Recombinant Pseudomonas syringae pv. syringae Glycerol-3-phosphate dehydrogenase [NAD (P)+] (gpsA)

What is the primary function of gpsA in Pseudomonas syringae metabolism?

GpsA in Pseudomonas syringae functions as a glycerol-3-phosphate dehydrogenase that catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) using the reducing power of NADH or NADPH. This enzyme represents a critical bidirectional oxidoreductase node that connects glycolytic energy production to glycerol metabolism and lipid biosynthesis . The reaction proceeds as follows:

Dihydroxyacetone phosphate + NADH/NADPH → Glycerol-3-phosphate + NAD+/NADP+

GpsA essentially performs the reverse reaction of GlpD (another glycerol-3-phosphate dehydrogenase), which oxidizes G3P to DHAP. Together, these enzymes create a metabolic cycle that helps maintain redox balance and provides precursors for membrane phospholipid synthesis .

How does genetic disruption of gpsA affect bacterial viability and virulence?

Based on studies in other bacterial systems like Borrelia burgdorferi, disruption of gpsA can have profound effects on bacterial viability and virulence. In B. burgdorferi, a gpsA mutant exhibited decreased survival under nutrient stress conditions and reduced infectivity in mouse models . The mutant showed pleiotropic phenotypes including morphological changes and metabolic imbalances of NADH and glycerol-3-phosphate .

Interestingly, deletion of glpD in a gpsA mutant background restored viability under nutrient stress, suggesting a suppressor mutation effect. This indicates that the metabolic imbalance created by gpsA deletion, rather than the absence of the enzyme itself, may be responsible for the observed phenotypes .

For P. syringae specifically, research would need to examine:

• Growth characteristics in minimal vs. rich media

• Survival under various environmental stresses

• Plant infection capacity and symptom development

• Metabolite profiles compared to wild-type strains

What is known about the genomic context and evolution of gpsA in Pseudomonas syringae?

While specific information about the genomic context of gpsA in P. syringae is not directly provided in the search results, understanding the evolutionary context requires analysis of P. syringae genomics and population structure. P. syringae is known to have extensive genetic diversity organized into several phylogroups .

Recombination appears to occur at a moderately high rate over many loci in P. syringae . This suggests that gpsA, like other genes, may have been subject to horizontal gene transfer events during the evolution of different P. syringae lineages. Understanding the genomic context would involve:

• Identifying conserved regions surrounding gpsA across P. syringae strains

• Analyzing GC content and codon usage patterns for evidence of horizontal acquisition

• Examining synteny with related bacterial species

• Constructing phylogenetic trees to determine the evolutionary history of gpsA

Patterns of recombination could be used to define functional populations, as recombination occurs more frequently among closely related strains interacting in the same niche .

What expression systems and strategies work best for recombinant P. syringae gpsA?

For recombinant expression of P. syringae gpsA, researchers have several options based on general recombineering techniques applied to Pseudomonas proteins:

• Pseudomonas-based expression systems:

  • Native expression in P. syringae using vectors like pUCP24, which has been successfully used for recombinant protein expression in Pseudomonas

  • These vectors can incorporate promoters with controlled expression levels

  • The pUCP24/47 vector contains a Gateway cassette for efficient cloning and a sacB gene for counterselection

• E. coli-based expression systems:

  • Standard BL21(DE3) strains for high-level expression

  • Rosetta strains if rare codon usage is an issue

  • Cold-inducible systems if protein solubility is problematic

When expressing in Pseudomonas, research has demonstrated that plasmid vectors can be successfully eliminated from cells after recombination using counterselectable markers like sacB , which is important for preparing recombinants for downstream experiments.

What purification challenges are specific to recombinant gpsA and how can they be addressed?

