Recombinant Pseudomonas syringae pv. syringae Cardiolipin synthase (cls)

What is cardiolipin synthase (cls) and what role does it play in Pseudomonas syringae?

Cardiolipin synthase catalyzes the synthesis of cardiolipin, an essential phospholipid in bacterial membranes. In Pseudomonas syringae, as in other bacteria, this enzyme mediates the condensation of cytidinediphosphate-diacylglycerol and phosphatidylglycerol to form cardiolipin . This phospholipid is critical for membrane structure and function, particularly in the bacterial inner membrane. Cardiolipin plays important roles in energy metabolism, membrane organization, and potentially in stress responses, which may contribute to P. syringae's adaptability to various environmental conditions. The enzyme typically requires divalent cations for activity and functions optimally under alkaline pH conditions, similar to cardiolipin synthases characterized in other organisms .

How is the cls gene conserved across different strains and pathovars of Pseudomonas syringae?

The cardiolipin synthase gene shows varying degrees of conservation across the P. syringae complex. While specific data on cls gene conservation is limited in the provided search results, we can infer patterns based on general genomic characteristics of P. syringae. The species complex demonstrates considerable genetic diversity across its 13 recognized phylogroups and 23 clades . Certain "housekeeping" genes undergo recombination within the species, which may influence the evolution of genes like cls . The conservation pattern of cls likely follows phylogenetic relationships established through Multi Locus Sequence Typing (MLST) analyses. For accurate assessment of cls gene conservation, researchers should examine the gene sequence within the context of the broader phylogenetic framework established for P. syringae, particularly considering the robust phylogroup classification system based on the citrate synthase (cts) gene .

What expression systems are most effective for producing recombinant P. syringae cardiolipin synthase?

Based on available research data, effective expression systems for recombinant P. syringae cardiolipin synthase include E. coli-based systems with histidine tags for purification purposes . For functional studies, heterologous expression in yeast systems deficient in cardiolipin synthase (such as crd1Δ yeast strains) can be particularly valuable, as this approach has been successfully used for human cardiolipin synthase characterization . When designing expression systems, researchers should consider:

• Codon optimization for the host organism to improve translation efficiency

• The addition of affinity tags (such as His-tags) positioned to minimize interference with enzyme activity

• Inducible promoter systems to control expression levels

• Growth conditions that maximize protein solubility and proper folding

• The potential need for membrane fraction isolation, as cardiolipin synthase is typically membrane-associated

The choice of expression system should be guided by the specific research objectives, whether focused on structural studies, enzymatic characterization, or functional complementation assays.

What are the key structural features of cardiolipin synthase from P. syringae?

P. syringae cardiolipin synthase shares fundamental structural characteristics with other bacterial cardiolipin synthases, though specific structural data for the P. syringae enzyme is limited in the search results. The enzyme likely features:

• Transmembrane domains that anchor it within the bacterial membrane

• Active site residues that coordinate substrate binding and catalysis

• Regions for recognition of cytidinediphosphate-diacylglycerol and phosphatidylglycerol substrates

• Binding sites for essential divalent cations that facilitate catalysis

The recombinant full-length P. syringae pv. syringae cardiolipin synthase spans 479 amino acids , providing sufficient length for multiple functional domains. Researchers should note that, like other phospholipid synthases, the enzyme likely adopts a conformation that positions the active site appropriately with respect to the membrane interface where substrates are recruited and product is released. Structural predictions using homology modeling based on related bacterial cardiolipin synthases would be a valuable approach for researchers investigating this enzyme's structure-function relationships.

What methodologies are most effective for assessing the enzymatic activity of recombinant P. syringae cardiolipin synthase?

For accurate assessment of recombinant P. syringae cardiolipin synthase activity, researchers should implement a multi-faceted approach:

• In vitro activity assays: Using purified mitochondrial fractions (if expressing in yeast) or membrane fractions (if expressing in bacteria) containing the recombinant enzyme, measure the conversion of radiolabeled or fluorescently labeled substrates to cardiolipin. Optimal reaction conditions would include:

  • Alkaline pH buffer system (pH 8.0-9.0)

  • Divalent cations (Mg²⁺, Mn²⁺)

  • Controlled temperature (typically 30-37°C)

  • Substrate concentrations at or above Km values

• Complementation studies: Express the P. syringae cls gene in cardiolipin synthase-deficient organisms (such as crd1Δ yeast strains) to assess functional complementation . This approach allows for in vivo assessment of activity.

