Recombinant Pseudomonas syringae pv. tomato Urease subunit alpha (ureC), partial

What is the ureC gene in Pseudomonas syringae pv. tomato and why is it significant for research?

The ureC gene in Pseudomonas syringae pv. tomato encodes the alpha subunit of urease, an enzyme that catalyzes the hydrolysis of urea to ammonia and carbon dioxide. This gene is particularly significant for research because it is the largest of the genes encoding urease functional subunits and contains several highly conserved regions that are suitable as PCR priming sites . These conserved regions make ureC an excellent marker for studying urease diversity and function across bacterial species.

The significance of ureC in P. syringae pv. tomato research extends to understanding bacterial adaptation mechanisms in plant environments. Urease activity contributes to nitrogen metabolism and potentially to pathogenicity by altering the local pH environment during plant colonization. Studies of recombinant ureC enable researchers to investigate the role of this enzyme in bacterial survival and virulence, particularly in the context of host-pathogen interactions that characterize P. syringae pv. tomato infections in plants such as tomato and Arabidopsis thaliana .

The conserved nature of urease across different species, with similarities detected even between bacterial and plant ureases, suggests a common ancestral urease gene . This evolutionary conservation makes ureC an interesting target for comparative genomic studies across different bacterial pathogens.

How does the structure of urease in Pseudomonas syringae compare to other bacterial ureases?

While the specific crystal structure of P. syringae urease has not been fully characterized in the provided sources, insights can be drawn from studies of other bacterial ureases that share significant homology. Bacterial ureases typically exhibit a conserved structure with a barrel-like shape covered by a mobile flap that controls access to the active site . The active site contains nickel ions that are essential for catalytic activity.

Based on studies of Helicobacter pylori urease, which shares structural similarities with other bacterial ureases, the enzyme contains both penta- and hexacoordinate nickel ions with specific amino acid residues forming the coordination sphere . The binding pocket is predominantly lined with hydrophobic amino acids, and the enzyme contains a mobile flap covering the active site that plays a crucial role in substrate access and product release.

Comparative analysis suggests that P. syringae urease likely shares these fundamental structural features, with the urease subunit alpha (encoded by ureC) forming part of the multimeric enzyme complex. The amino acid sequence conservation among ureases from different species supports this structural similarity, despite variations in subunit organization across species . For instance, while H. pylori urease has a structure with two different subunits in a 1:1 ratio forming a larger complex, other species may have one or three distinct subunits, yet they maintain similar functional properties.

What PCR primers and conditions are recommended for amplifying the ureC gene from Pseudomonas syringae?

For amplifying the ureC gene from Pseudomonas syringae or related bacteria, researchers commonly use degenerate primers designed to target the conserved regions of this gene. Based on the literature, the primer set L2F (5′-ATHGGYAARGCNGGNAAYCC-3′) and L2R (5′-GTBSHNCCCCARTCYTCRTG-3′) has been successfully employed to amplify ureC genes from both genomic DNA and cDNA .

The recommended PCR conditions for ureC amplification are as follows:

• Reaction mixture (20 μl):

  • 100 ng template DNA

  • 500 nmol of each primer

  • 10 μl 2×PCR Mix

  • 0.5 μl DNA polymerase

  • H₂O to reach 20 μl total volume

• Thermocycling parameters:

  • Initial denaturation: 94°C for 5 minutes

  • 30 cycles of:

    • Denaturation: 94°C for 1 minute

    • Annealing: 57°C for 1.5 minutes

    • Extension: 72°C for 2 minutes

  • Final extension: 72°C for 10 minutes

This protocol typically yields a PCR product of approximately 390 bp, which can be purified by electrophoresis on a 2% agarose gel followed by recovery using a gel purification kit . For recombinant work, researchers may need to design specific primers that incorporate appropriate restriction sites to facilitate subsequent cloning steps, depending on the expression vector system being used.

What recombineering techniques are most effective for creating recombinant ureC constructs in Pseudomonas syringae?

