Recombinant Pseudomonas syringae pv. tomato Nicotinate-nucleotide--dimethylbenzimidazole phosphoribosyltransferase (cobT)

What is Nicotinate-nucleotide--dimethylbenzimidazole phosphoribosyltransferase (CobT) in Pseudomonas syringae pv. tomato?

CobT in Pseudomonas syringae pv. tomato is a phosphoribosyltransferase enzyme that plays a crucial role in the biosynthesis of cobalamin (vitamin B12) by catalyzing the activation of benzimidazole-type lower ligands. Similar to CobT in other bacterial species, this enzyme catalyzes the transfer of a phosphoribosyl group from nicotinate mononucleotide to form phosphoribosylated benzimidazole derivatives. CobT is structurally homologous to the well-characterized CobT from Salmonella typhimurium, which contributes to the synthesis of alpha-ribazole, a key component of the lower ligand of cobalamin . The enzyme demonstrates regiospecificity in the formation of isomers arising from the attachment of the C1' of ribose with nitrogen atoms of asymmetric benzimidazolyl lower ligands, producing compounds essential for cobamide structure and function .

How does CobT function in cobamide biosynthesis pathways?

CobT functions as a phosphoribosyltransferase in the cobamide biosynthesis pathway, catalyzing the regiospecific activation of benzimidazole-type compounds. In related bacterial systems such as Mycobacterium thermoacetica, CobT regiospecifically activates 5-hydroxybenzimidazole (5-OHBza) to form the 5-OHBza-ribotide (5-OHBza-RP) isomer as the sole product . This reaction represents a critical step in the biosynthesis of the lower ligand of cobamides. The enzyme demonstrates substrate specificity, showing varying regioselectivity in the formation of isomers arising from the attachment of the C1' of ribose with different nitrogen atoms of asymmetric benzimidazolyl lower ligands . This regiospecificity is essential for determining the final structure and function of cobamides in bacterial metabolism.

What structural features characterize the CobT enzyme?

The structure of CobT has been extensively studied in Salmonella typhimurium, revealing key insights applicable to the Pseudomonas syringae homolog. X-ray crystallography studies at 1.9 Å resolution have captured CobT in complex with both its substrate (5,6-dimethylbenzimidazole) and its reaction products (nicotinate and alpha-ribazole-5'-phosphate) . The enzyme typically crystallizes in the space group P2(1)2(1)2 with unit cell dimensions of a = 72.1 Å, b = 90.2 Å, and c = 47.5 Å, with one protomer per asymmetric unit . Homology modeling approaches similar to those used for human nicotinate phosphoribosyltransferase can be applied to predict the three-dimensional structure of P. syringae CobT, identifying conserved residues involved in substrate recognition and catalysis . These structural insights are valuable for understanding the enzyme's mechanism and designing experiments to probe its function.

How can I express and purify recombinant P. syringae CobT for enzymatic studies?

To express and purify recombinant P. syringae CobT, a molecular cloning approach involving PCR amplification of the cobT gene from P. syringae pv. tomato genomic DNA is recommended. The amplified gene can be cloned into an expression vector (such as pET series) containing an N- or C-terminal His-tag for affinity purification. For optimal expression conditions, transform the construct into E. coli BL21(DE3) or similar expression strains.

Expression protocol:

• Grow transformed E. coli cells in LB medium with appropriate antibiotics at 37°C until OD600 reaches 0.6-0.8

• Induce protein expression with 0.5-1.0 mM IPTG

• Continue cultivation at 18-25°C for 16-18 hours to enhance soluble protein production

• Harvest cells by centrifugation at 4,000 × g for 20 minutes at 4°C

Purification procedure:

• Resuspend cell pellet in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT)

• Lyse cells by sonication or French press

• Clarify lysate by centrifugation at 20,000 × g for 30 minutes at 4°C

• Apply supernatant to Ni-NTA column equilibrated with lysis buffer

• Wash with buffer containing 20-30 mM imidazole

• Elute with buffer containing 250-300 mM imidazole

• Perform size exclusion chromatography for higher purity

This approach has been successfully applied to similar phosphoribosyltransferases, yielding proteins of sizes approximately 39-40 kDa, as observed with Mycobacterium thermoacetica CobT (39.7 kDa) .

