Recombinant Pseudomonas syringae pv. tomato Cardiolipin synthase (cls)

What is Cardiolipin Synthase (CLS) and what is its role in Pseudomonas syringae pv. tomato?

Cardiolipin synthase (CLS) is an enzyme that catalyzes the final step in cardiolipin synthesis by transferring a phosphatidyl residue from cytidine diphosphate-diacylglycerol (CDP-DAG) to phosphatidylglycerol (PG) . In Pseudomonas syringae pv. tomato, which causes bacterial speck disease in tomato plants, CLS plays a crucial role in maintaining membrane integrity and function . The enzyme is particularly important for bacterial survival under various environmental stresses that pathogens encounter during host colonization and infection processes.

Functionally, CLS contributes to the formation of cardiolipin, a dimeric phospholipid that constitutes a significant component of bacterial membranes, particularly in the inner mitochondrial membrane of eukaryotes . In P. syringae pv. tomato, cardiolipin affects membrane fluidity, stability, and the organization of respiratory chain complexes, thus influencing energy metabolism and adaptability to changing environmental conditions during pathogenesis.

Unlike the extensively characterized human cardiolipin synthase (hCLS1), which has been shown to localize to mitochondria and is highly expressed in metabolically active tissues, the bacterial CLS in P. syringae has unique properties that make it relevant to understanding bacterial persistence and virulence mechanisms in plant hosts .

How does recombinant P. syringae pv. tomato CLS differ from native CLS?

Recombinant P. syringae pv. tomato CLS differs from the native enzyme in several important aspects that researchers must consider. Recombinant CLS is typically produced by expressing the cls gene in heterologous expression systems such as Escherichia coli or eukaryotic cell lines like COS-7 cells . This approach allows for protein purification and characterization outside the native bacterial environment.

The recombinant enzyme often includes additional features not present in the native form, such as affinity tags (His-tags, GST-tags) that facilitate purification, or reporter proteins that enable visualization and tracking of the enzyme in experimental settings. These modifications can potentially affect enzyme kinetics, substrate specificity, or structural properties compared to the native enzyme. Researchers have demonstrated that recombinant CLS enzymes can catalyze the synthesis of cardiolipin efficiently in vitro using CDP-DAG and PG as substrates, similar to findings with human CLS1 expressed in COS-7 cells .

Expression levels of recombinant CLS can be manipulated to exceed native concentrations, which has been shown to significantly increase cardiolipin synthesis in transfected cells . This overexpression system provides valuable insights into enzyme function but may not perfectly replicate the natural activity levels or regulatory mechanisms present in P. syringae pv. tomato.

What are the preferred methods for detecting viable P. syringae pv. tomato cells in experimental settings?

Detection of viable P. syringae pv. tomato cells is critical for accurate experimental results when studying bacterial-host interactions or evaluating antimicrobial treatments. The propidium monoazide-quantitative PCR (PMA-qPCR) assay has emerged as a particularly effective method for this purpose . This technique selectively quantifies viable bacterial cells by using PMA to bind to the chromosomal DNA of dead cells, thereby preventing DNA amplification during qPCR.

For optimal results with the PMA-qPCR assay, researchers should use the Pst3F/Pst3R primer pair, which targets the hrpZ gene specific to P. syringae pv. tomato . The assay's detection limit has been established at 10² CFU/ml in bacterial suspensions and 11.86 CFU/g in artificially contaminated tomato seed, making it sufficiently sensitive for most experimental applications .

In addition to PMA-qPCR, traditional culture-based methods remain valuable for confirming bacterial viability through demonstration of metabolic activity and cell division capability. These methods typically involve plating bacterial suspensions on selective media such as King's B medium supplemented with appropriate antibiotics to quantify colony-forming units (CFU).

For researchers conducting experiments with naturally contaminated plant material, it is noteworthy that viable P. syringae pv. tomato cells have been quantified at infestation levels of approximately 10² to 10⁴ CFU/g in tomato seed samples . This baseline information helps in experimental design and interpretation of results in pathogen-host interaction studies.

How can researchers express and purify recombinant P. syringae pv. tomato CLS for in vitro studies?

Expression and purification of recombinant P. syringae pv. tomato CLS requires a systematic approach involving several critical steps. Researchers typically begin by PCR-amplifying the cls gene from P. syringae pv. tomato genomic DNA using primers designed based on the published sequence . The amplified gene is then cloned into an appropriate expression vector containing a strong promoter (such as T7 or tac) and an affinity tag to facilitate purification.

For bacterial expression systems, the recombinant plasmid is transformed into an E. coli expression strain (BL21(DE3) or similar) optimized for protein production . Expression is induced using IPTG (isopropyl β-D-1-thiogalactopyranoside) at concentrations typically ranging from 0.1-1.0 mM at mid-log phase (OD600 of 0.6-0.8). Alternatively, mammalian expression systems like COS-7 cells can be used for expression through transient transfection methods, which has been successful for similar enzymes such as human CLS1 .

