Recombinant Photorhabdus luminescens subsp. laumondii Urease subunit alpha (ureC), partial

What is Photorhabdus luminescens and why is its urease subunit alpha of research interest?

Photorhabdus luminescens is a Gammaproteobacterium belonging to the family Morganellaceae that functions as a lethal insect pathogen. It naturally exists in a symbiotic relationship with entomopathogenic nematodes of the Heterorhabditidae family . The urease enzyme in P. luminescens, like in other bacteria, is a nickel-containing enzyme that catalyzes the hydrolysis of urea to ammonia and carbon dioxide. The ureC gene encodes the alpha subunit of urease, which forms part of the catalytic core of the enzyme. Research interest in this protein stems from its potential role in the bacteria's pathogenicity, symbiotic relationships, and unique biochemical properties that differ from better-studied urease systems like those in Helicobacter pylori.

How does the urease gene organization in P. luminescens compare to other bacterial species?

The urease gene cluster in P. luminescens shows phylogenetic similarities to urease genes in Brucella species, Yersinia species, and other Photorhabdus luminescens subspecies . Similar to these bacteria, the P. luminescens urease is likely composed of multiple subunits. In most bacterial species, urease comprises three subunits (α, β, and γ) encoded by ureA, ureB, and ureC genes, respectively . Accessory proteins encoded by ureD, ureE, ureF, ureG, and sometimes ureI are also commonly present and assist in the assembly of the active enzyme by incorporating nickel ions into the active site. P. luminescens maintains this general structure, though specific regulatory mechanisms may differ between bacterial species.

What are the recommended vectors and host systems for recombinant expression of P. luminescens ureC?

For optimal expression of P. luminescens ureC, the pRSET vector system (particularly pRSET version "a") has shown good results with E. coli BL21(DE3) pLysS as the expression host . This combination provides tight control of expression through the T7 promoter system. Alternative approaches include:

When designing your cloning strategy, it's important to note that the position of affinity tags can affect protein functionality. For example, in studies with UreA from H. pylori, C-terminal tagging preserved function better than N-terminal tagging , which might also apply to P. luminescens ureC.

What are the optimal conditions for expressing soluble recombinant P. luminescens ureC protein?

Based on analogous studies with urease subunits, the following conditions typically yield optimal soluble expression:

• Growth temperature: Initial growth at 37°C until OD600 reaches 0.5, followed by induction and expression at lower temperatures (20-25°C) for 12-18 hours .

• Induction: IPTG concentration of 0.25-1.0 mM, with lower concentrations favoring solubility .

• Growth media: Enriched media like LB supplemented with appropriate antibiotics based on the expression vector.

• Cell lysis: Sonication in Tris-HCl buffer (50 mM, pH 8.0) containing protease inhibitors .

• Solubility enhancement: Addition of 0.1% Triton X-100 to lysis buffer may improve solubilization .

For particularly challenging expressions, co-expression with molecular chaperones or expression as a fusion protein with solubility enhancers like GST or MBP may be beneficial.

What purification protocols are most effective for recombinant P. luminescens ureC?

For His-tagged recombinant ureC, immobilized metal affinity chromatography (IMAC) using nickel nitrilotriacetic acid (Ni-NTA) resin is the method of choice . A detailed purification workflow:

• Bind the clarified cell lysate to Ni-NTA resin pre-equilibrated with binding buffer (typically 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole).

• Wash extensively with binding buffer containing 20-30 mM imidazole to remove non-specifically bound proteins.

• Elute the target protein with elution buffer containing 250-300 mM imidazole.

• For higher purity, implement secondary purification steps such as ion exchange chromatography or size exclusion chromatography.

For GST-tagged constructs, glutathione Sepharose 4B beads provide an effective purification matrix . The purified protein should be analyzed by SDS-PAGE and Western blotting to confirm identity and assess purity.

How can I verify the structural integrity and functional activity of purified recombinant ureC?

Multiple complementary methods should be employed:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to analyze secondary structure elements

  • Size exclusion chromatography to verify oligomeric state

  • Mass spectrometry for accurate mass determination and potential post-translational modifications

  • Limited proteolysis to assess proper folding

• Functional characterization:

  • For assembled urease complexes containing ureC, enzyme activity can be measured using colorimetric assays based on detecting ammonia production

  • Native PAGE with activity staining can detect urease activity directly in gels

  • Surface Plasmon Resonance (SPR) to assess binding to known interaction partners like Hsp60 (if such interactions are conserved from other bacterial species)

Note that the isolated ureC subunit alone may not display catalytic activity without the other urease subunits and assembly factors.