• Mass spectrometry analysis: Employ lipidomic approaches to quantify cardiolipin production and characterize the acyl chain composition of newly synthesized cardiolipin.

• Substrate preference determination: Systematically vary the cytidinediphosphate-diacylglycerol and phosphatidylglycerol species to determine substrate preferences, which may differ between species as observed with human cardiolipin synthase .

Each methodological approach should include appropriate positive controls (known active cardiolipin synthases) and negative controls (inactive enzyme variants or reaction mixes lacking essential components).

How does genetic diversity within the P. syringae complex influence cardiolipin synthase function and evolution?

The P. syringae complex exhibits remarkable genetic diversity, comprising at least 13 phylogroups and 23 clades identified through MLST analysis . This diversity likely influences cardiolipin synthase function and evolution in several key ways:

• Adaptive variation: Different environmental niches occupied by P. syringae strains (including agricultural and non-agricultural habitats) may select for variations in membrane phospholipid composition, potentially driving functional diversity in cardiolipin synthase .

• Recombination effects: P. syringae undergoes moderately high rates of recombination across multiple loci, including housekeeping genes . This recombination could contribute to genetic exchange affecting the cls gene, potentially creating functional variants adapted to specific ecological contexts.

• Horizontal gene transfer influence: The presence of plasmids, prophages, and other mobile genetic elements in P. syringae genomes facilitates horizontal gene transfer . While the cls gene is likely chromosomally encoded, its expression and function might be modulated by horizontally acquired elements.

• Evolutionary pressures: Differential selection pressures across diverse habitats could drive lineage-specific adaptations in membrane phospholipid metabolism. Researchers studying cls evolution should examine sequence variations within the phylogenetic framework established for P. syringae, potentially revealing correlations between cls sequence and habitat type or host range.

To investigate these influences, researchers should conduct comparative genomic analyses of cls genes across phylogroups, complemented by functional characterization of diverse variants.

What is the relationship between cardiolipin synthase activity and P. syringae virulence or environmental adaptation?

The relationship between cardiolipin synthase activity and P. syringae virulence or environmental adaptation represents a complex interplay that researchers should investigate through multiple approaches:

• Stress response correlation: Cardiolipin content in bacterial membranes often increases under stress conditions. Researchers should examine whether P. syringae strains modulate cardiolipin synthase activity in response to environmental stressors (temperature, osmotic pressure, pH) or plant defense responses. This could be particularly relevant given that P. syringae occupies diverse habitats including agricultural contexts and water-associated environments .

• Membrane organization and protein function: Cardiolipin creates specialized membrane microdomains that may influence the localization and function of virulence-associated protein complexes. Investigate whether cardiolipin-rich domains co-localize with secretion systems or other virulence machinery in P. syringae.

• Co-infection dynamics: Studies have shown that co-infections with different P. syringae strains influence disease outcomes . Researchers should explore whether cardiolipin synthesis plays a role in competitive fitness during co-infection events.

• Phylogroup-specific patterns: Analyze whether cardiolipin synthase sequence variations correlate with the 13 established phylogroups of P. syringae , potentially revealing adaptive patterns associated with specific ecological niches or host specialization.

Experimental approaches should include creating cls gene knockout or overexpression strains, followed by comprehensive phenotyping across multiple environmental conditions and virulence assays in appropriate plant hosts.

How can structural biology approaches be applied to understand the catalytic mechanism of P. syringae cardiolipin synthase?

Advancing our understanding of P. syringae cardiolipin synthase catalytic mechanisms requires integrating multiple structural biology approaches:

• X-ray crystallography or cryo-EM studies: These techniques can reveal the three-dimensional structure of the enzyme, though membrane protein crystallization presents challenges. Researchers should consider:

  • Using detergent solubilization or nanodiscs to maintain protein stability

  • Employing lipidic cubic phase crystallization methods

  • Creating fusion constructs with crystallization-promoting proteins

  • Capturing different conformational states using substrate analogs or inhibitors

• Site-directed mutagenesis coupled with activity assays: Systematically mutate predicted catalytic residues based on homology models with other cardiolipin synthases. Analyze the impact on:

  • Substrate binding (Km values)

  • Catalytic efficiency (kcat)

  • pH dependence

  • Divalent cation requirements

• Molecular dynamics simulations: Model the enzyme within a phospholipid bilayer to understand:

  • Conformational changes during catalysis

  • Substrate approach pathways

  • Product release mechanisms

  • Membrane interactions that stabilize the enzyme

• HDX-MS (Hydrogen-Deuterium Exchange Mass Spectrometry): This technique can identify flexible regions and conformational changes upon substrate binding without requiring crystallization.

