supplementation

This approach allows for stable complementation without the complications associated with plasmid-based systems, such as plasmid loss or variable copy number .

How can pyrE-based systems be used for targeted gene modifications in C. violaceum?

The pyrE-based allelic exchange system provides a powerful tool for precise genetic manipulations in C. violaceum, similar to systems established in other bacteria. The general workflow involves:

• Starting with a pyrE deletion mutant (uracil auxotroph, 5-FOA resistant)

• Constructing an allelic exchange vector containing:

  • Homology arms flanking the target gene

  • The desired modification (deletion, point mutation, etc.)

  • A heterologous pyrE allele as a counter/negative selection marker

• Selection process:

  • First selection: Integration of the plasmid (typically on media containing uracil)

  • Second selection: Resolution of the plasmid (on media containing 5-FOA and uracil)

  • Screening for the desired modification among 5-FOA resistant colonies

• Restoration of pyrE prototrophy:

  • After confirming the target gene modification, the pyrE function can be restored using ACE

  • This allows characterization of mutant phenotypes in a PyrE proficient background

This system has been successfully used to create in-frame deletions in multiple genes in Clostridium difficile and could be adapted for C. violaceum, targeting genes of interest such as those involved in violacein production, virulence, or biofilm formation .

What advantages does pyrE-based genetic manipulation offer over other methods for C. violaceum?

The pyrE-based genetic manipulation system offers several distinct advantages compared to other methods:

• Precision and flexibility:

  • Allows for precise, marker-less modifications including:

    • In-frame deletions

    • Point mutations

    • Gene replacements

    • Insertions of heterologous genes

• Efficiency:

  • Higher efficiency than many traditional homologous recombination approaches

  • Robust counter-selection with 5-FOA provides strong selection for desired recombination events

• Complementation advantages:

  • Enables single-copy complementation at the pyrE locus

  • Avoids gene dosage issues associated with plasmid-based complementation

  • Provides stable complementation without antibiotic selection pressure

• Iterative engineering:

  • Once a modification is made, the strain can be quickly returned to uracil prototrophy

  • The process can be repeated for multiple sequential modifications

  • Rapid "correction" method (5-7 days) makes pyrE⁻ strains attractive hosts for mutagenesis studies

• Addressing biological questions:

  • Particularly useful for studying genes involved in violacein production, which is regulated by complex signaling networks in C. violaceum

  • Enables investigation of the interplay between ribosomal perturbation and violacein production

How does pyrE function relate to violacein production in C. violaceum?

While pyrE itself is not directly involved in violacein biosynthesis, research suggests interesting connections between translation processes and violacein production that may be relevant when studying pyrE mutants:

• Translation inhibition and violacein production:

  • Sublethal doses of antibiotics that target the polypeptide elongation step of translation induce violacein production in C. violaceum ATCC31532

  • This includes antibiotics like hygromycin A, blasticidin S, spectinomycin, hygromycin B, apramycin, tetracycline, erythromycin, and chloramphenicol

• Regulatory connections:

  • An antibiotic-induced response (air) two-component regulatory system has been identified that is required for violacein production in response to translation inhibition

  • This system consists of a sensor histidine kinase (AirS), a response regulator (AirR), and an oxidoreductase molybdopterin-binding domain protein (AirM)

• Link to quorum sensing:

  • The Air system appears to be connected to quorum-dependent signaling through the CviI/CviR system

  • The negative regulator VioS is also involved in this regulatory network

• Implications for pyrE research:

  • Since pyrE mutations affect nucleotide metabolism and can potentially impact translation processes, researchers should consider possible effects on violacein production when working with pyrE mutants

  • This connection provides an interesting avenue for investigating the relationship between central metabolism and secondary metabolite production in C. violaceum

What role might pyrE play in C. violaceum virulence and host interactions?

The relationship between pyrE and C. violaceum virulence is complex and worth considering in research contexts:

• Direct metabolic impacts:

  • As a key enzyme in pyrimidine biosynthesis, pyrE function affects bacterial growth and survival

  • Pyrimidine auxotrophy resulting from pyrE deficiency would likely attenuate virulence in infection models

• Connection to virulence factors:

  • C. violaceum is known to cause fatal septicemia in humans and animals

  • The organism possesses at least two distinct type III secretion systems (T3SSs) encoded by Chromobacterium pathogenicity islands (Cpi-1/1a and Cpi-2)

  • Research suggests connections between metabolic status and virulence factor expression

• Biofilm and virulence relationships:

  • Translation-inhibiting antibiotics induce not only violacein production but also biofilm formation and virulence against Drosophila melanogaster

  • These responses involve the Air regulatory system that connects translation status to virulence traits

• Experimental considerations:

  • When studying pyrE mutations or using pyrE-based tools, researchers should monitor potential effects on:

    • Virulence factor expression

    • Biofilm formation capabilities

    • Host interaction outcomes

    • Antibiotic susceptibility profiles

This understanding is crucial when interpreting results from genetic studies using pyrE-based tools or when investigating the relationship between metabolism and virulence in C. violaceum.

What are common challenges when working with C. violaceum pyrE systems and how can they be addressed?

