Recombinant Pseudomonas syringae pv. tomato Molybdenum cofactor guanylyltransferase (mobA)

What recombineering approaches are most effective for genetic manipulation of the mobA gene in Pseudomonas syringae pv. tomato?

Recombineering in Pseudomonas syringae pv. tomato requires species-specific recombination systems for optimal efficiency. The RecTE system identified in P. syringae pv. syringae B728a has been demonstrated to function effectively in P. syringae pv. tomato DC3000 .

For targeted manipulation of the mobA gene, the following methodology is recommended:

• Single-stranded DNA recombination: When making point mutations or small insertions in mobA, the Pseudomonas RecT homolog alone is sufficient. This protein promotes recombination of single-stranded DNA oligonucleotides with the bacterial chromosome .

• Double-stranded DNA recombination: For larger modifications such as gene replacements or insertions, both the RecT and RecE homologs should be expressed together. The RecE homolog provides exonuclease activity that processes double-stranded DNA, while RecT mediates strand invasion and annealing .

Experimental Protocol:

• Clone the P. syringae recT (recTpsy) and recTE genes into an expression vector such as pUCP24/47

• Transform the expression vector into P. syringae pv. tomato

• Design recombination substrates with 40-50 bp homology arms flanking the mobA target region

• Electroporate the linear DNA substrate into cells expressing the recombination proteins

• Select for recombinants using appropriate markers

• Use counterselection with sacB to eliminate the expression vector after recombination

This approach has shown greater success in Pseudomonas species compared to heterologous systems like lambda Red, which demonstrates the importance of using species-compatible recombination machinery .

How does Pseudomonas syringae pv. tomato infection progress in plant hosts, and how might mobA function relate to this process?

Pseudomonas syringae pv. tomato infection follows a specific progression that may involve mobA-related metabolic processes:

• Entry phase: The bacteria must first locate and enter the plant apoplast. This process is driven by chemotaxis towards plant-derived compounds, with specific chemoreceptors like PsPto-PscC responding to plant signals such as GABA and l-Pro .

• Colonization phase: Once inside the apoplast, bacteria establish colonies and initiate pathogenic activities.

• Virulence expression: The bacteria deploy virulence factors that contribute to disease development.

While the provided research doesn't specifically address mobA's role, molybdenum cofactor-dependent enzymes often participate in metabolic processes that may influence bacterial fitness during infection. The enzyme mobA catalyzes the conversion of molybdopterin to molybdenum cofactor, which is essential for the activity of several enzymes involved in nitrate reduction, sulfur metabolism, and other processes that might contribute to bacterial survival in the plant environment.

Data from host infection studies:

Researchers studying mobA function could adopt similar experimental designs, comparing wild-type and mobA mutant strains at different timepoints to assess the contribution of this enzyme to infection dynamics.

What is the optimal expression system for producing recombinant Pseudomonas syringae pv. tomato Molybdenum cofactor guanylyltransferase (mobA)?

Based on successful recombinant protein expression strategies for Pseudomonas proteins, the following approach is recommended:

Vector selection and construction:

• Use a broad-host-range vector like pUCP24, which has been successfully employed for expression in Pseudomonas species .

• Replace native promoters with constitutive promoters such as the BAD promoter with the constitutive nptII promoter for consistent expression .

• Include a Gateway cassette for efficient cloning of the mobA gene .

Expression optimization protocol:

• PCR amplify the mobA gene from P. syringae pv. tomato genomic DNA using primers that add appropriate restriction sites

• Clone the amplified gene into the expression vector

• Transform the constructed vector into the expression host

• Test expression under various conditions (temperature, induction time, media composition)

• Include a purification tag (His-tag or GST) for downstream purification

Expression host considerations:

• For functional studies: Use a Pseudomonas strain with a mobA deletion to avoid interference from native protein

• For structural studies: E. coli expression systems may provide higher yields, but folding and activity should be carefully verified

How can the RecTE recombineering system be optimized for creating precise modifications in the mobA gene?

Advanced recombineering of the mobA gene can benefit from these optimized approaches:

• Homology length optimization: While the RecTE system can work with relatively short homology regions (40-50 bp), efficiency increases with longer homology arms. For critical modifications, consider using homology regions of 500-1000 bp .