What are the kinetic parameters of P. syringae CobT and how do they compare to homologs?

While specific kinetic parameters for P. syringae pv. tomato CobT have not been directly reported in the search results, comparative analysis with homologous enzymes provides valuable insights. Research on human nicotinate phosphoribosyltransferase (NaPRT), which catalyzes a similar reaction, reveals a comprehensive kinetic profile that can serve as a reference .

Table 1: Comparative Kinetic Parameters of Phosphoribosyltransferases

The enzyme likely exhibits negative cooperativity with ATP, showing stimulation at low substrate concentrations and inhibition at high substrate concentrations . For accurate determination of P. syringae CobT kinetic parameters, researchers should employ spectrophotometric assays monitoring the formation of the ribosylated product at 300-320 nm or HPLC-based methods to detect product formation with potential LC-MS confirmation of product identity .

How does P. syringae CobT contribute to bacterial pathogenicity and virulence?

P. syringae pv. tomato CobT likely contributes to bacterial pathogenicity through ensuring cobalamin biosynthesis, which supports bacterial metabolism during infection. While direct evidence for CobT's role in P. syringae pathogenicity is limited in the search results, we can draw parallels with other bacterial systems and metabolic pathways important during infection.

P. syringae pv. tomato infection of tomato plants involves complex chemotactic responses to plant-derived compounds, enabling bacteria to locate plant openings and access the apoplast . The perception of GABA and L-Pro through the PsPto-PscC chemoreceptor drives bacterial entry into the tomato apoplast and is crucial for virulence . While CobT is not directly implicated in this chemotactic response, its contribution to cobalamin biosynthesis likely supports the metabolic requirements for bacterial growth during infection.

In Mycobacterium avium subsp. paratuberculosis, CobT has been shown to function as a DC maturation-inducing antigen that drives Th1 polarized-naive/memory T cell expansion in a TLR4-dependent cascade . This immunomodulatory role positions CobT as a potential virulence factor that shapes host immune responses. Investigation of similar immunomodulatory properties in P. syringae CobT could reveal novel aspects of plant-pathogen interactions.

What techniques are most effective for analyzing CobT regiospecificity and substrate selectivity?

The regiospecific activity of CobT enzymes is critical for determining the structure and function of cobamides. Based on research with Mycobacterium thermoacetica CobT, several techniques have proven effective for analyzing regiospecificity and substrate selectivity :

HPLC-based analysis:

• React purified CobT with various benzimidazole substrates (e.g., 5-OHBza, 5-OMeBza) and nicotinate mononucleotide

• Analyze reaction products using reversed-phase HPLC with a C18 column

• Monitor elution profiles at multiple wavelengths (260-280 nm for nucleotides, 300-320 nm for benzimidazole compounds)

• Compare retention times and peak profiles to identify specific isomers

LC-MS confirmation:

• Collect HPLC fractions or directly couple HPLC to mass spectrometry

• Analyze product masses to confirm identity (e.g., fragment of 135 m/z corresponding to 5-OHBza)

• Compare fragmentation patterns between isomers to distinguish regiospecific products

Comparative enzymatic analysis:

• Express and purify CobT homologs known to produce different isomers (e.g., E. coli CobT produces both 5-OHBza-RP and 6-OHBza-RP isomers)

• Compare reaction products from P. syringae CobT with those from the reference enzymes

• Analyze elution profiles and spectral characteristics to determine regiospecificity

This approach revealed that Mycobacterium thermoacetica CobT exclusively produces the 5-OHBza-RP isomer, while E. coli CobT produces both 5-OHBza-RP and 6-OHBza-RP isomers . Similar analysis of P. syringae CobT would elucidate its regiospecificity and substrate preferences.

How can recombineering approaches be used to study CobT function in P. syringae?

Recombineering offers powerful approaches for targeted genetic modification of P. syringae to study CobT function. The identification of RecTE homologs from P. syringae pv. syringae B728a provides a foundation for efficient site-directed mutagenesis of chromosomal loci in P. syringae .