Purification of the recombinant enzyme is typically achieved through affinity chromatography, taking advantage of the engineered tags. For His-tagged proteins, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is the method of choice. The bound protein is eluted using an imidazole gradient (20-250 mM), followed by buffer exchange to remove imidazole. Additional purification steps may include ion exchange chromatography and size exclusion chromatography to achieve higher purity.

For functional studies, it is crucial to verify the integrity and activity of the purified enzyme. This is typically accomplished through enzyme activity assays using CDP-DAG and PG as substrates, with the production of cardiolipin monitored using thin-layer chromatography (TLC) or mass spectrometry . Researchers should expect to see efficient catalysis of cardiolipin synthesis only in the presence of both CDP-DAG and radiolabeled PG, which serves as a specificity control .

What are the optimal conditions for assaying recombinant P. syringae pv. tomato CLS activity?

The optimal assay conditions for recombinant P. syringae pv. tomato CLS activity involve careful consideration of several parameters to ensure maximum enzyme performance. Based on studies with similar cardiolipin synthases, the assay buffer typically consists of 50 mM Tris-HCl (pH 8.0), 50 mM KCl, and 0.1 mM MnCl₂ . The presence of divalent cations, particularly Mn²⁺, is critical for optimal enzyme activity.

Substrate concentrations must be carefully optimized, with typical reactions containing 50-100 μM CDP-DAG and 50-100 μM phosphatidylglycerol (PG) . For detection and quantification purposes, one of the substrates is often radiolabeled (e.g., [¹⁴C]PG) to track product formation . The enzyme concentration should be adjusted to ensure linear reaction kinetics, typically in the range of 0.1-1.0 μg of purified protein per reaction.

Temperature and pH significantly impact enzyme activity, with most bacterial CLS enzymes showing optimal activity at 30-37°C and pH 7.5-8.5 . Reaction times generally range from 15-60 minutes, during which the reaction should proceed linearly before reaching a plateau. To terminate the reaction, a mixture of chloroform:methanol (2:1, v/v) is typically added.

For product analysis, thin-layer chromatography (TLC) using silica gel 60 plates with a chloroform:methanol:water (65:25:4, v/v/v) solvent system allows separation of cardiolipin from other phospholipids . Radiolabeled products can be visualized and quantified using a phosphorimager, while non-radiolabeled products may require staining with iodine vapor or phosphomolybdic acid spray.

It is important to include appropriate controls in each assay, such as reactions lacking one substrate, heat-inactivated enzyme preparations, or known CLS inhibitors. These controls help validate the specificity of the observed enzyme activity and ensure reliable results.

How do researchers analyze cardiolipin production in bacterial systems expressing recombinant CLS?

Analysis of cardiolipin production in bacterial systems expressing recombinant CLS involves a combination of lipid extraction, separation, and detection techniques. Researchers typically begin by extracting total lipids from bacterial cultures using established protocols such as the Bligh and Dyer method, which employs a chloroform:methanol:water system to efficiently separate lipids from other cellular components .

For quantitative analysis of cardiolipin synthesis in intact cells, researchers often employ metabolic labeling approaches. This involves culturing bacteria in the presence of radiolabeled precursors such as [¹⁴C]oleate or [³H]glycerol, which become incorporated into newly synthesized phospholipids including cardiolipin . This approach allows for direct measurement of cardiolipin synthesis rates in response to recombinant CLS expression.

Thin-layer chromatography (TLC) represents a fundamental separation technique for phospholipid analysis. Using silica gel 60 plates and appropriate solvent systems (chloroform:methanol:acetic acid, 65:25:10, v/v/v), researchers can effectively separate cardiolipin from other phospholipids . The radiolabeled lipids are then visualized by autoradiography or phosphorimaging, with cardiolipin identified by its characteristic Rf value and comparison to authentic standards.

For more detailed structural analysis, mass spectrometry (MS) provides powerful capabilities. Electrospray ionization mass spectrometry (ESI-MS) and tandem MS techniques enable researchers to characterize cardiolipin molecular species based on their fatty acid composition and identify structural modifications . These advanced analytical approaches have revealed that manipulation of CLS expression can alter not only the total amount of cardiolipin but also lead to accumulation of precursors like phosphatidylglycerol .

The data from these analyses typically show that increased expression of recombinant CLS correlates with elevated cardiolipin levels, often in a dose-dependent manner relative to the amount of expression plasmid used in transfection experiments . This relationship provides strong evidence for the functional activity of the recombinant enzyme in cellular contexts.

How does cardiolipin synthesis affect P. syringae pv. tomato pathogenicity and host interactions?