Recombineering (recombination-mediated genetic engineering) offers powerful tools for generating precise genetic modifications in bacterial genomes, including the creation of recombinant ureC constructs in Pseudomonas syringae. Research indicates that the most effective recombineering approaches for P. syringae utilize homologous recombination systems derived from Pseudomonas itself, particularly the RecTE system from P. syringae pv. syringae B728a .

The RecTE system consists of two key components: RecE, an exonuclease that processes double-stranded DNA to generate single-stranded overhangs, and RecT, a single-stranded DNA binding protein that promotes strand invasion and annealing to complementary sequences. Studies have demonstrated that the P. syringae RecT homolog alone is sufficient to promote recombination with single-stranded DNA oligonucleotides, while efficient recombination with double-stranded DNA requires expression of both RecT and RecE homologs .

For practical implementation, expression vectors carrying the RecTE genes have been developed specifically for use in Pseudomonas. These vectors typically include:

• A constitutive promoter (such as the BAD nptII promoter) to ensure stable expression of the recombineering proteins

• The RecT and/or RecTE genes from P. syringae pv. syringae B728a

• Selectable markers for maintaining the vector during recombineering

• Counterselectable markers (such as sacB) to facilitate subsequent removal of the vector

A particularly useful vector system described in the literature is based on pUCP24/47, which includes a Gateway cassette and the sacB gene. This allows for versatile cloning of recombineering genes and subsequent elimination of the vector after recombination has occurred . The efficiency of this recombineering system makes it valuable for creating targeted gene disruptions, insertions, or precise modifications of the ureC gene in P. syringae.

How can researchers verify urease activity in recombinant Pseudomonas syringae strains expressing modified ureC?

Verification of urease activity in recombinant P. syringae strains expressing modified ureC genes requires both qualitative and quantitative approaches. One reliable qualitative method involves using a colorimetric assay based on pH indicators that change color in response to ammonia production from urea hydrolysis .

The procedure for qualitative verification can be performed as follows:

• Prepare agar plates containing a pH indicator such as phenol red and urea

• Coat the plates with a slurry or liquid culture of the recombinant P. syringae strain

• Incubate the plates and observe for color change (typically from yellowish to pink for phenol red, indicating alkalization due to ammonia production)

• Include appropriate positive controls (such as known urease-positive bacteria) and negative controls (such as culture medium alone)

For quantitative assessment of urease activity, spectrophotometric methods can be employed to measure ammonia production rates. Additionally, researchers can determine enzyme kinetic parameters such as Km values. For instance, the Km for urea in Helicobacter pylori urease was estimated at 0.2 mM, providing a reference point for comparison .

Molecular confirmation of the recombinant ureC expression should accompany enzymatic assays. This can be achieved through:

• RT-PCR to verify transcription of the recombinant ureC gene

• Western blotting with antibodies against the urease alpha subunit

• Mass spectrometry to confirm the protein sequence of the expressed recombinant protein

Researchers should be aware that urease activity may be affected by multiple factors including the expression level of other urease subunits, the availability of nickel ions (essential cofactors), and the proper assembly of the multisubunit enzyme complex. Therefore, comprehensive characterization should address these aspects to fully validate the functionality of recombinant ureC constructs.

What regulatory considerations must be addressed when working with recombinant Pseudomonas syringae ureC in a laboratory setting?

Laboratory work involving recombinant P. syringae ureC falls under the regulations governing research with recombinant or synthetic nucleic acid molecules. Researchers must comply with institutional biosafety protocols and national guidelines such as the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules .

Several key regulatory considerations must be addressed:

• Risk assessment and biosafety level determination:

  • P. syringae pv. tomato is primarily a plant pathogen, causing bacterial speck disease in tomato and serving as a model pathogen for Arabidopsis thaliana

  • Work typically requires at least BSL-1 or BSL-2 containment depending on the specific experiments

  • The risk assessment should consider whether genetic modifications might alter pathogenicity or host range

• Institutional Biosafety Committee (IBC) approval:

  • Experiments involving recombinant DNA in P. syringae require registration and approval from the institutional biosafety committee

  • Documentation should include detailed experimental protocols, safety measures, and containment procedures

• Specific types of experiments requiring additional scrutiny:

  • Introduction of antibiotic resistance genes, particularly if they confer resistance to clinically relevant antibiotics

  • Creation of modifications that might alter pathogenicity, virulence, or host range

  • Large-scale culture (>10 liters)

• Plant protection and containment:

  • Since P. syringae is a plant pathogen, additional containment measures may be required for preventing environmental release

  • Work involving whole plants requires appropriate plant growth facility containment

• Disposal protocols:

  • Proper decontamination and disposal procedures for all materials containing recombinant organisms

  • Autoclaving or chemical treatment of cultures before disposal

Researchers should maintain detailed records of all experiments, strain constructions, and safety procedures. Regular monitoring for compliance with institutional and national guidelines is essential throughout the research project.