While the search results don't provide specific information about purification challenges for P. syringae gpsA, several strategies can be inferred based on the nature of the enzyme:

• Affinity tags selection:

  • His-tags are commonly used but may affect enzyme activity

  • Alternative tags like GST or MBP may improve solubility

  • Consider tag removal options using specific proteases

• Maintaining enzyme activity:

  • Include cofactors (NAD+/NADP+) in trace amounts during purification

  • Add reducing agents to prevent oxidation of critical cysteine residues

  • Include glycerol (10-20%) as a stabilizing agent

• Multi-step purification approach:

  • Initial capture using affinity chromatography

  • Secondary purification using ion exchange chromatography

  • Final polishing using size exclusion chromatography

• Activity monitoring:

  • Develop a rapid spectrophotometric assay to track activity during purification

  • Monitor specific activity at each purification step

  • Identify and mitigate steps that lead to activity loss

Based on research with other enzymes, maintaining reducing conditions and preventing aggregation are typically the most challenging aspects of purifying dehydrogenases.

How can recombineering techniques improve gpsA expression and modification?

Recombineering techniques can significantly enhance the expression and modification of gpsA in P. syringae. Based on research with RecTE from P. syringae, several approaches can be considered:

• Homologous recombination-based strategies:

  • The RecT protein from P. syringae promotes recombination of single-stranded DNA oligonucleotides

  • Combined RecT and RecE expression enables efficient recombination of double-stranded DNA

  • These systems can be used to introduce specific mutations or tags into the genomic copy of gpsA

• Promoter optimization:

  • Replacing the native promoter with constitutive or inducible promoters

  • The constitutive BAD nptII promoter has been successfully used in P. syringae studies

  • Gateway technology facilitates efficient promoter swapping

• Site-directed mutagenesis:

  • RecT-mediated recombineering allows for precise mutations without selection markers

  • This approach is ideal for structure-function studies of gpsA

• Chromosomal integration:

  • Stable expression can be achieved through chromosomal integration

  • This eliminates plasmid loss concerns and antibiotic selection requirements

The efficiency of recombineering in P. syringae pv. tomato DC3000 has been quantitatively assessed, demonstrating that these techniques provide a foundation for efficient site-directed mutagenesis in P. syringae .

What methods are available for assessing gpsA enzyme kinetics and substrate specificity?

To characterize the kinetic properties and substrate specificity of recombinant P. syringae gpsA, several methodological approaches can be employed:

• Spectrophotometric assays:

  • Direct measurement of NAD(P)H oxidation or NAD(P)+ reduction at 340 nm

  • Continuous monitoring to determine initial reaction rates

  • Determination of pH and temperature optima

• Kinetic parameter determination:

  • Michaelis-Menten kinetics to determine Km and Vmax for DHAP and NAD(P)H

  • Inhibition studies to identify regulatory molecules

  • Steady-state and pre-steady-state kinetics to elucidate reaction mechanism

• Substrate specificity analysis:

  • Testing structurally similar compounds to DHAP

  • Comparing activity with NADH versus NADPH

  • Determining specificity constants (kcat/Km) for different substrates

For example, based on studies with other G3P dehydrogenases, a typical experimental setup would include:

How does gpsA interact with other enzymes in glycerol metabolism, especially glpD?

The interaction between gpsA and glpD in P. syringae appears to be similar to that observed in other bacteria, where they form a bidirectional oxidoreductase node connecting glycolysis to lipid metabolism . Their relationship can be characterized as follows:

• Metabolic interplay:

  • GpsA reduces DHAP to G3P using NADH/NADPH

  • GlpD oxidizes G3P to DHAP, reducing NAD+ or FAD

  • Together they create a cycle that can respond to cellular redox and metabolic demands

• Genetic interaction evidence:

  • In B. burgdorferi, deletion of glpD restored wild-type phenotype to the pleiotropic gpsA mutant

  • This represents a suppressor mutation effect where removing glpD rescues defects caused by gpsA deletion

  • This suggests that accumulation of metabolic intermediates or redox imbalance, rather than the absence of end products, may cause the gpsA mutant phenotype

• Experimental approaches to study interactions:

  • Double knockout studies (ΔgpsA/ΔglpD) compared to single knockouts

  • Metabolomic profiling to track flux through these pathways

  • Transcriptional analysis to identify regulatory connections

  • Protein-protein interaction studies to detect physical associations

The finding that glycerol becomes bactericidal to cells lacking glpD demonstrates the importance of balanced activity between these enzymes . When glpD is absent, cells cannot metabolize glycerol-3-phosphate, leading to toxic accumulation when exposed to external glycerol.

