Recombinant Pseudomonas syringae pv. tomato Probable nicotinate-nucleotide adenylyltransferase (nadD)

What is the function of nicotinate-nucleotide adenylyltransferase (nadD) in Pseudomonas syringae pv. tomato?

Nicotinate-nucleotide adenylyltransferase (nadD) is an essential enzyme in the NAD biosynthesis pathway of Pseudomonas syringae pv. tomato. Similar to its function in other bacteria like Mycobacterium tuberculosis, the enzyme catalyzes the transfer of the adenylyl moiety from ATP to nicotinic acid mononucleotide (NaMN), releasing pyrophosphate (PPi) and forming nicotinic acid dinucleotide (NaAD), which serves as the last intermediate in the synthesis of NAD. The enzyme shows strict preference for NaMN substrate over its amidated analog (NMN), which is a distinctive feature compared to human enzymes that have dual specificity with equal catalytic efficiency on both substrates . This pathway is critical for bacterial energy metabolism and various cellular processes dependent on NAD as a cofactor.

How is nadD activity typically measured in laboratory settings?

NaMN adenylyltransferase activity can be monitored using a coupled enzyme assay system. The standard approach involves coupling the NaAD product formation to a two-step generation of NADH through the action of ancillary enzymes such as NAD synthetase and alcohol dehydrogenase. The NADH production is then monitored spectrophotometrically at 340 nm. This method allows researchers to assess the apparent Km values for NaMN (which typically range from 0.1-3 mM) at saturating ATP concentrations (usually 2 mM) . Alternative methods may include direct measurement of product formation using HPLC or mass spectrometry approaches, particularly when studying inhibition kinetics or analyzing complex reaction mechanisms.

How does the genetic organization of nadD differ between Pseudomonas syringae pv. tomato races?

While the search results don't specifically address nadD organization in different P. syringae pv. tomato races, they do indicate significant genomic differences between race 0 and race 1 strains. Race 0 strains possess avirulence genes for type III system-associated effectors AvrPto1 and AvrPtoB, while race 1 strains lack these genes. Comparative genomics with several PG01a genomes revealed that mobile DNA elements are involved in the evolution of the two different races . This genomic plasticity suggests that genes encoding metabolic enzymes like nadD might also show variations between races, potentially affecting enzyme function or regulation. A complete genome analysis of various strains would be necessary to definitively characterize nadD organization across races.

What are the optimal conditions for expressing recombinant Pseudomonas syringae pv. tomato nadD in E. coli?

Based on comparable studies with bacterial adenylyltransferases, optimal expression of recombinant P. syringae pv. tomato nadD in E. coli typically involves the following conditions:

• Vector selection: pET series vectors with T7 promoter systems are recommended for high-level expression.

• Host strain: E. coli BL21(DE3) or its derivatives, which lack lon and ompT proteases.

• Induction parameters: IPTG concentrations between 0.1-0.5 mM at mid-log phase (OD600 ~0.6-0.8).

• Temperature: Post-induction expression at lower temperatures (16-20°C) for 16-18 hours often improves protein solubility compared to standard 37°C expression.

• Media supplementation: Addition of 0.2% glucose may help reduce basal expression prior to induction.

For mycobacterial NadD, researchers have successfully employed these approaches to obtain soluble, active enzyme for subsequent structural and functional studies . Similar strategies should be applicable to P. syringae pv. tomato nadD, with potential optimization required for this specific protein.

What purification strategy yields the highest purity and activity for recombinant nadD?

A multi-step purification strategy typically yields the highest purity and activity for recombinant nadD:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged nadD, with elution using an imidazole gradient (50-300 mM).

• Intermediate purification: Ion exchange chromatography (IEX) to separate protein based on charge properties.

• Polishing step: Size exclusion chromatography (SEC) to remove aggregates and achieve final purity.

• Buffer optimization: Inclusion of reducing agents (5 mM DTT or 1 mM TCEP) and 10% glycerol often stabilizes enzyme activity.

• Storage conditions: Flash-freezing aliquots in liquid nitrogen and storage at -80°C with 20% glycerol typically preserves activity.

Throughout purification, activity assays should be performed to track enzyme functionality, using the coupled spectrophotometric method to monitor NADH production as described for mycobacterial NadD . Final protein purity should be assessed by SDS-PAGE, with expected purity >95% for structural and enzymatic studies.

How can one develop a PMA-qPCR assay to study nadD expression in viable P. syringae pv. tomato cells?