Practical recombineering workflow for CobT functional studies:

• Express recombineering proteins:

  • Transform P. syringae pv. tomato DC3000 with a plasmid expressing RecT (sufficient for single-stranded DNA recombination) or both RecE and RecT (required for double-stranded DNA recombination)

  • Induce expression of recombineering proteins before introducing targeting DNA

• Design targeting constructs:

  • For point mutations: Design single-stranded DNA oligonucleotides (60-90 nt) with the desired mutation flanked by 25-45 nt homology arms

  • For gene deletion/insertion: Prepare double-stranded DNA with antibiotic resistance marker flanked by 50-500 bp homology regions

• Introduce DNA by electroporation:

  • Grow cells expressing recombineering proteins to mid-log phase

  • Prepare electrocompetent cells and electroporate with targeting DNA

  • Allow cells to recover and select for recombinants

• Verify modifications:

  • Screen colonies by PCR, restriction digestion, or sequencing

  • Confirm phenotypic effects by assessing CobT function

This approach enables precise engineering of the P. syringae genome, allowing for the introduction of point mutations in catalytic residues, domain swapping between CobT homologs, or complete gene deletion to assess the physiological importance of CobT .

What site-directed mutagenesis approaches can identify critical residues in P. syringae CobT?

Identification of critical residues in P. syringae CobT can be achieved through rational site-directed mutagenesis approaches informed by structural modeling and sequence conservation analysis. The strategy employed for human nicotinate phosphoribosyltransferase provides an excellent template :

• Homology modeling:

  • Generate a three-dimensional model of P. syringae CobT using crystallographic data from homologous proteins (e.g., Salmonella CobT)

  • Identify potential substrate binding sites and catalytic residues through structural analysis

• Sequence conservation analysis:

  • Perform multiple sequence alignment of CobT homologs across bacterial species

  • Identify universally conserved residues likely to be functionally important

  • Focus on residues in predicted substrate-binding pockets and catalytic sites

• Molecular docking simulations:

  • Conduct docking simulations with substrates (nicotinate mononucleotide and benzimidazole derivatives)

  • Identify residues involved in substrate recognition and binding

• Targeted mutagenesis strategy:

  • Design mutants with substitutions at conserved residues (typically Ala substitutions)

  • Create additional mutants with conservative substitutions to probe specific interactions

  • Include mutations altering regiospecificity based on comparison with homologs having different specificity

Table 2: Potential Critical Residues for P. syringae CobT Mutagenesis

This systematic approach will generate a functional map of critical residues influencing substrate recognition, catalysis, and regiospecificity, providing mechanistic insights into P. syringae CobT function .

What crystallization conditions are optimal for structural studies of P. syringae CobT?

Structural studies of P. syringae CobT require careful optimization of crystallization conditions. Based on successful crystallization of Salmonella typhimurium CobT and other phosphoribosyltransferases, the following approach is recommended:

Protein preparation:

• Purify recombinant P. syringae CobT to >95% homogeneity using affinity chromatography followed by size exclusion chromatography

• Concentrate protein to 10-15 mg/mL in a buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT

• Filter through a 0.22 μm membrane before crystallization trials

Initial screening:

• Perform high-throughput screening using commercial crystallization kits (e.g., Hampton Research, Molecular Dimensions)

• Set up sitting-drop vapor diffusion experiments at 18°C and 4°C

• Screen with and without substrates or substrate analogs

Optimization strategies:

• Based on initial hits, optimize promising conditions by varying:

  • pH (± 1.0 pH unit around initial hit)

  • Precipitant concentration (± 10% around initial hit)

  • Protein concentration (5-20 mg/mL)

  • Additive screening with potential ligands

Co-crystallization approach:

• For substrate complexes, incubate protein with 5,6-dimethylbenzimidazole (5-10 mM) before setting up crystallization trials

• For product complexes, soak crystals containing benzimidazole substrate with nicotinate mononucleotide to allow the reaction to occur in the crystal lattice

Salmonella CobT successfully crystallized in space group P2(1)2(1)2 with unit cell dimensions of a = 72.1 Å, b = 90.2 Å, and c = 47.5 Å, with one protomer per asymmetric unit . Similar conditions may serve as a starting point for P. syringae CobT crystallization.