How can I design experiments to study the role of P. luminescens ureC in pathogenicity and symbiosis?

Several experimental approaches can be employed:

• Gene knockout studies:

  • Create a markerless deletion of the ureC gene using allelic exchange mutagenesis

  • Alternatively, use insertional inactivation if markerless deletion proves challenging

  • Complement the mutation with a wild-type copy to confirm phenotype specificity

• In vitro assays:

  • Assess growth rates in nitrogen-limited media with urea as the sole nitrogen source

  • Evaluate pH resistance in acidic environments

  • Measure urease activity using qualitative (urease test broth) and quantitative enzymatic assays

• In vivo studies:

  • Examine colonization of insect hosts by wild-type versus ureC mutant strains

  • Investigate nematode fitness when carrying wild-type versus ureC mutant bacteria

  • Study survival and persistence in environmental conditions

A comprehensive experimental design should include appropriate controls and a comparative approach with other urease systems when possible.

What methods are recommended for studying protein-protein interactions involving P. luminescens ureC?

Based on successful studies with other bacterial urease subunits, these approaches are recommended:

• In vitro protein interaction studies:

  • GST pull-down assays using purified recombinant proteins

  • Surface Plasmon Resonance (SPR) for quantitative binding kinetics

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters

  • Co-immunoprecipitation with specific antibodies

• Structural studies:

  • X-ray crystallography of protein complexes

  • Cryo-electron microscopy for larger assemblies

  • Molecular docking combined with site-directed mutagenesis to validate interaction interfaces

• In vivo interaction validation:

  • Bacterial two-hybrid assays

  • Fluorescence resonance energy transfer (FRET)

  • Cross-linking coupled with mass spectrometry

When designing interaction studies, it's important to consider tag positioning, as demonstrated in UreA studies where GST-UreA (N-terminal tag) failed to pull down Hsp60 while UreA-GST (C-terminal tag) successfully did so .

How can molecular docking and computational approaches be applied to study P. luminescens ureC structure and function?

Computational approaches provide valuable insights when structural data is limited:

• Homology modeling:

  • Generate a structural model of P. luminescens ureC using known structures of urease subunits from related species

  • Validate the model using energy minimization and Ramachandran plot analysis

• Molecular docking:

  • Predict binding interfaces with other urease subunits and accessory proteins

  • Identify key residues involved in protein-protein interactions

  • Screen for potential inhibitors or modulators of urease activity

• Molecular dynamics simulations:

  • Investigate conformational changes under different conditions

  • Study the stability of protein-protein interfaces

  • Analyze the impact of mutations on protein structure and dynamics

In a study of UreA-Hsp60 interactions, molecular docking successfully identified key interfacial residues that were subsequently validated through site-directed mutagenesis and binding studies . Similar approaches could be applied to P. luminescens ureC.

What are the considerations when using P. luminescens ureC as an immunogen or vaccine component?

Based on research with other bacterial urease components:

• Antigen design:

  • Express full-length recombinant ureC or identify immunodominant epitopes

  • Consider fusions with carrier proteins or adjuvants to enhance immunogenicity

  • Evaluate different delivery formats (soluble protein, nanocapsules, DNA vaccines)

• Production considerations:

  • Ensure high purity to avoid non-specific immune responses

  • Perform endotoxin removal to prevent reactogenicity

  • Verify correct folding to maintain conformational epitopes

• Evaluation methods:

  • Assess antibody production by ELISA and Western blotting

  • Determine protective efficacy in appropriate animal models

  • Analyze cellular immune responses through T-cell proliferation assays

Research with UreA nanocapsules has demonstrated that particle size and formulation significantly impact vaccine efficacy, with larger nanocapsules (approximately 510 nm) showing better protective efficacy than smaller ones (47 nm) when combined with adjuvants .

What strategies can address low expression or insolubility issues with recombinant P. luminescens ureC?

Several approaches can overcome expression and solubility challenges:

• Optimization of expression conditions:

  • Test multiple E. coli strains (BL21, Rosetta, Arctic Express)

  • Reduce induction temperature (16-20°C)

  • Decrease IPTG concentration (0.1-0.25 mM)

  • Use enriched media formulations (TB, 2xYT)

• Protein engineering approaches:

  • Try both N- and C-terminal fusion tags (His, GST, MBP, SUMO)

  • Remove flexible regions predicted to contribute to aggregation

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

• Alternative solubilization methods:

  • Add solubility enhancers to lysis buffer (Triton X-100, low concentrations of urea)

  • Consider mild denaturing conditions followed by on-column refolding

  • Explore detergent screens if the protein has hydrophobic regions

If expression in E. coli remains problematic, consider alternative expression hosts such as P. luminescens itself using the Pluγβα recombineering system, which allows for genome engineering in Photorhabdus and Xenorhabdus bacteria .