What expression systems are most suitable for producing recombinant Pseudomonas syringae ureC protein for structural studies?

For structural studies of recombinant P. syringae ureC protein, researchers must select expression systems that ensure proper folding, solubility, and yield of the target protein. Based on the literature and common practices in structural biology, the following expression systems are recommended:

• Escherichia coli expression systems:

  • The pET expression system under the control of T7 promoter offers high-level inducible expression

  • For improved solubility, fusion tags such as MBP (maltose-binding protein), GST (glutathione S-transferase), or SUMO can be employed

  • Expression in specialized E. coli strains such as BL21(DE3) or Rosetta 2(DE3) can enhance proper folding and address codon bias issues

• Pseudomonas-based expression systems:

  • Homologous expression in Pseudomonas provides the native cellular environment

  • Vectors such as pUCP24 derivatives with appropriate promoters (such as the BAD promoter system) have been successfully used for expression in Pseudomonas

  • This approach is particularly valuable if native post-translational modifications are required

• Considerations for urease-specific expression:

  • Urease requires nickel ions for activity, so expression media should be supplemented with NiCl₂

  • Co-expression with urease accessory proteins may be necessary for proper assembly

  • Since the native urease is a multimeric enzyme, co-expression of other subunits might be required for structural studies of the complete complex

For crystallization and structural studies, purification typically involves:

• Affinity chromatography using tags incorporated into the recombinant protein

• Size exclusion chromatography to ensure homogeneity

• Ion exchange chromatography for further purification

Special attention should be paid to maintaining protein stability during purification, as ureases can be sensitive to oxidation. The addition of reducing agents and protease inhibitors throughout the purification process is recommended to preserve protein integrity.

How can researchers assess the impact of ureC mutations on Pseudomonas syringae virulence in plant models?

Assessing the impact of ureC mutations on P. syringae virulence requires a comprehensive approach combining molecular, biochemical, and plant infection studies. The following methodology provides a systematic framework for such investigations:

• Generation of defined ureC mutants:

  • Site-directed mutagenesis targeting specific residues of interest

  • Recombineering techniques using the P. syringae RecTE system for chromosomal integration

  • Creation of complemented strains carrying wild-type ureC for validation

• In vitro characterization:

  • Quantitative urease activity assays to determine the enzymatic consequences of mutations

  • Growth curves in minimal media with urea as the sole nitrogen source

  • pH modulation assays to assess the ability of mutants to alkalinize their environment

• Plant infection assays:

  • Pathogenicity tests on tomato (Lycopersicon esculentum) and Arabidopsis thaliana, the primary host plants for P. syringae pv. tomato

  • Quantification of bacterial growth in planta over time

  • Assessment of disease symptom development (water-soaked lesions, chlorosis, necrosis)

• Microscopic analysis:

  • Confocal microscopy with fluorescently labeled bacteria to track colonization patterns

  • Histochemical staining to visualize plant defense responses (ROS production, callose deposition)

• Molecular analysis of host responses:

  • RT-qPCR to measure expression of plant defense genes

  • RNA-seq for genome-wide transcriptional profiling of host responses

  • Proteomics to identify changes in the plant proteome during infection

• Competition assays:

  • Mixed infections with wild-type and mutant strains to assess relative fitness

  • Calculation of competitive index to quantify virulence attenuation

When interpreting results, researchers should consider the potential pleiotropic effects of ureC mutations, as changes in urease activity might affect multiple aspects of bacterial physiology beyond direct virulence. Additionally, environmental conditions (such as pH, temperature, and plant nutritional status) may influence the significance of urease activity in pathogenesis and should be systematically varied in experimental designs.