How can I design activity-based probes to monitor CobT activity in complex biological samples?

Activity-based probes for monitoring CobT activity in complex biological samples can be designed based on the enzyme's catalytic mechanism and substrate specificity. The following approaches are recommended:

Modified substrate approach:

• Synthesize fluorescently labeled benzimidazole derivatives that maintain CobT substrate properties

• Design probes with fluorescence quenching that is relieved upon phosphoribosylation

• Validate probe specificity using purified recombinant CobT enzyme

Click chemistry strategy:

• Synthesize benzimidazole derivatives with alkyne or azide handles

• Allow CobT-catalyzed phosphoribosylation in biological samples

• Perform bioorthogonal click chemistry to attach fluorophores or affinity tags

• Analyze products by fluorescence imaging or pull-down followed by mass spectrometry

Competitive activity-based protein profiling:

• Design mechanism-based inhibitors that covalently modify the CobT active site

• Incorporate reporter tags or clickable groups for visualization

• Use these probes to assess CobT activity in plant samples during P. syringae infection

Analytical detection methods:

• HPLC-based assays monitoring the specific formation of ribosylated products

• LC-MS analysis to detect specific mass transitions corresponding to CobT products

• In-gel fluorescence for visualization of labeled CobT in complex mixtures

These approaches enable monitoring of CobT activity during P. syringae infection of plant tissues, providing insights into the temporal and spatial regulation of cobamide biosynthesis during pathogenesis.

How might inhibiting CobT function affect P. syringae virulence in planta?

Inhibiting CobT function could significantly impact P. syringae virulence through disruption of cobamide biosynthesis. The following experimental approaches can test this hypothesis:

• Generate conditional CobT mutants:

  • Create P. syringae strains with cobT under the control of an inducible promoter

  • Compare virulence with and without CobT expression during tomato infection

• Chemical inhibition studies:

  • Design competitive inhibitors based on structural studies of CobT

  • Test inhibitor effects on bacterial growth in minimal media requiring cobamide-dependent enzymes

  • Assess inhibitor impact on bacterial virulence in planta

• Metabolic bypass experiments:

  • Supplement infection sites with cobamide precursors that bypass CobT function

  • Determine if supplementation restores virulence in CobT-deficient strains

• Combined approaches:

  • Target multiple steps in cobamide biosynthesis simultaneously

  • Assess synergistic effects on bacterial fitness and virulence

Expected outcomes include reduced bacterial proliferation in planta, attenuated disease symptoms, and potential alterations in plant defense responses. The requirement for CobT likely varies depending on infection stage and environmental conditions, with potentially greater importance during early colonization when nutrient acquisition is critical.

What emerging technologies could advance our understanding of CobT function in P. syringae?

Several emerging technologies offer promising avenues for advancing our understanding of CobT function in P. syringae:

CRISPR-Cas9 genome editing:

• Develop efficient CRISPR-Cas9 systems for P. syringae based on recombineering principles

• Create precise mutations in cobT to study structure-function relationships

• Generate conditional knockdowns using CRISPRi approaches

Single-cell techniques:

• Apply single-cell RNA-seq to study cobT expression heterogeneity during infection

• Develop fluorescent reporters for monitoring cobT expression at the single-cell level

• Track spatial and temporal patterns of CobT activity during infection

Advanced imaging approaches:

• Use correlative light and electron microscopy (CLEM) to visualize CobT localization

• Apply expansion microscopy for improved resolution of bacterial structures

• Implement live-cell imaging to track cobamide biosynthesis dynamics

Systems biology integration:

• Construct metabolic models incorporating cobamide biosynthesis pathways

• Apply flux balance analysis to predict the impact of CobT inhibition

• Integrate transcriptomic, proteomic, and metabolomic data to build comprehensive models of P. syringae infection

Synthetic biology applications:

• Engineer CobT variants with altered substrate specificity

• Design biosensors based on CobT activity for detecting P. syringae infection

• Create synthetic cobamide biosynthesis pathways for studying metabolic requirements

These emerging technologies will enable unprecedented insights into the role of CobT in P. syringae metabolism and pathogenesis, potentially revealing new strategies for plant disease control.