Recombinant Pseudomonas syringae pv. tomato NADH pyrophosphatase (nudC)

What is NudC in Pseudomonas syringae pv. tomato?

NudC is a Nudix hydrolase enzyme encoded by the nudC gene in Pseudomonas syringae pv. tomato str. DC3000, a plant pathogen causing bacterial speck on Arabidopsis and tomato plants. It belongs to the Nudix pyrophosphatase family, enzymes found ubiquitously across organisms that hydrolyze pyrophosphate bonds in nucleoside diphosphate derivatives. In P. syringae, NudC plays a crucial role in regulating intracellular redox balance and is essential for normal bacterial growth, motility, and biofilm formation .

What is the primary enzymatic function of NudC?

The primary enzymatic function of NudC is to act as an NADH pyrophosphatase. Biochemical studies have demonstrated that NudC preferentially hydrolyzes NADH in vitro . This enzymatic activity helps maintain proper NADH/NAD+ ratios within bacterial cells, which in turn affects numerous metabolic processes. The catalytic mechanism involves the hydrolysis of the pyrophosphate bond in NADH, and this function requires specific conserved residues in the enzyme's active site .

How does NudC differ between P. syringae and P. aeruginosa?

Despite being homologous enzymes with similar biochemical activities, NudC proteins from P. syringae and P. aeruginosa exhibit striking functional differences:

These differences suggest that despite structural and enzymatic similarities, these homologous proteins have evolved divergent roles in their respective bacterial species .

What phenotypic changes occur in P. syringae when nudC is deleted?

Deletion of the nudC gene in P. syringae results in multiple phenotypic defects:

• Accumulation of NADH within bacterial cells

• Significant growth defects (reduced growth rate)

• Impaired cellular motility

• Defective biofilm formation

These phenotypic changes can be reversed by complementation with a wild-type copy of the nudC gene with its native promoter delivered in trans, confirming that the observed defects are specifically due to the absence of functional NudC .

What is the structural organization of NudC?

NudC proteins from both P. syringae and P. aeruginosa exist as homodimers in their native state . This quaternary structure appears important for the proper functioning of the enzyme. The protein contains the characteristic Nudix box motif (GX5EX7REUXEEXGU, where U represents a hydrophobic residue), which is critical for substrate binding and catalytic activity. Structural studies suggest that dimerization likely affects both enzyme stability and catalytic properties by potentially creating optimal conformations of the active sites.

How should experiments be designed to evaluate NudC's role in redox balance regulation?

To effectively evaluate NudC's role in redox balance regulation, researchers should implement a comprehensive experimental design strategy:

Experimental Design Strategy:

• Genetic Manipulation Studies:

  • Create precise deletion mutants of nudC using allelic exchange

  • Generate complemented strains with wild-type nudC under native and inducible promoters

  • Develop point mutants affecting catalytic residues to distinguish protein presence from enzymatic activity

  • Create strains with tagged versions of NudC for localization and interaction studies

• Metabolomic Analysis:

  • Quantify NADH/NAD+ ratios in wild-type, ΔnudC, and complemented strains using fluorometric assays

  • Perform global metabolite profiling to identify downstream metabolic changes

  • Measure changes in redox-sensitive metabolites under various growth conditions

• Physiological Assays:

  • Assess growth curves in different media compositions and stress conditions

  • Quantify motility using swimming, swarming, and twitching assays

  • Evaluate biofilm formation using crystal violet staining and confocal microscopy

  • Measure ROS (reactive oxygen species) levels using fluorescent probes

• Transcriptomic Analysis:

  • Perform RNA-seq comparing wild-type and ΔnudC strains

  • Identify differentially expressed genes involved in redox homeostasis

  • Validate findings with RT-qPCR for selected genes

This multi-faceted approach allows for a comprehensive understanding of how NudC regulates redox balance, connecting molecular function to physiological outcomes .

What molecular mechanisms explain the differential importance of NudC in P. syringae versus P. aeruginosa?