Cardiolipin synthesis has profound implications for P. syringae pv. tomato pathogenicity through multiple mechanisms affecting bacterial membrane function and stress responses. The presence of cardiolipin in bacterial membranes influences membrane fluidity, permeability, and the organization of membrane proteins involved in pathogenesis . These properties directly impact the bacterium's ability to survive host defense responses and environmental stresses encountered during infection.

Research on cardiolipin's role in bacterial pathogens suggests that it contributes to the proper assembly and function of protein complexes involved in secretion systems, particularly the Type III Secretion System (T3SS) that delivers effector proteins into host cells . In P. syringae pv. tomato, disruptions in cardiolipin synthesis could potentially compromise T3SS function, reducing bacterial virulence and ability to suppress host immune responses.

Interestingly, studies have shown that alterations in lipopolysaccharide (LPS) synthesis pathways, which can interact with cardiolipin metabolism, affect bacterial resistance to bacteriophages in P. syringae . This relationship between membrane lipid composition and phage susceptibility represents an important aspect of bacterial ecology that may influence pathogen population dynamics in agricultural settings.

For researchers investigating P. syringae pv. tomato-host interactions, cardiolipin synthesis represents a potential target for developing novel control strategies that could disrupt bacterial membrane function and reduce virulence without necessarily killing the pathogen outright.

What are the current challenges in analyzing contradictory data from CLS expression studies?

Analyzing contradictory data from CLS expression studies presents several significant challenges that researchers must navigate carefully. One primary source of contradiction arises from differences in experimental systems and expression platforms . Studies using heterologous bacterial expression systems (E. coli) may yield different results compared to eukaryotic expression systems (COS-7 cells) due to differences in post-translational modifications, protein folding machinery, and membrane composition .

Methodological variations in enzyme activity assays represent another significant challenge. Differences in assay conditions (pH, temperature, ionic strength), detection methods (radioactive labeling versus mass spectrometry), and data analysis approaches can yield contradictory results even when studying the same enzyme . Researchers need to carefully evaluate methodological details when comparing studies.

The biological context of CLS function introduces additional complexity. Cardiolipin synthesis occurs within a broader network of phospholipid metabolism, and perturbations in CLS expression can have indirect effects on other lipid synthesis pathways . These network effects may manifest differently across experimental systems, leading to apparently contradictory observations about the consequences of CLS manipulation.

To address these challenges, researchers should employ computational models that integrate data from multiple sources and account for context-dependent variables. Multi-omics approaches that combine transcriptomics, proteomics, and lipidomics data can provide a more comprehensive view of how CLS expression affects cellular physiology across different experimental systems .

How can phage resistance mechanisms in P. syringae pv. tomato inform research on CLS function?

Phage resistance mechanisms in P. syringae pv. tomato offer valuable insights into CLS function through their connections to membrane lipid composition and modification. Research has demonstrated that P. syringae evolves resistance to bacteriophages primarily through modifications in lipopolysaccharide (LPS) synthesis pathways . These findings are significant for CLS research because both LPS and cardiolipin are key components of bacterial membranes that contribute to membrane organization and function.

The observation that modifications affecting membrane lipids can confer phage resistance suggests that membrane composition plays a critical role in phage-host interactions . Cardiolipin, as a major phospholipid that influences membrane curvature and organization of membrane proteins, may indirectly affect phage adsorption, DNA injection, or other stages of the phage infection cycle.

Molecular genetic approaches used to study phage resistance can be adapted for investigating CLS function. Deletion of LPS-associated genes has revealed that LPS serves as the main receptor for multiple phages targeting P. syringae . Similar gene deletion or modification approaches targeting the cls gene could help elucidate the specific contributions of cardiolipin to membrane structure and function in this bacterium.

Coevolutionary experiments between P. syringae and phages provide a powerful model system for studying bacterial adaptation through membrane modifications . Such experimental evolution approaches could be extended to investigate how alterations in cardiolipin synthesis affect bacterial fitness and adaptation to environmental stresses or antimicrobial compounds.

The finding that bacterial fitness and virulence were affected in only a few LPS mutants resistant to phages suggests that bacteria can evolve resistance mechanisms with minimal fitness costs. This observation may have implications for understanding the potential consequences of targeting cardiolipin synthesis as an antimicrobial strategy against P. syringae pv. tomato.

What are the optimal primers and conditions for detecting the cls gene in P. syringae pv. tomato?

Optimal primer design for detecting the cls gene in P. syringae pv. tomato requires careful consideration of sequence specificity and amplification efficiency. Based on research approaches for similar bacterial genes, researchers should design primers targeting conserved regions of the cls gene with minimal homology to other genes . Typically, primers of 18-25 nucleotides with a GC content of 40-60% and a melting temperature (Tm) between 55-65°C yield the best results.

