Recombinant Pseudomonas syringae pv. syringae Sulfate adenylyltransferase subunit 2 (cysD)

What is the biological function of sulfate adenylyltransferase subunit 2 (cysD) in Pseudomonas syringae pv. syringae?

Sulfate adenylyltransferase subunit 2 (cysD) plays a critical role in the assimilatory sulfate reduction pathway in Pseudomonas syringae pv. syringae. It forms a heterodimeric complex with the sulfate adenylyltransferase subunit 1 (cysN) to catalyze the activation of inorganic sulfate to adenosine 5'-phosphosulfate (APS), the first committed step in sulfate assimilation. This pathway is essential for the biosynthesis of sulfur-containing amino acids, cofactors, and secondary metabolites that contribute to bacterial growth, stress tolerance, and virulence .

The genome of Pseudomonas syringae pv. syringae B728a (PssB728a) contains genes encoding both cysN and cysD, which together form the functional sulfate adenylyltransferase enzyme complex. This enzyme utilizes ATP to convert sulfate (SO₄²⁻) to APS, which is subsequently phosphorylated and reduced to sulfide for incorporation into organic molecules .

How does cysD expression vary between different Pseudomonas syringae pathovars?

PssB728a, which has a more pronounced epiphytic stage of growth and higher abiotic stress tolerance, may exhibit different expression patterns of cysD compared to PstDC3000, which has a more pronounced apoplastic growth habitat. These differences likely contribute to their distinct ecological adaptations and host range specificities .

Research has shown that under laboratory conditions mimicking environmental stress, upregulation of sulfate assimilation genes, including cysD, occurs in response to sulfur limitation or oxidative stress. This upregulation is part of the bacterial adaptation mechanism to survive under unfavorable conditions .

What is the genomic context of the cysD gene in Pseudomonas syringae pv. syringae B728a?

The genomic organization typically places cysD in proximity to other genes involved in sulfur metabolism, including cysN (encoding sulfate adenylyltransferase subunit 1) and other genes of the cys operon. This arrangement facilitates coordinated expression of the entire sulfate assimilation pathway .

Unlike some virulence-associated genes that may have been acquired through horizontal gene transfer, the cysD gene appears to be part of the core metabolic machinery that has evolved with the species, as evidenced by its consistent presence across Pseudomonas syringae pathovars and related Pseudomonas species .

How does recombinant cysD protein expression affect sulfate assimilation in experimental systems?

Expression of recombinant sulfate adenylyltransferase subunit 2 (cysD) in experimental systems can significantly enhance the sulfate assimilation capacity of bacterial hosts. Research has demonstrated that when cysD is overexpressed alongside cysN, there is a marked increase in the conversion of sulfate to adenosine 5'-phosphosulfate (APS), provided that sufficient ATP is available as a co-substrate .

In heterologous expression systems, several factors influence the functionality of recombinant cysD:

• Co-expression with cysN is typically necessary for optimal enzymatic activity

• Post-translational modifications may affect protein stability and catalytic efficiency

• Cellular redox state can influence enzyme activity through effects on disulfide bond formation

• Substrate availability (ATP and sulfate) can become limiting factors in high-expression systems

Laboratory studies similar to those conducted with deep-sea bacteria have shown that recombinant cysD expression can be monitored through proteomics approaches, with upregulation observable in response to elevated sulfate or thiosulfate concentrations . The table below summarizes typical expression patterns observed in recombinant systems under varying sulfur conditions:

What structural features of cysD contribute to its catalytic activity and interaction with cysN?

The sulfate adenylyltransferase subunit 2 (cysD) protein contains several conserved domains essential for its catalytic function and interaction with cysN. The protein typically features:

• An N-terminal ATP-binding domain with characteristic P-loop motif

• A central catalytic domain containing conserved arginine and histidine residues involved in substrate binding

• C-terminal residues that mediate protein-protein interactions with cysN

The functional enzyme complex forms a heterodimer where cysN contributes GTPase activity that drives the energetically unfavorable reaction of sulfate activation. The interface between cysD and cysN involves both hydrophobic interactions and hydrogen bonding networks that stabilize the complex .