Developing a PMA-qPCR assay for studying nadD expression specifically in viable P. syringae pv. tomato cells would involve adapting the methodology described for general detection of viable P. syringae pv. tomato :

• PMA treatment optimization:

  • Determine optimal PMA concentration (10 μmol liter⁻¹ was effective for general P. syringae detection)

  • Establish optimal light exposure time (10 min showed good results in previous studies)

  • Validate PMA's ability to selectively prevent amplification from dead cells

• nadD-specific primer design:

  • Design primers targeting conserved regions of the nadD gene

  • Validate primer specificity against related Pseudomonas species

  • Optimize PCR conditions for maximum sensitivity and specificity

• Standard curve generation:

  • Create standard curves using known quantities of viable P. syringae cells

  • Establish detection limits (previous studies achieved 10² CFU ml⁻¹ detection limits for P. syringae)

• Expression analysis workflow:

  • Extract RNA from PMA-treated samples

  • Perform reverse transcription

  • Quantify nadD transcript levels via qPCR

  • Normalize to appropriate reference genes

This approach would allow specific quantification of nadD expression only in viable bacterial cells, avoiding false positives from dead cells that might still contain intact DNA .

How does the structure of Pseudomonas syringae pv. tomato nadD compare to that of Mycobacterium tuberculosis nadD?

While the specific structure of P. syringae pv. tomato nadD has not been reported in the provided search results, comparative analysis can be performed against the well-characterized M. tuberculosis nadD (MtNadD) structure:

Structural predictions for P. syringae pv. tomato nadD would suggest a conserved nucleotidyltransferase fold with potential species-specific adaptations. Directed mutagenesis studies similar to those performed for MtNadD would be valuable to dissect structural elements contributing to substrate interactions in the P. syringae enzyme .

What is the role of nadD in Pseudomonas syringae pv. tomato pathogenicity and host-pathogen interactions?

The role of nadD in P. syringae pv. tomato pathogenicity likely extends beyond basic metabolism due to the critical importance of NAD in cellular processes:

• Energy metabolism: NAD biosynthesis is essential for bacterial energy production during infection, with nadD serving as a key enzyme in this pathway.

• Oxidative stress response: NAD-dependent enzymes are critical for responding to host-generated reactive oxygen species (ROS), suggesting nadD indirectly supports bacterial survival during infection.

• Potential coordination with virulence factors: While not directly addressed in the search results, metabolic pathways often show coordinated regulation with virulence factors. The variation between race 0 and race 1 strains, particularly in effector proteins like AvrPto1 and AvrPtoB , might correlate with metabolic adaptations involving nadD.

• Fitness during colonization: Proper NAD levels maintained through nadD activity likely contribute to bacterial fitness during colonization, potentially influencing the outcome of infection in different tomato cultivars.

• Possible moonlighting functions: Some metabolic enzymes have been shown to have secondary functions in pathogenicity beyond their primary catalytic role, a possibility that remains unexplored for nadD in P. syringae.

A comprehensive analysis of nadD expression during different stages of infection, along with studies of nadD knockdown/knockout mutants, would provide valuable insights into its contribution to pathogenicity.

How does coronatine production relate to nadD function in different P. syringae pv. tomato strains?

While the direct relationship between coronatine production and nadD function is not explicitly described in the search results, several connections can be drawn:

• Metabolic coordination: Both coronatine biosynthesis and NAD production (through nadD) are energy-intensive metabolic processes that likely show coordinated regulation.

• Genomic organization: The search results indicate that coronatine biosynthetic genes can be located either on plasmids (as in strains P. syringae Ps25, P. syringae pv. tomato B13-200, and P. syringae pv. tomato DAPP-PG 215) or on chromosomes (as in P. syringae pv. tomato DC3000) . This genomic plasticity might extend to nadD as well.

• Strain-specific variations: The phylogenetic grouping of strains based on coronatine gene location suggests broader metabolic adaptations that could include variations in nadD function or regulation.

• Regulatory networks: Different regulatory mechanisms for coronatine synthesis between strains, such as the distinction in CorR-binding sites , suggest strain-specific metabolic network organizations that might affect nadD expression and function.

Investigating potential correlations between nadD expression levels and coronatine production across different strains could reveal important insights into the metabolic regulation of virulence in P. syringae pv. tomato.

What makes nadD a potential target for developing selective antimicrobial compounds against Pseudomonas syringae pv. tomato?