• Selection strategy design:

  • For gene deletions: Replace mobA with a selectable marker

  • For point mutations: Incorporate a selectable marker near the desired mutation, then remove it in a second recombineering step

  • For scarless modifications: Use counterselectable markers like sacB that allow selection for marker loss

• Expression timing control:

  • Temporary expression of RecTE proteins reduces potential off-target recombination

  • The pUCP24 vector containing sacB allows for counterselection to cure the plasmid after recombination is complete

Experimental design table for mobA modifications:

The recombineering efficiency can be quantitatively assessed through a recombination frequency assay, allowing researchers to optimize their protocols for the specific mobA modifications desired .

What methods are most effective for analyzing the phenotypic impacts of mobA mutations in Pseudomonas syringae pv. tomato during plant infection?

When investigating the functional role of mobA in P. syringae pv. tomato pathogenicity, comprehensive phenotypic analysis requires multiple approaches:

• Entry and colonization assays:

  • Spray-inoculation followed by bacterial recovery at 2 hours post-inoculation to quantify entry efficiency

  • Measurement of bacterial populations at later timepoints (e.g., 6 days) to assess in planta growth

  • Comparison between spray-inoculation and direct infiltration to distinguish between entry and growth phenotypes

• Molecular characterization of infection dynamics:

  • RT-qPCR analysis of virulence gene expression in wild-type versus mobA mutant strains

  • Transcriptomic profiling to identify downstream effects of mobA mutation

  • Metabolomic analysis to detect changes in molybdenum cofactor-dependent pathways

• Complementation studies:

  • Expression of wild-type mobA gene in trans to confirm phenotype specificity

  • Domain-specific mutations to identify critical regions for enzyme function

  • Heterologous expression of mobA from other bacterial species to assess functional conservation

Experimental design for plant infection assays:

This comprehensive approach allows researchers to distinguish between effects on entry, growth, and virulence expression, providing mechanistic insights into mobA function during infection .

What are the common challenges in obtaining active recombinant Molybdenum cofactor guanylyltransferase (mobA) and how can they be addressed?

Researchers working with recombinant mobA often encounter several challenges:

• Protein solubility issues:

  • Molybdenum cofactor guanylyltransferase may form inclusion bodies when overexpressed

  • Mitigation strategy: Optimize expression conditions (lower temperature, 16-20°C; lower IPTG concentration, 0.1-0.5 mM; rich media supplementation)

  • Alternative approach: Express as a fusion protein with solubility-enhancing tags (MBP, SUMO, TrxA)

• Cofactor incorporation:

  • Proper folding and activity may require molybdopterin binding during expression

  • Solution: Supplement expression media with molybdate (1-10 μM sodium molybdate)

  • Consider co-expression with molybdopterin biosynthesis genes if necessary

• Enzymatic activity assay development:

  • Direct assays for mobA activity can be technically challenging

  • Approach: Use coupled enzyme assays that monitor the conversion of GTP to molybdopterin guanine dinucleotide

  • Alternative: Develop a complementation assay using a mobA-deficient bacterial strain

• Protein stability during purification:

  • mobA may be sensitive to oxidation during purification procedures

  • Mitigation: Include reducing agents (DTT or β-mercaptoethanol) in all buffers

  • Consider anaerobic purification techniques for optimal enzyme activity preservation

How can the RecTE recombineering system from Pseudomonas syringae be adapted for difficult genetic modifications?

The RecTE recombineering system from Pseudomonas syringae offers powerful capabilities for genetic modification, but challenging modifications require specialized approaches:

• For modifications with toxic intermediates:

  • Tightly regulated expression systems are essential

  • Use the BAD promoter system for stringent control of recombination protein expression

  • Implement two-step recombineering strategies that avoid toxic intermediate states

• For large insertions or replacements:

  • Standard recombineering efficiency decreases with insert size

  • Use both RecE and RecT proteins together, as they are required for efficient double-stranded DNA recombination

  • Increase homology arm length proportionally to insert size (e.g., 1kb homology for large inserts)

  • Consider implementing selection schemes that place selective pressure throughout the recombination region

• For precise edits without markers:

  • Implement CRISPR-Cas9 in combination with RecTE recombineering

  • Design a two-step process where the first recombination incorporates a counterselectable marker, and the second removes it

  • Use the sacB counterselection system, which has been validated in Pseudomonas species

Recombineering efficiency data for different modification types:

These efficiency values are approximate and based on similar recombineering systems, as specific data for mobA modifications in P. syringae pv. tomato would require experimental determination .