An integrated structural biology approach should aim to answer key questions including:

• How does the enzyme coordinate its two substrates (cytidinediphosphate-diacylglycerol and phosphatidylglycerol)?

• What is the molecular basis for the alkaline pH optimum observed in cardiolipin synthases ?

• How do divalent cations participate in the catalytic mechanism?

• What structural features determine substrate preferences for specific acyl chain compositions?

What methodologies are most effective for investigating P. syringae cls gene regulation under different environmental conditions?

To comprehensively investigate P. syringae cls gene regulation under different environmental conditions, researchers should implement a multi-layered experimental approach:

• Transcriptional analysis:

  • qRT-PCR to quantify cls transcript levels under varying conditions (pH, temperature, osmolarity, nutrient limitation)

  • RNA-seq for genome-wide transcriptional responses, contextualizing cls regulation within broader adaptation networks

  • 5' RACE to precisely map transcription start sites and identify potential alternative promoters

• Promoter characterization:

  • Reporter gene fusions (luciferase, GFP) to monitor promoter activity in real-time

  • Electrophoretic mobility shift assays (EMSAs) to identify transcription factors binding to the cls promoter region

  • ChIP-seq to identify regulatory proteins interacting with the cls locus in vivo

  • DNA footprinting to precisely map protein-DNA interaction sites

• Environmental condition matrix:

  • Create a systematic matrix of environmental variables relevant to P. syringae ecological niches, including:

    • Plant-associated conditions (apoplast-mimicking media)

    • Water cycle-associated conditions (low nutrient, varying temperatures)

    • Stress conditions (oxidative stress, membrane perturbations)

    • Co-infection scenarios with other microbial strains

• Phylogroup comparisons:

  • Compare cls regulation across strains representing different phylogroups to identify conserved and lineage-specific regulatory mechanisms

  • Correlate regulatory patterns with ecological niches and host ranges

This multi-faceted approach will reveal how P. syringae modulates cardiolipin synthesis in response to environmental cues, providing insights into the adaptive significance of cardiolipin in bacterial membrane homeostasis.

What are the methodological considerations for investigating the role of cardiolipin in P. syringae membrane organization and protein localization?

Investigating cardiolipin's role in P. syringae membrane organization and protein localization requires specialized approaches addressing the unique challenges of bacterial membrane biology:

• Visualization techniques:

  • Fluorescent cardiolipin-specific probes (e.g., 10-N-nonyl acridine orange) for live-cell imaging

  • Super-resolution microscopy (STORM, PALM) to visualize cardiolipin domains at nanoscale resolution

  • Correlative light and electron microscopy to link functional observations with ultrastructural features

  • Freeze-fracture electron microscopy to examine membrane organization

• Protein-lipid interaction methods:

  • Crosslinking of cardiolipin to interacting proteins followed by mass spectrometry identification

  • Lipidomic analysis of membrane microdomains isolated by detergent-resistant membrane fractionation

  • Reconstitution of purified proteins into liposomes with defined cardiolipin content to assess functional impacts

  • Microscale thermophoresis or isothermal titration calorimetry to measure binding affinities between cardiolipin and membrane proteins

• Genetic manipulation approaches:

  • Generation of cls gene knockout or conditional expression strains

  • Complementation with cls variants producing cardiolipins with modified acyl chain compositions

  • Introduction of heterologous cardiolipin-binding proteins as biosensors

• Specific membrane function assays:

  • Membrane fluidity measurements using environment-sensitive fluorescent probes

  • Proton permeability assays to assess membrane integrity

  • Protein mobility tracking using fluorescence recovery after photobleaching (FRAP)

  • Assessment of membrane potential using voltage-sensitive dyes

Researchers should specifically investigate whether cardiolipin-enriched domains in P. syringae membranes serve as organization centers for virulence-associated protein complexes, potentially linking membrane composition to pathogenicity mechanisms that vary across the diverse P. syringae phylogroups .