What structural features determine the NAD(P)+ specificity of gpsA?

While specific structural information about P. syringae gpsA is not provided in the search results, the determinants of NAD(P)+ specificity in dehydrogenases are well-established and can be investigated through:

• Sequence analysis approaches:

  • Comparison with related G3P dehydrogenases of known specificity

  • Identification of the Rossmann fold motif characteristic of nucleotide-binding domains

  • Presence of signature motifs associated with NADH vs. NADPH preference

• Structural analysis methods:

  • Homology modeling based on related enzyme structures

  • Identification of residues interacting with the 2'-phosphate of NADPH

  • Structural comparison of the cofactor binding pocket with NAD+ or NADP+-specific enzymes

• Experimental confirmation:

  • Site-directed mutagenesis of predicted specificity-determining residues

  • Kinetic analysis with both NADH and NADPH as cofactors

  • Determination of binding affinities for different cofactors

The typical structural determinants of NAD(P)+ specificity include:

How do environmental conditions affect gpsA expression and activity in P. syringae?

Environmental regulation of gpsA expression and activity is critical for understanding P. syringae adaptation to different niches, including plant hosts. While specific information about gpsA regulation in P. syringae is not provided in the search results, several approaches can be used to investigate this:

• Transcriptional regulation analysis:

  • qRT-PCR to measure gpsA expression under different conditions

  • Promoter-reporter fusions to visualize expression patterns

  • ChIP-seq to identify transcription factors binding the gpsA promoter

• Environmental conditions to test:

  • Nutrient limitation (similar to conditions where gpsA was essential in B. burgdorferi)

  • Plant-mimicking conditions vs. epiphytic growth conditions

  • Temperature shifts (relevant for plant-associated bacteria)

  • Different carbon sources, especially glycerol vs. sugars

• Post-translational regulation:

  • Phosphorylation state analysis under different conditions

  • Redox state of the enzyme during oxidative stress

  • Allosteric regulation by metabolic intermediates

• Ecological relevance:

  • Expression during different stages of plant infection

  • Correlation with virulence and persistence

  • Role in environmental stress resistance

The study of P. syringae evolution, genomics, and epidemiology indicates that adaptation to different environments drives the diversification of this species . Understanding how gpsA responds to these selective pressures would provide insights into its role in P. syringae ecology.

How can recombinant gpsA be used to study P. syringae pathogenesis and metabolism?

Recombinant gpsA can serve as a powerful tool for investigating P. syringae pathogenesis and metabolism through several experimental approaches:

• Metabolic profiling studies:

  • Compare metabolite levels between wild-type and gpsA-deficient strains

  • Identify metabolic bottlenecks using labeled substrates

  • Map changes in redox balance during infection

• Plant infection experiments:

  • Generate gpsA mutants using recombineering techniques

  • Compare virulence with wild-type strains on various host plants

  • Test complementation with recombinant gpsA to confirm phenotypes

• Stress response analysis:

  • Examine survival under nutrient limitation and oxidative stress

  • Test response to antimicrobial compounds

  • Investigate biofilm formation capabilities

• Co-infection studies:

  • Perform co-infections with wild-type and gpsA mutant strains

  • Assess competitive fitness during plant colonization

  • Examine population dynamics during infection

Research on co-infections has shown that the interactions between distinct P. syringae lineages can strongly influence disease outcomes and epidemiological dynamics . For example, co-infections of environmental and outbreak P. syringae isolates on kiwifruit resulted in the same total population sizes but reduced disease symptoms compared to single infections .