How might mobA function in Pseudomonas syringae pv. tomato relate to plant immune response evasion?

Emerging research suggests potential connections between molybdenum cofactor-dependent enzymes and bacterial virulence that researchers should consider:

• Nitrate metabolism and plant defense evasion:

  • Molybdenum cofactor is essential for nitrate reductase activity

  • Hypothesis: mobA-dependent enzymes may help bacteria metabolize nitrite, potentially interfering with plant nitric oxide (NO) signaling during immune responses

  • Experimental approach: Compare NO levels in plant tissues infected with wild-type versus mobA mutant strains

• ROS detoxification pathways:

  • Several molybdenum-containing enzymes participate in redox reactions

  • Hypothesis: mobA-dependent enzymes may contribute to detoxification of reactive oxygen species produced during plant defense responses

  • Methodology: Measure survival of wild-type versus mobA mutant strains when exposed to oxidative stress in vitro and in planta

• Intersection with chemotaxis and entry mechanisms:

  • Plant GABA and l-Pro levels increase during infection and regulate defense responses

  • Research question: Do mobA-dependent metabolic pathways interact with bacterial chemotaxis systems that sense these plant compounds?

  • Experimental design: Analyze chemotactic responses of mobA mutants toward plant-derived compounds like GABA and l-Pro

What structural and functional insights can genomic analysis provide about mobA in Pseudomonas syringae pv. tomato?

Genomic analysis can provide valuable insights for researchers working with mobA:

• Comparative genomic approaches:

  • Whole-genome sequence analysis of P. syringae pathovars reveals evolutionary relationships and functional adaptations

  • Methodology: Compare mobA gene sequences, promoter regions, and genomic context across multiple P. syringae pathovars to identify selection pressures and functional importance

  • Analysis tool recommendation: Use progressive Mauve alignment for multi-genome comparisons of the mobA region

• Structural prediction and domain analysis:

  • Homology modeling based on crystal structures of mobA from other organisms

  • Identification of catalytic residues through sequence conservation analysis

  • Structure-guided mutagenesis to validate functional predictions

• Transcriptional regulation investigation:

  • Analysis of mobA promoter regions across Pseudomonas species

  • ChIP-seq to identify transcription factors regulating mobA expression

  • RNA-seq under various conditions to map the mobA regulon

Genomic comparison of mobA across Pseudomonas species:

This genomic comparison would need to be experimentally determined for specific research applications.

How can high-throughput approaches accelerate research on mobA function in Pseudomonas syringae pv. tomato?

Modern high-throughput methodologies offer powerful approaches for investigating mobA function:

• Transposon sequencing (Tn-seq) applications:

  • Create a saturated transposon library in wild-type and mobA mutant backgrounds

  • Identify genetic interactions by screening for differential fitness effects between backgrounds

  • Methodology: Compare growth/survival of the libraries under various conditions (plant infection, oxidative stress, nutrient limitation)

• Metabolomics integration:

  • Profile metabolic changes in mobA mutants using LC-MS/MS

  • Focus on molybdenum cofactor-dependent pathways (nitrate metabolism, sulfur oxidation)

  • Data analysis: Use pathway enrichment analysis to identify key affected metabolic networks

• Protein-protein interaction mapping:

  • BioID or proximity labeling approaches to identify interacting partners of mobA

  • Bacterial two-hybrid screening to map the mobA interactome

  • Verification: Co-immunoprecipitation and reciprocal pull-down assays

• CRISPR interference screens:

  • Deploy CRISPRi libraries targeting the P. syringae pv. tomato genome in wild-type and mobA mutant backgrounds

  • Identify synthetic lethal and synthetic rescue interactions

  • Application: Map genetic pathways dependent on or compensating for mobA function

The integration of these high-throughput approaches can accelerate discovery by generating comprehensive datasets that reveal the functional context of mobA in bacterial physiology and pathogenesis.