What techniques are recommended for studying the interaction between recombinant ureC and other urease subunits in Pseudomonas syringae?

Understanding the interactions between recombinant ureC and other urease subunits in P. syringae requires sophisticated protein-protein interaction techniques. The following approaches are recommended for comprehensive analysis:

• Co-immunoprecipitation (Co-IP):

  • Generate antibodies against the ureC product or use epitope-tagged versions

  • Perform pull-down assays followed by Western blotting or mass spectrometry to identify interacting partners

  • Compare wild-type versus mutant interactions to map critical interaction domains

• Bacterial two-hybrid system:

  • Adapt bacterial two-hybrid systems for use in Pseudomonas or use E. coli-based systems

  • Screen for interactions between ureC and other urease subunits

  • Map interaction domains through truncation and point mutation analysis

• Structural biology approaches:

  • X-ray crystallography of co-expressed and co-purified urease complexes

  • Cryo-electron microscopy for visualization of complex assembly

  • NMR spectroscopy for studying dynamic interactions

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Molecular dynamics simulations:

  • Computational modeling of urease subunit interactions

  • Simulation of conformational changes during complex assembly

  • This approach can be particularly informative when integrated with experimental structural data, as demonstrated in studies of H. pylori urease

• Crosslinking studies:

  • In vivo crosslinking to capture transient protein-protein interactions

  • Chemical crosslinking combined with mass spectrometry to identify interaction sites

  • Validation of crosslinking results through mutational analysis

• Fluorescence-based techniques:

  • Förster resonance energy transfer (FRET) with fluorescently tagged urease subunits

  • Bimolecular fluorescence complementation (BiFC) to visualize interactions in living cells

  • Fluorescence recovery after photobleaching (FRAP) to study the dynamics of complex assembly

The data from these complementary approaches can be integrated to develop a comprehensive model of urease complex assembly and function in P. syringae. Researchers should particularly focus on understanding how the unique features of P. syringae ureC might influence complex formation compared to well-characterized ureases from other species such as H. pylori .

How does the ureC gene of Pseudomonas syringae pv. tomato compare with ureC genes from other bacterial pathogens?

The ureC gene of Pseudomonas syringae pv. tomato shares significant evolutionary conservation with ureC genes from diverse bacterial species, reflecting the fundamental importance of urease in bacterial metabolism. Comparative analyses reveal both conserved features and species-specific adaptations.

At the sequence level, bacterial ureC genes typically contain highly conserved regions that serve as reliable PCR priming sites for identification and amplification . These conserved regions correspond to functionally critical domains involved in catalysis and subunit interactions. The conservation extends across different bacterial phyla, suggesting strong selective pressure to maintain urease function throughout bacterial evolution.

Structural comparisons of urease proteins show remarkable conservation in the catalytic core, particularly in the amino acid residues coordinating the essential nickel ions in the active site. For instance, studies of Helicobacter pylori urease reveal specific coordination patterns for pentacoordinate and hexacoordinate nickel ions that are likely conserved in P. syringae urease as well . The table below summarizes the typical coordination environment for urease nickel centers:

Despite this conservation, there are notable differences in urease organization across bacterial species. While some bacteria possess three separate genes encoding the three structural subunits of urease (ureA, ureB, and ureC), others have gene fusions resulting in fewer subunits with equivalent function. For example, H. pylori urease consists of just two subunits with a stoichiometry of (29.5 kDa-66 kDa)₆, suggesting a fusion of what would be separate subunits in other bacteria . These organizational differences provide insights into the evolutionary history of urease genes through fusion, fission, and duplication events.

Furthermore, antigenic analyses have demonstrated that at least some antigenic determinants are conserved among ureases from different species, as antisera raised against the 66-kDa subunit of H. pylori urease specifically recognize urease subunits from Morganella morganii and jack bean . This immunological cross-reactivity further supports the evolutionary conservation of key structural features.

How has the knowledge of ureC gene function in Pseudomonas syringae contributed to our understanding of bacterial pathogenesis mechanisms?