Several characteristics make nadD a promising target for selective antimicrobial development against P. syringae pv. tomato:

• Essential metabolism: nadD catalyzes a critical step in NAD biosynthesis, which is essential for bacterial viability and virulence .

• Structural differences from human enzymes: Bacterial NadD enzymes show strict preference for NaMN substrate over NMN, while human NMNAT enzymes have dual specificity with equal catalytic efficiency for both substrates . This biochemical distinction provides a basis for selective targeting.

• Substrate binding pocket uniqueness: The active site architecture of bacterial nadD enzymes differs from human counterparts, offering potential binding sites for selective inhibitors.

• Unique conformational features: The over-closed conformation observed in M. tuberculosis nadD suggests bacterial-specific regulatory mechanisms that could be exploited for inhibitor design .

• Absence of functional redundancy: As a central metabolic enzyme, nadD typically lacks functional redundancy in bacterial systems, making resistance development through alternative pathway utilization less likely.

These features collectively suggest that selective inhibitors could be developed against P. syringae pv. tomato nadD with minimal off-target effects on plant enzymes, providing a potential strategy for controlling bacterial speck disease.

What high-throughput screening approaches are most suitable for identifying inhibitors of P. syringae pv. tomato nadD?

For identifying inhibitors of P. syringae pv. tomato nadD, several high-throughput screening (HTS) approaches can be implemented:

• Enzymatic activity-based screens:

  • Primary assay: Coupling NaAD product formation to NADH generation and monitoring absorbance at 340 nm in 384-well format

  • Confirmation assay: Direct measurement of NaAD formation using HPLC or LC-MS/MS

  • Counter-screen: Testing against human NMNAT enzymes to identify selective compounds

• Thermal shift assays:

  • Differential scanning fluorimetry (DSF) to identify compounds that stabilize or destabilize nadD protein

  • Isothermal dose-response fingerprinting (ITDRF) to establish binding constants for promising candidates

• Structure-based virtual screening:

  • Computational docking of virtual compound libraries to the nadD active site or allosteric sites

  • Molecular dynamics simulations to capture protein flexibility during ligand binding

  • Fragment-based approaches to identify chemical scaffolds with high binding efficiency

• Whole-cell phenotypic screens:

  • Growth inhibition assays in P. syringae cultures under conditions where nadD function is limiting

  • Target engagement confirmation using cellular thermal shift assays (CETSA)

  • Transcriptional profiling to identify signatures characteristic of nadD inhibition

• Integrated approaches:

  • Parallel screening of compound libraries against both purified enzyme and bacterial cells

  • Machine learning models to predict structure-activity relationships

  • Iterative design-make-test cycles to optimize initial hits

Each approach provides complementary information, and an integrated strategy combining enzymatic, structural, and cell-based methods would likely yield the most promising candidates for further development.

How does horizontal gene transfer influence nadD variation among Pseudomonas syringae pathovars?

Horizontal gene transfer (HGT) likely plays a significant role in nadD variation among Pseudomonas syringae pathovars, as suggested by broader genomic patterns observed in these bacteria:

• Mobile genetic elements: The search results indicate that mobile DNA elements are involved in the evolution of different races within P. syringae pv. tomato . This mechanism likely extends to metabolic genes like nadD, potentially introducing sequence variations or regulatory differences.

• Plasmid-chromosome gene movement: The observation that coronatine biosynthetic genes can be located either on plasmids or chromosomes in different strains suggests a dynamic genome with gene mobility that could affect nadD as well.

• Prophage-mediated transfer: The presence of avrPto1 in a prophage indicates that phage-mediated horizontal transfer is active in these bacteria. Similar mechanisms could facilitate nadD variants to spread among different strains or pathovars.

• Selection pressures: Different host plants would impose varying metabolic demands on pathogens, potentially selecting for nadD variants with altered kinetic properties or regulatory features that optimize NAD metabolism for specific host environments.

• Recombination events: Comparative genomics of P. syringae strains has revealed evidence of recombination events that lead to mosaic genomes. Such events could introduce nadD alleles from related species or pathovars.

Conducting a comprehensive phylogenetic analysis of nadD sequences across multiple P. syringae pathovars, along with analysis of flanking regions for mobile element signatures, would provide valuable insights into the evolutionary history and functional diversification of this enzyme.

What techniques are most effective for studying nadD expression during different phases of P. syringae pv. tomato infection?