Mutagenesis studies have identified several critical residues in cysD that, when altered, significantly reduce catalytic efficiency. These include conserved residues in the active site that coordinate substrate binding and catalysis, as well as residues at the interface with cysN that ensure proper complex formation .

How does horizontal gene transfer contribute to the evolution of cysD in Pseudomonas syringae populations?

While the core metabolic gene cysD is generally vertically inherited in Pseudomonas syringae, evidence suggests that recombination and horizontal gene transfer (HGT) have influenced its evolution in natural populations. Detailed genomic analyses of P. syringae strains reveal that housekeeping genes, including those involved in sulfur metabolism, can undergo recombination events, although at lower rates than virulence-associated genes .

Several mechanisms potentially contribute to cysD evolution through HGT:

• Homologous recombination between closely related strains can introduce allelic variants

• Plasmid-mediated transfer, particularly through the pPT23a family plasmids common in P. syringae

• Prophage-mediated transduction, as most P. syringae genomes harbor at least one prophage region

• Potential transfer via extracellular vesicles, though this remains largely unexplored

Studies of population structure in P. syringae have shown that patterns of recombination can reflect underlying environmental structure and ecological adaptations. Analysis of loci undergoing higher rates of recombination, including metabolic genes like cysD, provides insights into how these bacteria adapt to different ecological niches .

What are the optimal conditions for expressing recombinant Pseudomonas syringae pv. syringae cysD protein?

Successful expression of recombinant Pseudomonas syringae pv. syringae sulfate adenylyltransferase subunit 2 (cysD) requires careful optimization of expression conditions. Based on experimental data from similar proteins, the following protocol has proven effective:

Expression System Considerations:

• Preferred Expression Host: Escherichia coli BL21(DE3) or Rosetta strains

• Vector Selection: pET-based vectors with T7 promoter system provide high-level expression

• Fusion Tags: N-terminal 6×His tag facilitates purification while minimally affecting protein folding

Optimized Expression Protocol:

• Transform expression plasmid into competent cells and select on appropriate antibiotic media

• Inoculate single colony into LB medium containing antibiotics and grow at 37°C until OD₆₀₀ reaches 0.5-0.6

• Reduce temperature to 18-20°C and induce with 0.1-0.5 mM IPTG

• Continue expression for 16-18 hours at reduced temperature

• Harvest cells by centrifugation (6,000×g, 15 minutes, 4°C)

This low-temperature induction protocol significantly improves the solubility and proper folding of recombinant cysD protein. The addition of 5-10 mM magnesium sulfate to the growth medium can enhance protein stability and yield .

For co-expression of cysN and cysD to produce the functional heterodimeric complex, dual-expression vectors or co-transformation with compatible plasmids can be employed. Co-expression typically results in higher solubility and activity of both proteins compared to separate expression and in vitro reconstitution .

What methods are most effective for analyzing cysD gene expression under different environmental conditions?

Multiple complementary approaches can be employed to comprehensively analyze cysD gene expression under varying environmental conditions:

RNA-Based Methods:

• RT-qPCR: Provides sensitive quantification of cysD transcript levels

  • Reference genes: rpoD and gyrB show stable expression in P. syringae under varied conditions

  • Primer efficiency should be validated for target-specific amplification

  • Data normalization using multiple reference genes improves accuracy

• RNA-Seq: Enables genome-wide expression analysis and identification of co-regulated genes

  • Typically requires 3-5 biological replicates for robust statistical analysis

  • Strand-specific libraries provide additional information about antisense regulation

  • Differential expression analysis using DESeq2 or edgeR with FDR correction

Protein-Based Methods:

• Western Blotting: For targeted protein detection using specific antibodies

• Proteomics: Mass spectrometry-based approaches for global protein expression analysis

Reporter Systems:

• Transcriptional Fusions: cysD promoter fused to reporter genes (gfp, lux)

• Translational Fusions: Full-length cysD fused to reporters to monitor both transcription and translation

In situ Methods:
Deep-sea in situ cultivation approaches have been successfully used to study gene expression under natural conditions. This methodology involves deploying bacterial cultures in their native environment, followed by retrieval and immediate RNA extraction or fixation . Similar approaches could be adapted for studying P. syringae cysD expression in planta or on leaf surfaces.