The study of ureC gene function in Pseudomonas syringae has made significant contributions to our broader understanding of bacterial pathogenesis mechanisms, particularly in plant-pathogen interactions. This research has illuminated several important aspects of bacterial physiology and virulence that extend beyond the specific role of urease itself.

First, investigations of ureC have highlighted the complex interplay between basic metabolism and virulence. While much attention in P. syringae pathogenesis has focused on specialized virulence factors like type III secretion systems and effector proteins , urease represents a metabolic enzyme that potentially bridges primary metabolism and pathogenic capability. This reinforces the concept that bacterial virulence often depends on metabolic adaptations that enable survival in host environments rather than solely on dedicated virulence factors.

Second, research on urease has contributed to our understanding of the genomic organization and regulation of virulence-associated genes in bacterial pathogens. Though ureC in P. syringae is not typically regulated by the HrpL alternative sigma factor that controls many virulence genes , studies of its regulation provide insights into how bacteria coordinate the expression of metabolic functions with pathogenesis-related activities under different environmental conditions.

Third, the conservation of urease genes across diverse bacterial pathogens has facilitated comparative genomic analyses that reveal evolutionary relationships and potential horizontal gene transfer events. These comparative approaches have strengthened our understanding of how pathogenic capabilities evolve and spread among bacteria, potentially contributing to the emergence of new plant pathogens.

Fourth, the development of recombineering techniques for genetic manipulation of P. syringae, including modification of genes like ureC, has established important methodological frameworks that benefit research on many bacterial pathogens . These technical advances enable more precise investigations of gene function and have accelerated progress throughout the field of bacterial pathogenesis.

Finally, the molecular mechanisms underlying urease function—including protein complex assembly, metal cofactor incorporation, and catalytic activity—provide insights into fundamental aspects of bacterial protein biochemistry. These insights are valuable not only for understanding urease itself but also for elucidating the function of other multiprotein complexes involved in bacterial virulence.

What emerging technologies could enhance our understanding of ureC function in Pseudomonas syringae?

Several cutting-edge technologies show promise for advancing our understanding of ureC function in Pseudomonas syringae. These innovative approaches could address existing knowledge gaps and provide unprecedented insights into urease biology in this important plant pathogen.

CRISPR-Cas genome editing technology offers transformative potential for precise manipulation of the ureC gene in P. syringae. Unlike traditional recombineering approaches , CRISPR-Cas systems allow for scarless gene editing, facilitating the creation of single nucleotide mutations, domain swaps, or regulatory element modifications with minimal disruption to genomic context. This precision could enable detailed structure-function analyses of ureC without the confounding effects of marker genes or remaining scars from homologous recombination.

Single-cell techniques represent another frontier in ureC research. Single-cell RNA sequencing could reveal cell-to-cell variability in ureC expression during plant infection, potentially uncovering subpopulations with distinct metabolic states or virulence profiles. Similarly, single-cell protein analysis using mass cytometry or similar approaches could track urease protein levels in individual bacteria during different stages of plant colonization, providing insights into the heterogeneity of bacterial populations during pathogenesis.

Advanced imaging technologies also hold great promise. Super-resolution microscopy could visualize the subcellular localization of urease complexes within P. syringae cells, potentially revealing unexpected associations with other cellular structures. Correlative light and electron microscopy (CLEM) could connect urease localization with ultrastructural features of the bacteria and surrounding plant tissues during infection.

Finally, structural biology techniques like cryo-electron microscopy could provide high-resolution structures of the complete P. syringae urease complex, including accessory proteins involved in nickel incorporation and complex assembly. These structural insights would complement existing knowledge from other bacterial ureases and could guide rational design of inhibitors or other tools for functional studies.

What are the potential applications of recombinant Pseudomonas syringae ureC in agricultural biotechnology?

Recombinant Pseudomonas syringae ureC holds significant potential for innovative applications in agricultural biotechnology, presenting opportunities to address current challenges in crop protection, soil health, and sustainable farming practices.