Researchers working with C. violaceum pyrE systems may encounter several challenges:

• Growth limitations with pyrE mutants:

  Challenge	Solution
  Slow growth of pyrE mutants	Supplement media with optimal uracil concentration (typically 50 μg/mL)
  Variable colony morphology	Use freshly prepared media and consistent uracil concentrations
  Reversion to prototrophy	Maintain 5-FOA selection pressure when working with auxotrophs

• Transformation efficiency issues:

  • C. violaceum can have lower transformation efficiency compared to model organisms

  • Optimize electroporation parameters specifically for C. violaceum

  • Consider using conjugation as an alternative method for introducing DNA

  • Ensure DNA is free of methylation if C. violaceum possesses restriction systems

• Selection challenges:

  • False positives on 5-FOA media can occur due to spontaneous mutations

  • Verify all putative mutants by PCR and sequencing

  • Include proper controls at each step of the selection process

  • Use appropriate 5-FOA concentration (typically 500 μg/mL) to balance selection stringency

• Complementation verification:

  • Confirm restoration of uracil prototrophy by growth tests on minimal media

  • Verify gene sequence integrity after complementation

  • Check for any unintended polar effects on neighboring genes

  • Consider whole genome sequencing to confirm no additional mutations have occurred

How can recombinant C. violaceum OPRTase be optimally expressed and purified for biochemical studies?

For researchers interested in biochemical and structural studies of C. violaceum OPRTase:

• Expression system optimization:

  Parameter	Recommendation
  Expression host	E. coli BL21(DE3) or similar strain optimized for protein expression
  Vector	pET-based vectors with T7 promoter and appropriate affinity tag (His6 is commonly used)
  Induction	IPTG 0.1-0.5 mM at OD600 0.6-0.8; lower temperature (16-25°C) may improve solubility
  Media	Rich media (LB) for general expression; minimal media for isotope labeling if needed

• Purification strategy:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

  • Consider adding 5-10 mM Mg²⁺ in buffers to stabilize the enzyme

  • Include size exclusion chromatography as a polishing step to ensure homogeneity

  • Verify purity by SDS-PAGE and activity by enzymatic assays

• Activity assay considerations:

  • Monitor the formation of OMP spectrophotometrically

  • Include appropriate controls for non-enzymatic reaction rates

  • Consider the equilibrium between different tautomeric forms of orotate substrate

  • Ensure the presence of Mg²⁺ as a cofactor for optimal activity

• Structural studies preparation:

  • For crystallography, screen various buffer conditions with and without substrates/substrate analogs

  • For molecular dynamics studies, consider the flexible catalytic loop and its conformational changes

  • For enzyme kinetics, determine optimal pH, temperature, and substrate concentration ranges

How might CRISPR-Cas systems be integrated with pyrE-based tools for C. violaceum genetic engineering?

The integration of CRISPR-Cas systems with pyrE-based tools represents an exciting frontier for C. violaceum genetic engineering:

• Combined approach strategy:

  • Use pyrE counter-selection to introduce CRISPR-Cas components into C. violaceum

  • Employ CRISPR-Cas for precise targeting and pyrE for selection of edited cells

  • Leverage the rapid restoration of uracil prototrophy via ACE after CRISPR-mediated editing

• Potential advantages of integration:

  • Increased precision of genetic modifications

  • Improved efficiency for multiplexed gene editing

  • Reduced occurrence of off-target effects by combining two selection mechanisms

  • Ability to target previously challenging genomic regions

• Technical considerations:

  • Optimization of guide RNA design specific to C. violaceum genome

  • Selection of appropriate Cas variants (Cas9, Cas12a, etc.) for optimal activity in C. violaceum

  • Development of delivery methods for CRISPR components into pyrE-deficient strains

  • Establishment of protocols to verify editing outcomes without introducing unwanted mutations

• Proof-of-concept targets:

  • Focus initial efforts on well-characterized genes like those involved in violacein production

  • Target the Air regulatory system components to further elucidate their role

  • Engineer biosynthetic gene clusters for novel secondary metabolite production

  • Modify type III secretion system components to investigate virulence mechanisms

What potential applications exist for engineered C. violaceum strains in biotechnology and medicine?

Engineered C. violaceum strains created using pyrE-based systems offer promising applications:

• Violacein production optimization:

  • Engineered strains with enhanced or regulated violacein production

  • Exploitation of violacein's antimicrobial and anti-parasitic properties

  • Development of controlled production systems responding to specific environmental cues

• Biosensor development:

  • Utilizing the Air regulatory system's response to translation inhibitors

  • Creating reporter strains for antibiotic discovery programs

  • Developing environmental sensors for antimicrobial compounds

• Therapeutic protein production:

  • Leveraging C. violaceum's unique metabolic capabilities for heterologous protein expression

  • Engineering strains with modified glycosylation or other post-translational modifications

  • Development of novel biopharmaceuticals with specific activity profiles

• Ecological applications:

  • Understanding and potentially modifying C. violaceum's interactions with other microorganisms

  • Exploring the "competition sensing" mechanism to develop strains with enhanced competitive fitness

  • Creating modified strains for environmental applications such as bioremediation

• Gene therapy vector development:

  • Investigating the potential of modified, attenuated C. violaceum strains as delivery vehicles

  • Utilizing type III secretion systems for targeted protein delivery to eukaryotic cells

  • Engineering non-pathogenic variants with specific tissue tropisms

These diverse applications highlight the importance of developing robust genetic tools like pyrE-based systems for C. violaceum manipulation, opening new avenues for both basic research and applied biotechnology.