What role does recombination play in gpsA evolution among P. syringae strains?

• Recombination patterns and rates:

  • P. syringae undergoes a moderately high rate of recombination over many loci

  • This recombination helps maintain genetic cohesion between lineages

  • Patterns of recombination could reflect underlying environmental structure and define functional populations

• Mechanisms of genetic exchange:

  • While P. syringae is not known to be naturally competent for transformation, recombination clearly occurs

  • Plasmid and phage-mediated gene exchange mechanisms contribute to horizontal transfer

  • Extracellular vesicles may provide an additional, less explored mechanism

• Evolutionary implications:

  • Recombination may help disseminate adaptive variants of gpsA between strains

  • Selection pressure on gpsA function could drive patterns of conservation or divergence

  • Comparing gpsA sequences across phylogroups could reveal signatures of selection

• Experimental approaches to study recombination:

  • Sequence analysis of gpsA across diverse P. syringae strains

  • Identification of recombination breakpoints using bioinformatic tools

  • Construction of phylogenetic trees to identify incongruence suggestive of recombination

Understanding recombination patterns is particularly relevant as they can be used to define species boundaries in P. syringae based on genetic cohesion and ecological adaptation .

How can site-directed mutagenesis of gpsA inform our understanding of enzyme mechanism?

Site-directed mutagenesis of gpsA, facilitated by recombineering techniques described for P. syringae , can provide valuable insights into the enzyme's catalytic mechanism:

• Key residues to target:

  • Catalytic triad/dyad residues identified through sequence alignments

  • Cofactor binding residues that determine NAD(P) specificity

  • Substrate binding residues that interact with DHAP

  • Residues potentially involved in allosteric regulation

• Experimental design approach:

  • Generate a library of single amino acid substitutions

  • Express and purify mutant proteins

  • Perform comprehensive kinetic characterization

  • Analyze structural changes using biophysical techniques

• Parameters to measure for each mutant:

  • Km and kcat values for DHAP and NAD(P)H

  • pH-rate profiles to identify ionizable groups

  • Temperature dependence to determine activation parameters

  • Inhibition patterns to identify binding mode changes

• Data interpretation framework:

What potential applications exist for using gpsA in metabolic engineering of Pseudomonas?

The strategic position of gpsA in P. syringae metabolism offers several opportunities for metabolic engineering applications:

• Redox balance engineering:

  • Manipulating NAD(P)H/NAD(P)+ ratios by altering gpsA expression

  • Creating strains with enhanced oxidative stress resistance

  • Engineering metabolic pathways that depend on redox cofactor availability

• Glycerol utilization improvement:

  • Enhancing growth on glycerol as a carbon source

  • Creating strains for glycerol valorization in industrial applications

  • Optimizing the glycerol-3-phosphate node for metabolic flux

• Lipid production optimization:

  • Increasing glycerol-3-phosphate availability for phospholipid biosynthesis

  • Engineering membrane composition for stress tolerance

  • Developing strains for biotechnological lipid production

• Biosensor development:

  • Creating biosensors based on gpsA activity to monitor metabolic state

  • Developing screening tools for identifying metabolic engineering targets

  • Designing reporter systems for environmental monitoring

Recombineering techniques established for P. syringae, including the use of RecT for single-stranded DNA recombination and RecT/RecE for double-stranded DNA recombination , provide the genetic tools necessary for precision engineering of metabolic pathways involving gpsA.

What are the best approaches for studying the in vivo dynamics of gpsA in P. syringae?