Engineered biocontrol agents represent one promising application area. By modifying ureC expression or activity in non-pathogenic Pseudomonas strains, researchers could develop biocontrol agents with enhanced competitive ability against pathogenic P. syringae pv. tomato strains. These biocontrol agents could effectively colonize plant surfaces and exclude pathogens without causing disease themselves. The manipulation of urease activity could provide these beneficial bacteria with metabolic advantages in the plant environment, improving their persistence and protective effects.

Soil health management offers another potential application. Engineered P. syringae strains with modified urease activity could be developed to optimize nitrogen cycling in agricultural soils. By controlling the rate of urea hydrolysis, these strains could reduce nitrogen loss through ammonia volatilization and improve nitrogen use efficiency in cropping systems. This application would be particularly valuable in sustainable agriculture systems seeking to reduce synthetic fertilizer inputs while maintaining productivity.

Plant immunity priming represents an innovative frontier. Recent research suggests that bacterial metabolites can trigger plant immune responses that enhance resistance to subsequent pathogen attack. Recombinant P. syringae strains producing modified urease-derived peptides or controlled amounts of urease could potentially serve as immunity-priming agents, preparing plants for improved defense against multiple pathogens without causing disease symptoms themselves.

Bioremediation applications are also promising. Urease activity influences soil pH and nitrogen cycling, which affect the mobility and bioavailability of various contaminants in soil. Engineered P. syringae with enhanced or modified urease activity could potentially assist in bioremediation of agricultural soils contaminated with certain pesticides or heavy metals by altering local chemical conditions to favor degradation or immobilization of these pollutants.

Finally, diagnostic tools based on recombinant ureC could be developed for rapid detection of P. syringae pv. tomato in field settings. Antibodies raised against unique epitopes of the P. syringae urease alpha subunit could be incorporated into immunochromatographic tests for pathogen detection, enabling farmers to implement timely disease management strategies before significant crop damage occurs.

How might computational approaches advance our ability to predict the functional impacts of ureC mutations in Pseudomonas syringae?

Computational approaches offer powerful tools for predicting the functional consequences of ureC mutations in Pseudomonas syringae, potentially accelerating research by focusing experimental efforts on the most promising targets. A multi-layered computational strategy could significantly enhance our predictive capabilities in this area.

Molecular dynamics (MD) simulations represent a foundational approach for studying the structural and dynamic properties of the urease enzyme. Building on methodologies applied to Helicobacter pylori urease , researchers can develop detailed atomic-level models of P. syringae urease and simulate the effects of specific mutations on protein structure, flexibility, and substrate binding. These simulations can reveal how mutations might affect the coordination of essential nickel ions in the active site or disrupt critical interactions between protein subunits. Modern MD approaches incorporating enhanced sampling techniques can access longer timescales relevant to enzyme catalysis, providing insights into the dynamic aspects of urease function.

Quantum mechanics/molecular mechanics (QM/MM) methods offer even greater precision for modeling the chemical reactions catalyzed by urease. By treating the active site with quantum mechanical methods while representing the rest of the protein with classical molecular mechanics, researchers can calculate activation energies for urea hydrolysis and predict how specific mutations might alter reaction barriers and catalytic efficiency. These calculations could identify key residues whose mutation would have the greatest impact on enzymatic activity.

Machine learning approaches trained on existing mutagenesis data could develop predictive models for the effects of novel mutations. By integrating features such as sequence conservation, structural context, and physicochemical properties, these models could rapidly screen thousands of potential mutations to identify those most likely to yield specific phenotypic outcomes. As experimental data accumulates, these models would become increasingly accurate and valuable for guiding targeted mutagenesis studies.

Network analysis of protein-protein interactions could predict how ureC mutations might propagate effects throughout the bacterial proteome. By modeling the urease interaction network, researchers could identify mutations that disrupt not only enzymatic activity but also key protein-protein interactions involved in complex assembly or regulation. This systems-level perspective is essential for understanding the full biological consequences of ureC mutations beyond direct effects on catalysis.

Evolutionary analysis through approaches such as ancestral sequence reconstruction and coevolutionary analysis can identify functionally coupled residues within ureC. Mutations affecting these co-evolving networks of amino acids are particularly likely to have significant functional impacts. By combining evolutionary information with structural and biochemical data, researchers can develop more robust predictions about which mutations will affect urease function in P. syringae.