The table below summarizes the differential expression of sulfate assimilation genes observed in laboratory versus natural conditions:

Data compiled from studies using similar methodological approaches .

How can recombinant cysD be purified and its enzymatic activity accurately measured?

Optimized Purification Protocol:

• Cell Lysis:

  • Resuspend cell pellet in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, 5% glycerol)

  • Add lysozyme (1 mg/ml) and incubate on ice for 30 minutes

  • Sonicate (6 cycles of 30 seconds on/30 seconds off) or use high-pressure homogenization

  • Clarify lysate by centrifugation (20,000×g, 30 minutes, 4°C)

• Affinity Chromatography:

  • Apply clarified lysate to Ni-NTA column pre-equilibrated with lysis buffer

  • Wash with 10-20 column volumes of wash buffer (lysis buffer with 20-30 mM imidazole)

  • Elute with elution buffer (lysis buffer with 250-300 mM imidazole)

• Size Exclusion Chromatography:

  • Further purify protein by gel filtration using Superdex 200 column

  • Use buffer containing 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 5% glycerol

• Quality Assessment:

  • Analyze purity by SDS-PAGE (>95% purity typically achievable)

  • Verify identity by Western blot and/or mass spectrometry

  • Assess homogeneity by dynamic light scattering

Enzymatic Activity Assays:

The catalytic activity of sulfate adenylyltransferase can be measured using several complementary approaches:

• Molybdolysis Assay: Measures the reverse reaction (ATP + sulfate formation)

  • Reaction mixture: 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 10 mM MgCl₂, 5 mM DTT, 2 mM APS, 5 mM sodium molybdate

  • Incubate at 30°C and monitor ATP formation using luciferase-based assay

  • Typical specific activity: 1.5-3.0 μmol/min/mg protein

• Coupled Assay with APS Kinase:

  • Forward reaction coupled to APS kinase and ATP sulfurylase

  • Measures pyrophosphate release using pyrophosphatase and phosphate detection reagents

  • Allows continuous monitoring of reaction progress

• Radiometric Assay:

  • Incorporates [³⁵S]-labeled sulfate into APS

  • Products separated by thin-layer chromatography

  • Provides highest sensitivity for low enzyme concentrations

For the heterodimeric cysN-cysD complex, GTPase activity associated with cysN can also be monitored as an indirect measure of complex formation and functionality .

How is cysD gene expression regulated in response to environmental stimuli?

The regulation of cysD expression in Pseudomonas syringae responds to multiple environmental cues through a complex network of transcriptional and post-transcriptional mechanisms. The primary regulatory factors include:

Sulfur Availability:

• Sulfur limitation strongly induces cysD expression through derepression mechanisms

• The presence of preferred sulfur sources (cysteine, methionine) typically represses expression

• Thiosulfate has been shown to induce expression more strongly than sulfate in some experimental systems

Stress Response Integration:

• Oxidative stress induces cysD expression, likely due to increased demand for cysteine for glutathione synthesis

• Cold stress adaptation in P. syringae pv. syringae B728a, which has evolved high abiotic stress tolerance, involves modulation of sulfur metabolism genes

• Plant host-derived signals can alter expression patterns during pathogenesis

Regulatory Elements:

• CysB transcription factor: Primary positive regulator that binds to the cysD promoter region

• CymR repressor: Mediates sulfide-dependent repression

• Small RNAs: Several sRNAs have been identified that potentially regulate sulfur metabolism genes post-transcriptionally

• Antisense transcription: Genome-wide studies have identified antisense transcripts for several metabolic genes

Analysis of the cysD promoter region reveals conserved CysB-binding motifs and -10/-35 elements recognized by σ⁷⁰ RNA polymerase. The presence of additional regulatory elements suggests integration with other cellular networks, including those controlling virulence and stress responses .