How can I distinguish between true protein-protein interactions and non-specific binding when studying ureC interactions?

Distinguishing specific from non-specific interactions requires multiple controls and validation methods:

• Essential controls:

  • Include GST-only (or appropriate tag-only) controls in pull-down experiments

  • Use unrelated proteins of similar size and properties as negative controls

  • Perform competition assays with unlabeled proteins

• Validation through mutagenesis:

  • Identify potential interface residues through computational prediction

  • Generate site-directed mutants of these residues

  • Quantitatively measure how mutations affect binding kinetics

• Analytical approaches:

  • Use SPR to determine binding kinetics (kon, koff) and affinity (KD)

  • Employ microscale thermophoresis for interaction studies in solution

  • Consider native mass spectrometry to verify complex formation

In the UreA-Hsp60 interaction study, the investigators confirmed specificity by showing that UreB-GST did not pull down Hsp60, while UreA-GST did. Additionally, they validated the interaction interface by creating alanine substitutions of predicted interface residues and measuring their effects on binding .

How does P. luminescens urease structure and function compare to well-studied bacterial ureases?

This comparative analysis helps contextualize research findings:

Notable differences include:

• P. luminescens urease genes show phylogenetic relationship to those in Brucella species, Yersinia species, and other Photorhabdus subspecies

• Unlike Brucella suis, which has two urease operons with the ure1 operon being functionally dominant , the organization in P. luminescens may differ

• While H. pylori urease is essential for gastric colonization , the specific role of urease in P. luminescens symbiosis and insect pathogenicity remains to be fully characterized

What can we learn from urease studies in other bacteria that might apply to P. luminescens ureC research?

Key transferable insights include:

• Structural insights:

  • The UreA-Hsp60 interaction in H. pylori reveals specific binding interfaces that could be conserved in other bacteria

  • The relative positioning of affinity tags significantly impacts protein-protein interactions, as seen with GST-UreA vs. UreA-GST

• Functional roles:

  • In Brucella suis, urease contributes to acid resistance and in vivo persistence

  • In H. pylori, urease-generated ammonia increases gastric pH, creating an environment permissive for colonization

• Methodological approaches:

  • The combined use of molecular docking and site-directed mutagenesis to map protein interaction interfaces

  • Native gel electrophoresis for detection of urease activity

  • Qualitative (urease test broth) and quantitative measurements of urease activity

These insights provide a foundation for hypothesis generation and experimental design in P. luminescens ureC research.

How might global regulators affect P. luminescens ureC expression and function?

Understanding regulatory mechanisms provides insights into urease function in different conditions:

• Global regulators:

  • The LysR-type transcriptional repressor HexA regulates secondary metabolite production in P. luminescens, and its knockout (ΔhexA) leads to upregulation of small molecules

  • Other global regulators like Lrp (leucine-responsive protein) might affect metabolic switching and potentially urease expression

• Environmental sensing:

  • L-proline transport and metabolism may contribute to derepression of HexA and subsequent gene regulation

  • pH, nitrogen availability, and oxygen levels might regulate urease expression

• Experimental approaches:

  • Transcriptional profiling under different conditions

  • Reporter gene fusions to study promoter activity

  • Chromatin immunoprecipitation to identify direct regulatory interactions

Research with P. luminescens has shown that proline transporter mutants (ΔproU and ΔputP) exhibit differential effects on secondary metabolite production , suggesting complex regulatory networks that might also affect urease expression.

What novel applications of recombinant P. luminescens ureC are being explored in current research?

Emerging research areas include:

• Biotechnological applications:

  • Development of biosensors for environmental monitoring

  • Use as a model system for studying protein-protein interactions

  • Applications in protein nanocapsule technology for targeted delivery

• Therapeutic potential:

  • Exploration as vaccine components against related pathogens

  • Investigation of immunomodulatory properties

  • Identification of novel antimicrobial targets

• Symbiosis studies:

  • Role in establishing and maintaining symbiotic relationships with nematodes

  • Contribution to insect pathogenicity and bioconversion of insect cadavers

  • Interactions with other microbial components in complex ecological systems

The unique properties of P. luminescens as both a symbiont and pathogen make its molecular components, including urease, valuable subjects for diverse research applications.