Recombinant Pseudomonas syringae pv. tomato Fatty acid oxidation complex subunit alpha (fadB), partial

How does the fadB gene organization in P. syringae pv. tomato differ from other Pseudomonas species?

While detailed genomic organization varies between species, P. syringae pv. tomato, like P. putida, contains multiple chromosomal copies of the β-oxidation genes. The genome of P. syringae pv. tomato DC3000 includes at least two copies of the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (fadB) gene, often located in operons alongside the 3-ketoacyl-CoA thiolase (fadA) gene . This differs from some other Pseudomonas species which may have different copy numbers or genomic arrangements, suggesting potentially specialized metabolic roles in this plant pathogen .

What is the relationship between fadB and bacterial virulence in P. syringae pv. tomato?

Although direct evidence linking fadB to virulence is limited, metabolic adaptability is crucial for successful plant infection. The β-oxidation pathway contributes to the bacterium's ability to utilize alternative carbon sources during infection. Studies with plant pathogenic bacteria, including P. syringae pv. tomato DC3000, suggest that mutations affecting metabolism impact colonization and disease progression. For instance, disruption of fatty acid metabolism can affect the production of biosurfactants like syringafactin, which influences motility and potentially host colonization . A comprehensive understanding requires examining fadB in the context of the bacterium's environmental adaptation and pathogenesis strategies.

What are the most effective methods for cloning and expressing recombinant fadB from P. syringae pv. tomato?

For optimal recombinant fadB expression:

• Gene Amplification and Vector Selection:

  • Use high-fidelity DNA polymerase to amplify the fadB gene with appropriate restriction sites

  • For protein production, pET vectors in E. coli BL21(DE3) provide high expression levels

  • For complementation studies, use broad-host-range vectors like pBBR1MCS series

• Expression Optimization:

  • Culture temperature: 16-20°C after induction reduces inclusion body formation

  • IPTG concentration: 0.1-0.5 mM is typically sufficient

  • Induction timing: Mid-log phase (OD600 = 0.6-0.8) generally yields best results

• Purification Strategy:

  • N-terminal His6-tag allows efficient purification via nickel affinity chromatography

  • Include protease inhibitors in lysis buffer to prevent degradation

  • Consider ion exchange chromatography as a secondary purification step

For functional studies, the pComb3 phagemid vector system has been demonstrated effective for expressing recombinant proteins from Pseudomonas species .

What approaches can be used to generate and verify fadB knockout mutants in P. syringae pv. tomato?

Generation Methods:

Verification Protocols:

• PCR Verification: Use primers that flank the deletion site to confirm appropriate fragment size

• Functional Verification: Measure growth on fatty acids as sole carbon source

• Metabolic Verification: Analyze fatty acid utilization using [³H]acetate radiolabeling to quantify fatty acid metabolism

• Transcriptional Verification: Perform RT-PCR or RNA-Seq to confirm absence of fadB transcript

The recombineering approach using RecTE from P. syringae is particularly effective for creating markerless deletions in pseudomonads, as it allows for precise genomic modifications with high efficiency .

How can researchers accurately measure fadB enzyme activity in recombinant systems?

FadB enzyme activity can be measured through several complementary approaches:

• Spectrophotometric Assays:

  • 3-hydroxyacyl-CoA dehydrogenase activity: Monitor NAD⁺ reduction at 340 nm using 3-hydroxyacyl-CoA as substrate

  • Enoyl-CoA hydratase activity: Follow the hydration of crotonyl-CoA at 263 nm

• Coupled Enzyme Assays:

  • Link FadB activity to a secondary reaction that produces a measurable output

  • Ensure coupling enzymes are in excess to prevent rate-limiting effects

• Metabolic Flux Analysis:

  • Trace carbon flow through β-oxidation using labeled substrates

  • Quantify intermediates via LC-MS/MS to determine pathway activity

For normalization of results:

• Express activity as μmol substrate converted per minute per mg protein

• Use purified components for kinetic parameter determination (Km, Vmax)

• Include appropriate controls (heat-inactivated enzyme, known inhibitors)

How can fadB deletion enhance the production of valuable compounds in Pseudomonas species?