The differential importance of NudC in P. syringae versus P. aeruginosa represents an intriguing case of functional divergence between homologous proteins. Several molecular mechanisms may explain this phenomenon:

Experimental approaches to investigate these mechanisms should include comparative genomics, heterologous expression studies, and chimeric protein analysis to identify the specific domains responsible for the functional differences .

How might NudC function relate to pathogenicity in P. syringae?

NudC function may relate to P. syringae pathogenicity through several interconnected mechanisms:

• Metabolic Adaptation During Infection:

  • Proper NADH/NAD+ balance is critical for bacterial metabolism during plant colonization

  • NudC may help P. syringae adapt to changing nutrient availability in plant tissues

• Stress Response and Survival:

  • Plant defense responses often include oxidative burst; NudC-mediated redox balance may enhance bacterial survival

  • NADH metabolism impacts resistance to environmental stresses encountered during infection

• Biofilm Formation:

  • NudC deficiency impairs biofilm formation, which is important for epiphytic survival and plant colonization

  • Biofilms provide protection against antimicrobial compounds produced by plants

• Virulence Factor Expression:

  • NADH levels may influence expression of key virulence factors through redox-sensitive regulators

  • Recent research has identified a diazeniumdiolate signal (leudiazen) in P. syringae that upregulates virulence factors and promotes survival in plants, which may be influenced by redox balance

• Type III Secretion System Function:

  • The type III secretion system, critical for P. syringae virulence, delivers effector proteins like HopPtoD2 into plant cells to suppress host defenses

  • Proper redox balance maintained by NudC may be necessary for optimal function of this complex secretion apparatus

To investigate these relationships, researchers should compare the virulence of wild-type and ΔnudC strains in plant infection models and analyze how NudC affects the expression and function of known virulence factors .

What are the technical challenges in expressing and purifying active recombinant NudC?

Expressing and purifying active recombinant NudC presents several technical challenges that researchers should address:

• Expression System Selection:

  • Heterologous expression in E. coli can lead to inclusion body formation or toxicity, especially considering the toxic effect observed when P. syringae nudC is expressed under strong promoters

  • Selection of appropriate expression vectors and promoters is critical for optimal yields

• Protein Solubility and Folding:

  • NudC may require specific chaperones for proper folding

  • Optimization of induction conditions (temperature, IPTG concentration, induction time)

  • Use of solubility-enhancing fusion tags (MBP, SUMO, thioredoxin)

• Protein Stability:

  • NudC may be prone to degradation during purification

  • Inclusion of protease inhibitors and optimization of buffer conditions

  • Careful consideration of pH and ionic strength for maintaining enzyme stability

• Maintaining Enzymatic Activity:

  • Presence of required cofactors during purification

  • Avoiding oxidation of catalytic cysteine residues

  • Verification of proper oligomeric state (homodimer)

Recent studies with similar NADH pyrophosphatases have shown that strategies focusing on expression regulation, including screening vectors, optimizing promoters and ribosome binding sites, can enhance productivity (up to 1.8 U/mL for E. coli NudC) .

How can protein engineering approaches be applied to enhance NudC activity and stability?

Protein engineering offers powerful strategies to enhance NudC's activity and stability:

• Rational Design Approaches:

  • Structure-guided mutagenesis targeting residues in the active site to improve catalytic efficiency

  • Modification of surface residues to enhance solubility

  • Introduction of disulfide bridges or salt bridges to increase thermostability

  • Analysis of the dimerization interface to strengthen quaternary structure

• Directed Evolution Strategies:

  • Error-prone PCR to generate libraries of NudC variants

  • DNA shuffling between P. syringae and P. aeruginosa NudC genes

  • Development of high-throughput screening assays for NADH hydrolysis activity

  • Iterative rounds of selection under desired conditions (temperature, pH, etc.)