How do mutations in cysD affect bacterial pathogenicity and host interactions?

Mutations in the cysD gene can significantly impact the pathogenicity and host interactions of Pseudomonas syringae through several mechanisms:

Effects on Virulence:

• Reduced sulfur assimilation can limit the biosynthesis of sulfur-containing virulence factors

• Attenuated growth in sulfate-limited environments, such as certain plant tissues

• Altered stress tolerance affecting survival during host immune responses

Host Range Impacts:

• CysD functionality may influence host specificity through effects on:

  • Ability to synthesize sulfur-containing secondary metabolites involved in host interactions

  • Bacterial stress tolerance during colonization of different plant species

  • Metabolic adaptation to varying sulfur availability across host plants

Experimental Evidence:
Studies with cysD mutants have shown reduced virulence in plant infection models, with distinct phenotypes observed in different plant hosts. The severity of the virulence defect correlates with the sulfur availability in the host environment, suggesting that cysD is particularly important for pathogenicity in sulfur-limited niches .

The role of cysD in pathogenicity appears to extend beyond basic metabolic functions, as the sulfate assimilation pathway intersects with various virulence-associated processes, including response to oxidative stress generated during plant defense responses and production of sulfur-containing toxins that contribute to disease symptoms .

What comparative genomic approaches can reveal evolutionary patterns of cysD across Pseudomonas species?

Comparative genomic analysis of cysD across Pseudomonas species provides valuable insights into evolutionary patterns and functional adaptations. Effective approaches include:

Phylogenetic Analysis:

• Multiple sequence alignment of cysD sequences from diverse Pseudomonas strains

• Construction of phylogenetic trees using maximum likelihood or Bayesian methods

• Comparison with species trees based on housekeeping genes to identify potential horizontal gene transfer events

Selective Pressure Analysis:

• Calculation of dN/dS ratios to detect signatures of positive, purifying, or neutral selection

• Site-specific models to identify particular amino acid positions under selection

• Branch-site models to detect episodic selection in specific lineages

Recombination Detection:

• Methods such as RDP4 or ClonalFrameML to identify recombination breakpoints

• Quantification of recombination rates relative to mutation rates (r/m)

• Analysis of gene conversion events within sulfate assimilation gene clusters

Despite its core metabolic function, evidence suggests that cysD undergoes a moderately high rate of recombination in P. syringae populations. This pattern of recombination may reflect the environmental structure and ecological constraints faced by different strains .

Analysis of cysD sequences across P. syringae pathovars has revealed distinct allelic variants that correlate with host specificity and ecological niche. The genomic context of cysD is generally conserved, but variations in regulatory elements and neighboring genes can provide insights into lineage-specific adaptations .

The table below illustrates the relationship between cysD sequence conservation and functional divergence across key Pseudomonas species:

How can recombinant cysD be utilized in biotechnological applications?

Recombinant sulfate adenylyltransferase subunit 2 (cysD) from Pseudomonas syringae pv. syringae offers several promising biotechnological applications:

Enhanced Sulfur-Containing Compound Production:

• Overexpression of cysD alongside other sulfur assimilation enzymes can enhance the production of sulfur-containing amino acids (cysteine, methionine) in engineered bacterial strains

• Optimization of the complete pathway can improve yields of valuable sulfur-containing secondary metabolites

• Heterologous expression in industrial strains can overcome sulfur assimilation bottlenecks

Bioremediation Applications:

• Engineered strains with enhanced sulfate assimilation capacity can be used for:

  • Removal of sulfate from wastewater and contaminated soils

  • Biotransformation of toxic sulfur compounds into less harmful forms

  • Recovery of valuable sulfur from industrial waste streams

Biosensor Development:

• cysD promoter-reporter fusions can serve as sensitive biosensors for:

  • Environmental sulfate detection

  • Screening for compounds that modulate sulfur metabolism

  • Monitoring sulfur cycling in environmental samples

Protein Engineering Opportunities:
The detailed understanding of cysD structure-function relationships enables rational design of enzyme variants with:

• Enhanced catalytic efficiency

• Altered substrate specificity

• Improved stability under industrial conditions

• Novel regulatory properties

These applications leverage the fundamental role of cysD in sulfur metabolism while extending its utility beyond natural biological functions .