To study the dynamics of gpsA in living P. syringae cells, several advanced analytical techniques can be employed:

• Fluorescent protein fusions:

  • Creating gpsA-GFP fusions using recombineering techniques

  • Monitoring cellular localization under different conditions

  • Tracking expression levels in real-time during infection

• Transcriptomics approaches:

  • RNA-seq to measure gpsA expression across conditions

  • Single-cell RNA-seq to capture population heterogeneity

  • Ribosome profiling to assess translation efficiency

• Metabolic flux analysis:

  • 13C-labeled substrate experiments to track carbon flow

  • Isotope ratio analysis to determine pathway activities

  • Metabolic modeling to predict flux distributions

• In vivo activity probes:

  • Genetically encoded NAD(P)H sensors to monitor redox state

  • Fluorescence lifetime imaging to detect enzyme-substrate interactions

  • Activity-based protein profiling to assess functional state

These approaches would provide insights into how gpsA activity changes during P. syringae adaptation to different environments, including during plant infection and under stress conditions. They would also help elucidate the in vivo role of gpsA in the bacterial metabolic network.

How can structural biology techniques be applied to understand gpsA function?

Structural biology approaches provide crucial insights into gpsA function at the molecular level:

• X-ray crystallography strategy:

  • Expression and purification of highly pure recombinant gpsA

  • Screening crystallization conditions with and without substrates/cofactors

  • Structure determination at high resolution

  • Comparison with related G3P dehydrogenases

• Cryo-electron microscopy applications:

  • Visualization of gpsA in complex with interaction partners

  • Analysis of conformational changes during catalysis

  • Study of higher-order complexes or metabolons

• NMR spectroscopy approaches:

  • Characterization of protein dynamics during catalysis

  • Mapping of ligand binding sites

  • Analysis of protein-protein interactions

• Computational methods:

  • Molecular dynamics simulations to study conformational flexibility

  • Quantum mechanics/molecular mechanics to model reaction mechanisms

  • Molecular docking to identify potential inhibitors or regulators

The structural information obtained would facilitate rational design of gpsA variants with altered properties and provide insights into the molecular basis of substrate and cofactor specificity.

What bioinformatic approaches can predict gpsA function across different P. syringae strains?

Bioinformatic approaches can provide valuable insights into gpsA function and evolution across the diverse P. syringae species complex:

• Comparative genomic analysis:

  • Sequence alignment of gpsA across P. syringae phylogroups

  • Identification of conserved domains and variable regions

  • Detection of selection signatures using dN/dS ratios

• Phylogenetic methods:

  • Construction of gpsA phylogenetic trees

  • Comparison with species phylogeny to detect horizontal gene transfer

  • Ancestral sequence reconstruction to track evolutionary changes

• Structural prediction tools:

  • Homology modeling of gpsA from different strains

  • Prediction of functional effects of sequence variations

  • Virtual screening for strain-specific inhibitors

• Network analysis:

  • Identification of gpsA genetic interaction networks

  • Prediction of metabolic pathways influenced by gpsA

  • Comparison of regulatory networks across strains

These approaches would help understand how gpsA function may vary across the P. syringae phylogroups and how this variation might contribute to niche adaptation and host specificity.

How can systems biology integrate gpsA function into whole-cell metabolic models of P. syringae?

Integrating gpsA function into whole-cell metabolic models of P. syringae requires sophisticated systems biology approaches:

• Genome-scale metabolic model construction:

  • Annotation of all reactions involving gpsA

  • Integration of glycerol metabolism with central carbon pathways

  • Incorporation of redox balance constraints

• Flux balance analysis applications:

  • Prediction of metabolic fluxes under different conditions

  • Identification of essential reactions connected to gpsA

  • Simulation of knockout phenotypes

• Multi-omics data integration:

  • Correlation of transcriptomic, proteomic, and metabolomic data

  • Constraint-based modeling using experimental measurements

  • Regulatory network reconstruction

• Dynamic modeling approaches:

  • Kinetic modeling of the gpsA reaction and connected pathways

  • Simulation of system responses to perturbations

  • Prediction of metabolic adaptation during infection

The P. syringae pangenome and the extensive genetic diversity across phylogroups suggest that metabolic models may need to be strain-specific to accurately capture the role of gpsA in different genetic backgrounds.