Deleting fadB redirects carbon flux away from fatty acid degradation toward alternative pathways that produce valuable compounds:

• Enhanced PHA Production:

  • In P. putida, fadB deletion resulted in a 2.5-fold increase in medium-chain-length polyhydroxyalkanoate (mcl-PHA) production when grown on nitrogen-rich medium supplemented with heptanoate and octanoate

  • Combined deletion of fadBA1 and fadBA2 with other metabolic modifications further enhanced mcl-PHA yields from aromatic compounds

• Mechanistic Basis:

  • FadB deletion prevents the degradation of 3-hydroxyacyl-CoA intermediates

  • These intermediates accumulate and become available for PHA synthase (PhaC)

  • The blockage of β-oxidation forces carbon flux toward anabolic pathways

• Optimization Strategies:

  • Combine fadB deletion with overexpression of PHA biosynthetic genes (phaC1, phaC2)

  • Further enhance by eliminating PHA degradation (phaZ deletion)

  • Strengthen precursor supply by overexpressing phaG and alkK

These strategies can potentially be adapted to P. syringae pv. tomato, though consideration must be given to the specific metabolic network in this organism.

What are the effects of fadB mutations on bacterial motility and biofilm formation in Pseudomonas syringae pv. tomato?

FadB mutations can impact bacterial motility and biofilm formation through several mechanisms:

• Effects on Biosurfactant Production:

  • Fatty acid metabolism influences the production of biosurfactants like syringafactin

  • In P. syringae pv. tomato DC3000, syringafactin is essential for swarming motility

  • Metabolic alterations in β-oxidation can redirect precursors toward or away from biosurfactant synthesis

• Impact on Energy Availability:

  • Motility is energy-intensive; alterations in energy metabolism affect flagellar function

  • FadB mutations may alter ATP availability, influencing flagellar rotation

• Relationship with Regulatory Systems:

  • Fatty acid metabolism interacts with quorum sensing and cyclic-di-GMP signaling

  • These regulatory systems control both motility and biofilm formation

Research has shown that P. syringae pv. tomato DC3000 utilizes at least two different types of motility: flagellum-dependent swarming (requiring syringafactin) and flagellum-independent surface spreading or sliding (also requiring syringafactin) . Alterations in fadB likely affect both processes through changes in metabolic flux toward biosurfactant production.

How does the deletion of fadB affect the production of virulence factors in plant-pathogenic Pseudomonas species?

Deletion of fadB can influence virulence factor production through metabolic remodeling:

• Impact on Quorum Sensing:

  • Fatty acid metabolism provides precursors for acyl homoserine lactone (AHL) synthesis

  • AHLs regulate numerous virulence factors in Pseudomonas species

  • Similar to observations in P. aeruginosa, where fatty acid metabolism genes affect siderophore secretion and quorum-sensing signaling

• Effects on Type III Secretion System:

  • Energy metabolism changes can influence the expression and function of the Type III secretion system

  • This system is crucial for delivering effector proteins into plant cells

• Biosurfactant Production:

  • FadB deletion may increase precursor availability for biosurfactants like syringafactin

  • These compounds contribute to surface motility and potentially plant colonization

• Phytotoxin Production:

  • Metabolic changes might alter resources available for toxin synthesis

  • P. syringae pv. phaseolicola produces phaseolotoxin, and metabolic alterations could impact toxin levels

Transcriptomic studies in P. syringae have shown differential expression of numerous virulence-related genes when grown in plant extracts, indicating complex regulatory networks linking metabolism and virulence .

What strategies can overcome the challenges of low solubility when expressing recombinant fadB protein?