• Case Study Insights:
  Recent work with a related NADH pyrophosphatase from E. coli (EcNudc) demonstrated successful protein engineering:

  Mutation	Activity Improvement	Thermostability Improvement at 50°C
  R148A	1.8-fold increase	1.5-fold increase
  H149E	2.1-fold increase	2.2-fold increase
  R148A-H149E (combined)	3.3-fold increase	3.6-fold increase

  This combined mutant (EcNudc-M) achieved enzyme activity of 33.0 U/mL in fermentation, demonstrating the power of protein engineering approaches .

These approaches could be applied to P. syringae NudC to develop enzyme variants with improved properties for both research and potential biotechnological applications.

How does NudC's activity impact other metabolic pathways in Pseudomonas?

NudC's NADH pyrophosphatase activity has far-reaching impacts on multiple metabolic pathways in Pseudomonas:

• Central Carbon Metabolism:

  • NADH/NAD+ ratio affects the direction and flux through glycolysis, TCA cycle, and other central pathways

  • Glycolytic enzyme activities are often regulated by NAD+/NADH levels

  • Carbon flux distribution between different pathways may be altered in nudC mutants

• Respiratory Chain Function:

  • NADH serves as a primary electron donor to the respiratory chain

  • Altered NADH levels affect electron transport chain efficiency and energy generation

  • Impact on proton motive force and ATP synthesis

• Redox-Dependent Regulatory Networks:

  • Transcriptional regulators sensitive to cellular redox state

  • Global changes in gene expression patterns in response to altered NADH/NAD+ ratios

  • Similar RNA pyrophosphohydrolases in P. aeruginosa have been shown to affect the level of 537 transcripts involved in various cellular processes

• Secondary Metabolism:

  • Biosynthesis of many secondary metabolites requires NADH/NAD+

  • Production of virulence factors and toxins may be affected

  • In P. aeruginosa, Nudix-type RNA pyrophosphohydrolase has been shown to provide homeostasis of the virulence factor pyocyanin

• Biofilm Matrix Production:

  • Exopolysaccharide synthesis pathways utilize NAD+/NADH

  • Changes in redox balance affect production of biofilm matrix components

  • Connections between metabolism and biofilm development

Metabolomic profiling comparing wild-type and ΔnudC strains would provide valuable insights into these metabolic interactions .

What methods can be used to study the in vivo dynamics of NADH metabolism in relation to NudC activity?

Studying the in vivo dynamics of NADH metabolism in relation to NudC activity requires sophisticated methodological approaches:

• Real-time NADH/NAD+ Monitoring:

  • Genetically encoded fluorescent biosensors for real-time monitoring of NADH/NAD+ ratios

  • Microfluidic systems coupled with time-lapse fluorescence microscopy

  • Flow cytometry-based analysis of population heterogeneity in NADH levels

• Spatiotemporal Resolution Techniques:

  • Fluorescence lifetime imaging microscopy (FLIM) to distinguish free and protein-bound NADH

  • Subcellular fractionation with rapid quenching to preserve metabolic state

  • Single-cell analysis to capture cell-to-cell variability

• Isotope Tracing Approaches:

  • 13C-labeled substrate feeding and metabolite analysis

  • Metabolic flux analysis to quantify pathway activities

  • Isotopically non-stationary metabolic flux analysis for dynamic responses

• Genetic Reporter Systems:

  • Transcriptional fusions of redox-sensitive promoters with reporter genes

  • Engineered riboswitches responsive to NAD+/NADH levels

• Experimental Design Considerations:

  • Controlled environmental conditions to minimize variability

  • Appropriate time scales to capture both rapid and long-term effects

  • Careful selection of growth media to control metabolic state

  • Inclusion of appropriate controls (catalytically inactive NudC mutants)

These methodologies would need to be adapted to the specific research question and experimental system but collectively provide powerful approaches to understand how NudC activity shapes NADH metabolism dynamics in vivo .

How do environmental conditions affect NudC expression and activity?

Environmental conditions can significantly influence both NudC expression and activity:

Experimental approaches to study these effects should include qRT-PCR analysis of nudC expression under various conditions, reporter gene fusions to monitor transcriptional regulation, and enzyme activity assays under simulated environmental conditions .