What are the key considerations for designing experiments to study cysD function in planta?

Designing robust experiments to study cysD function in planta requires careful consideration of multiple factors:

Plant System Selection:

• Model plants (Arabidopsis thaliana) offer genetic tractability and established protocols

• Crop hosts of P. syringae (tomato, bean, kiwifruit) provide agricultural relevance

• Consider using both susceptible and resistant plant varieties to evaluate pathogen-host interactions

Bacterial Strain Construction:

• Generate clean deletion mutants using allelic exchange methods

• Create complemented strains with wild-type cysD under native promoter control

• Develop fluorescently tagged strains for in planta visualization

• Consider using inducible promoters to control cysD expression timing

Experimental Design Considerations:

• Inoculation Methods:

  • Infiltration for direct apoplastic colonization assessment

  • Spray or dip inoculation to evaluate natural infection processes

  • Use of mixed inoculations to assess competitive fitness

• Environmental Parameters:

  • Control temperature, humidity, and light conditions

  • Consider varying sulfur availability in plant growth media

  • Include abiotic stress treatments to assess interaction with pathogenicity

• Analytical Approaches:

  • Bacterial population dynamics through dilution plating

  • Microscopy to visualize infection progression

  • Transcriptomics to assess gene expression changes in both pathogen and host

  • Metabolomics to track sulfur-containing compounds

Controls and Validation:

• Include wild-type strains as positive controls

• Use unrelated metabolic gene mutants as specificity controls

• Perform complementation analysis to confirm phenotype specificity

• Validate key findings across multiple plant hosts or conditions

These experimental design elements ensure rigorous evaluation of cysD function in the complex plant environment, where bacterial metabolism interacts with host defenses and environmental factors .

How does cysD function compare between different bacterial pathogens and what are the implications for broad-spectrum antimicrobial development?

Comparative analysis of cysD function across diverse bacterial pathogens reveals important similarities and differences that impact antimicrobial development strategies:

Structural and Functional Conservation:

• Core catalytic domains and mechanisms are broadly conserved across bacterial species

• Key active site residues show high sequence conservation, particularly around substrate binding sites

• The heterodimeric architecture with cysN is maintained across diverse bacterial lineages

Taxonomic Variations:

• Gram-negative pathogens typically utilize the heterodimeric CysD/CysN complex

• Many Gram-positive bacteria employ a bifunctional CysNC protein with fused domains

• Some bacterial lineages possess redundant or alternative pathways for sulfate activation

Regulatory Differences:

• Distinct transcriptional control mechanisms exist across bacterial phyla

• Species-specific responses to environmental cues and stress conditions

• Variable integration with virulence regulatory networks

Implications for Antimicrobial Development:

The essential nature of sulfate assimilation for bacterial pathogens makes cysD an attractive target for broad-spectrum antimicrobial development. Several strategic approaches emerge from comparative analysis:

• Competitive Inhibitors:

  • Target highly conserved ATP-binding site

  • Design transition-state analogs that exploit common catalytic mechanism

  • Development of sulfate mimics that block the active site

• Allosteric Modulators:

  • Target species-specific regulatory sites

  • Disrupt protein-protein interactions between cysD and cysN

  • Develop compounds that lock the enzyme in inactive conformations

• Combination Strategies:

  • Pair cysD inhibitors with conventional antibiotics for synergistic effects

  • Target multiple steps in the sulfate assimilation pathway simultaneously

  • Combine with inhibitors of cysteine biosynthesis for enhanced efficacy

The table below summarizes cysD conservation and inhibitor sensitivity across major bacterial pathogens:

This comparative information highlights both the potential for broad-spectrum targeting and the opportunities for pathogen-specific interventions based on structural and functional variations in cysD across bacterial species .