Recombinant fadB proteins often exhibit solubility issues due to their hydrophobic nature and complex structure. Several strategies can improve solubility:

• Expression Optimization:

  • Lower induction temperature (16-20°C)

  • Reduce inducer concentration

  • Use specialized E. coli strains (Arctic Express, C41/C43) designed for membrane-associated proteins

• Fusion Tags:

  • MBP (maltose-binding protein) tag significantly enhances solubility

  • SUMO tag promotes proper folding

  • Thioredoxin fusion for disulfide bond formation

• Buffer Optimization:

  • Include mild detergents (0.05-0.1% Triton X-100 or 0.5-1% CHAPS)

  • Add stabilizing agents (5-10% glycerol, 100-500 mM NaCl)

  • Optimize pH based on protein's theoretical isoelectric point

• Co-expression Approaches:

  • Co-express with chaperones (GroEL/GroES, DnaK/DnaJ)

  • Express with natural binding partners (fadA) to promote complex formation

• Refolding Protocols:

  • If inclusion bodies form, optimize solubilization (8M urea or 6M guanidine HCl)

  • Employ step-wise dialysis for gentle refolding

  • Use additives like L-arginine (0.5-1M) during refolding

When working with membrane-associated proteins from Pseudomonas, periplasmic expression strategies have shown success, as demonstrated with antibody fragments in E. coli using the pComb3 phagemid vector .

How can researchers address genetic redundancy when studying fadB function in Pseudomonas syringae pv. tomato?

Genetic redundancy presents a significant challenge when studying fadB function. Several approaches can effectively address this issue:

• Comprehensive Gene Identification:

  • Perform thorough bioinformatic analysis to identify all fadB homologs

  • Use multiple search algorithms and profile hidden Markov models

  • Analyze synteny to identify operonic structures containing fadB genes

• Single and Combinatorial Knockouts:

  • Create individual gene knockouts to assess specific roles

  • Generate multiple gene deletions to address redundancy

  • Create a complete fadBA deletion strain by targeting all copies sequentially

• Transcriptional Profiling:

  • Use RNA-Seq to determine expression patterns under different conditions

  • Identify conditions where specific fadB homologs are predominantly expressed

  • Analyze co-expression networks to understand functional relationships

• Complementation Studies:

  • Express individual fadB homologs in a complete knockout background

  • Test functional complementation with homologs from other species

  • Use inducible promoters to control expression levels

• Biochemical Characterization:

  • Purify and characterize individual FadB proteins

  • Determine substrate specificities and kinetic parameters

  • Identify unique features that differentiate homologs

This multi-faceted approach has been successful in characterizing redundant systems in P. putida, where multiple fadBA copies were systematically deleted to redirect metabolic flux .

What methods can effectively track the metabolic flux through β-oxidation when studying fadB function?

Tracking metabolic flux through β-oxidation requires specialized techniques:

• Isotopic Labeling:

  • 13C-labeled fatty acids: Track carbon flow through central metabolism

  • Deuterium-labeled substrates: Monitor hydrogen exchange during β-oxidation

  • [³H]acetate radiolabeling: Quantify incorporation into fatty acids and other metabolites

• Mass Spectrometry Approaches:

  • Untargeted metabolomics: Identify unexpected metabolic shifts

  • Targeted analysis: Quantify specific β-oxidation intermediates

  • Flux ratio analysis: Determine relative pathway activities

• Computational Modeling:

  • Flux balance analysis (FBA): Predict optimal flux distributions

  • 13C-metabolic flux analysis (13C-MFA): Quantify intracellular fluxes

  • Kinetic modeling: Simulate dynamic responses to perturbations

• Real-time Monitoring:

  • Biosensors: Develop protein-based sensors for key intermediates

  • Reporter systems: Create transcriptional fusions to pathway-responsive promoters

  • Enzyme assays: Measure activities of pathway enzymes in cell extracts

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Correlate gene expression with metabolite levels and enzyme activities

  • Build comprehensive metabolic models specific to P. syringae

This approach has been successfully implemented in studies of fatty acid metabolism in Pseudomonas species, providing insights into carbon flow through complex metabolic networks .

How do environmental signals regulate fadB expression in plant-associated Pseudomonas species?