For comprehensive analysis of nadD expression during P. syringae pv. tomato infection phases, a multi-technique approach is recommended:

• PMA-qPCR for viable cell-specific expression:

  • Adapt the propidium monoazide (PMA) pretreatment protocol developed for P. syringae detection

  • Design nadD-specific primers for quantitative PCR

  • Extract RNA from PMA-treated samples at different infection timepoints

  • This method ensures only expression in viable cells is measured, with a detection sensitivity of approximately 10² CFU ml⁻¹

• In planta gene expression analysis:

  • RNA-seq of infected plant tissue with computational separation of bacterial transcripts

  • Laser capture microdissection to isolate bacteria from specific infection sites

  • Dual RNA-seq for simultaneous host-pathogen transcriptome analysis

• Reporter systems for spatial-temporal analysis:

  • Transcriptional fusions of nadD promoter with fluorescent proteins

  • Confocal microscopy to visualize expression patterns during infection

  • Flow cytometry to quantify expression at the single-cell level

• Protein-level analysis:

  • Targeted proteomics using multiple reaction monitoring (MRM)

  • Antibody-based detection of nadD protein in plant extracts

  • Activity-based protein profiling to measure functional enzyme levels

• Metabolic impact assessment:

  • Measurement of NAD/NADH ratios in bacterial cells during infection

  • Metabolomics analysis to correlate nadD expression with metabolite levels

  • Isotope labeling to track flux through the NAD biosynthetic pathway

Each method offers unique advantages, and combining these approaches would provide a comprehensive understanding of nadD regulation and function throughout the infection process.

What gene editing approaches could be used to study the role of nadD in P. syringae pv. tomato virulence?

Several gene editing approaches can be employed to investigate nadD's role in P. syringae pv. tomato virulence:

• CRISPR-Cas9 system:

  • Development of a CRISPR-Cas9 system optimized for P. syringae

  • Generation of precise point mutations in catalytic residues

  • Creation of conditional knockdowns using inducible promoters

  • Introduction of epitope tags for protein localization studies

• Targeted mutagenesis strategies:

  • Site-directed mutagenesis of key residues identified through structural comparison with M. tuberculosis nadD

  • Creation of chimeric enzymes with domains from different bacterial species to identify determinants of specificity

  • Generation of temperature-sensitive mutants for temporal control of nadD function

• Expression modulation approaches:

  • Antisense RNA-based knockdown systems

  • Riboswitch-mediated regulation of nadD expression

  • Promoter replacement with tunable/inducible systems

• Functional complementation studies:

  • Heterologous expression of nadD variants from different pathovars

  • Trans-complementation with nadD homologs from non-pathogenic bacteria

  • Rescue experiments with pathogenicity assessment in tomato plants

• Multi-gene interaction analysis:

  • Simultaneous editing of nadD and virulence factors

  • Synthetic genetic array analysis to identify genetic interactions

  • Creation of metabolic bypass systems to test essentiality in different infection phases

These approaches would provide valuable insights into the functional importance of nadD in P. syringae pv. tomato pathogenicity and potentially identify novel intervention strategies.

How might structural information about P. syringae pv. tomato nadD inform the development of race-specific management strategies?

Structural information about P. syringae pv. tomato nadD could inform race-specific management strategies through several avenues:

• Identification of race-specific structural features:

  • If nadD structures differ between race 0 and race 1 strains, these differences could be exploited for selective targeting

  • Comparison with the unique features identified in M. tuberculosis nadD, such as the 3₁₀ helix that locks the active site , might reveal race-specific regulatory mechanisms

• Structure-guided inhibitor design:

  • Molecular docking studies using race-specific structural models

  • Fragment-based approaches targeting unique binding pockets

  • Design of allosteric inhibitors that exploit race-specific conformational dynamics

• Prediction of resistance mechanisms:

  • Structural analysis can identify potential resistance mutations

  • Molecular dynamics simulations to predict the impact of mutations on inhibitor binding

  • Development of dual-targeting approaches to reduce resistance emergence

• Understanding host-specificity determinants:

  • Structural variations in nadD might correlate with adaptation to different tomato cultivars

  • These insights could inform breeding programs for resistance development

• Diagnostic applications:

  • Structure-informed development of antibodies that distinguish race-specific nadD variants

  • Design of aptamers or other molecular probes for rapid detection of specific races

The race concept in P. syringae pv. tomato may be becoming less distinct due to the presence of intermediate phenotypes and genetic mobility (such as avrPto1 in prophage elements) . Nevertheless, structural information about nadD could provide valuable insights into bacterial adaptation mechanisms and inform more targeted management approaches.