The regulation of fadB expression in plant-associated Pseudomonas involves complex networks responsive to environmental cues:

• Nutrient Availability:

  • Carbon catabolite repression controls fadB expression when preferred carbon sources are present

  • Nitrogen limitation can influence fatty acid metabolism gene expression

  • Phosphate availability affects membrane lipid composition and turnover

• Plant-Derived Signals:

  • Plant phenolic compounds may induce or repress fadB expression

  • Studies examining P. syringae transcriptional responses to plant extracts have identified differential expression of metabolic genes in response to host compounds

  • Apoplastic fluid components can trigger specific bacterial metabolic adaptations

• Regulatory Systems:

  • FleQ may regulate both flagellar synthesis and fatty acid metabolism genes

  • Pseudomonas species contain multiple transcription factors that respond to fatty acids and their derivatives

  • Two-component systems sense environmental changes and regulate metabolic gene expression

• Stress Responses:

  • Oxidative stress modulates fatty acid metabolism

  • The extracytoplasmic sensing system involving ChrR (PSPTO_1042) may connect oxidative stress responses to metabolic shifts and motility

  • Temperature fluctuations alter membrane fluidity and trigger compensatory changes in fatty acid metabolism

Understanding these regulatory networks is crucial for predicting bacterial behavior in plant hosts and designing effective intervention strategies.

What is the evolutionary significance of fadB duplication in Pseudomonas genomes?

The evolutionary significance of fadB duplication in Pseudomonas genomes reveals important adaptive strategies:

• Functional Specialization:

  • Duplicated fadB genes have diverged to handle different substrate ranges

  • Specialized paralogs may function optimally under different environmental conditions

  • This specialization allows metabolic flexibility across diverse niches

• Expression Divergence:

  • Different fadB copies are often under distinct regulatory control

  • This permits fine-tuned expression in response to specific environmental signals

  • Temporal separation of expression provides metabolic plasticity

• Horizontal Gene Transfer:

  • Phylogenetic analysis suggests some fadB copies were acquired through horizontal gene transfer

  • This is consistent with the extensive recombination observed in Pseudomonas genomes

  • Acquisition of novel metabolic capabilities may enhance colonization of new niches

• Selective Pressures:

  • Plant-associated Pseudomonas species face unique selection pressures

  • FadB duplication may facilitate adaptation to specific plant hosts

  • The ability to utilize diverse plant-derived fatty acids provides competitive advantages

• Genomic Context:

  • Different fadB paralogs often exist in distinct operonic structures

  • The genomic architecture suggests co-evolution with other metabolic genes

  • This arrangement facilitates coordinated regulation of related metabolic pathways

Comparative genomic analyses across Pseudomonas species have revealed significant genome-wide homologous recombination, particularly in pathways involved in ATP-dependent transport and metabolism of amino acids, bacterial motility, and secretion systems .

How can systems biology approaches integrate multi-omics data to fully characterize fadB function within the metabolic network of P. syringae pv. tomato?

Systems biology approaches can provide a comprehensive understanding of fadB function:

• Multi-omics Data Integration:

  • Genomics: Identify all fadB homologs and their genomic context

  • Transcriptomics: Determine expression patterns under diverse conditions

  • Proteomics: Quantify protein levels and post-translational modifications

  • Metabolomics: Map metabolite profiles and pathway activities

  • Fluxomics: Measure carbon flow through β-oxidation and connected pathways

• Network Analysis:

  • Construct protein-protein interaction networks

  • Build gene regulatory networks from transcriptomic data

  • Develop metabolic models incorporating all β-oxidation reactions

  • Identify key regulatory nodes controlling fatty acid metabolism

• Computational Modeling:

  • Use genome-scale metabolic models to predict phenotypic outcomes

  • Employ machine learning to identify patterns in multi-omics datasets

  • Develop dynamic models capturing temporal aspects of regulation

• Validation Strategies:

  • Test model predictions with targeted experiments

  • Use CRISPR interference to modulate gene expression

  • Employ metabolic engineering to redirect flux and confirm predictions

• Integration with Host Interaction Data:

  • Incorporate plant response data to bacterial infection

  • Model metabolic interactions at the host-pathogen interface

  • Predict metabolic adaptations during infection using dual-organism models