The following primer parameters have proven effective for similar bacterial gene detection:

For PCR amplification of the cls gene, a typical protocol would involve initial denaturation at 95°C for 5 minutes, followed by 30-35 cycles of denaturation (95°C, 30 seconds), annealing (58-62°C, 30 seconds), and extension (72°C, 30 seconds per 500 bp of expected amplicon), with a final extension at 72°C for 7 minutes . The specific annealing temperature should be optimized empirically, typically starting 5°C below the lowest Tm of the primer pair.

For quantitative real-time PCR (qPCR) detection, SYBR Green or TaqMan-based assays can be employed, with the latter offering higher specificity. Similar to the hrpZ gene-based detection of P. syringae pv. tomato, optimized qPCR conditions typically include 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute . Standard curves should be constructed using known quantities of purified cls gene to ensure accurate quantification.

To validate primer specificity, researchers should perform in silico analysis against genomic databases, followed by experimental validation using DNA from related bacterial species as negative controls. Sequencing of PCR products is recommended to confirm amplification of the correct target.

How can researchers differentiate between the effects of CLS expression and other factors on bacterial membrane function?

Differentiating between effects specifically attributable to CLS expression versus other factors affecting bacterial membrane function requires a multi-faceted experimental approach. One essential strategy involves creating isogenic bacterial strains that differ only in cls gene expression levels . This can be achieved through precise genetic manipulation techniques such as allelic exchange, CRISPR-Cas9 editing, or controlled expression systems using inducible promoters.

Complementation experiments provide critical evidence for CLS-specific effects. Researchers should reintroduce the wild-type cls gene into cls-deficient mutants to determine whether the observed phenotypes are reversed . Additionally, introducing site-specific mutations in the catalytic domain of CLS can help distinguish between effects dependent on enzymatic activity versus potential structural roles of the protein in the membrane.

Comprehensive lipid profiling using mass spectrometry allows researchers to quantify changes across the entire lipidome in response to CLS manipulation . This approach reveals not only changes in cardiolipin levels but also potential compensatory alterations in other phospholipids. The pattern of lipid changes can help distinguish direct effects of CLS activity from secondary adaptations in lipid metabolism pathways.

Time-course experiments following induction of CLS expression provide insights into the temporal sequence of membrane changes. Immediate effects are more likely to be direct consequences of CLS activity, while changes observed only after prolonged expression may represent adaptive responses or indirect effects .

Researchers should also employ membrane-specific functional assays to characterize the consequences of altered CLS expression. These include measurements of membrane fluidity using fluorescence anisotropy, membrane potential using voltage-sensitive dyes, proton permeability, and protein localization studies . Comparing these functional parameters across isogenic strains with different CLS expression levels helps establish causative relationships between cardiolipin synthesis and specific membrane properties.

What statistical approaches are most appropriate for analyzing enzyme kinetics data from recombinant CLS studies?

Statistical analysis of enzyme kinetics data from recombinant CLS studies requires specialized approaches to account for the complexities of multi-substrate reactions and potential sources of variability. For basic kinetic parameter estimation, non-linear regression analysis using the Michaelis-Menten equation or appropriate derivations for multi-substrate enzymes should be employed . Software packages such as GraphPad Prism, SigmaPlot, or R with specialized enzyme kinetics packages provide robust tools for fitting these models.

The kinetic data for CLS typically follows a bi-substrate reaction mechanism that can be analyzed using either rapid equilibrium or steady-state models . The most appropriate model can be determined by examining the pattern of Lineweaver-Burk or Eadie-Hofstee plots. For CLS, a sequential (ordered or random) bi-substrate mechanism is typically observed, requiring specialized equations that incorporate terms for both substrates.

To evaluate the quality of kinetic data and model fits, researchers should report R² values, residual plots, and confidence intervals for all estimated parameters . Additionally, the Akaike Information Criterion (AIC) or F-test comparisons help determine which kinetic model best describes the experimental data when multiple models are being considered.

For comparing kinetic parameters between different experimental conditions (e.g., pH, temperature) or between mutant forms of the enzyme, Analysis of Variance (ANOVA) with appropriate post-hoc tests or t-tests with Bonferroni correction for multiple comparisons should be applied . These statistical approaches account for experiment-wide error rates when making multiple comparisons.

When analyzing the effects of potential inhibitors on CLS activity, competitive, non-competitive, and uncompetitive inhibition models should be tested using global fitting approaches that simultaneously fit data from multiple inhibitor concentrations . This approach provides more robust estimates of inhibition constants and helps identify the mechanism of inhibition.

How might structural biology approaches advance our understanding of P. syringae pv. tomato CLS?

Structural biology approaches offer transformative potential for advancing our understanding of P. syringae pv. tomato CLS at the molecular level. X-ray crystallography represents a powerful technique for determining the three-dimensional structure of purified recombinant CLS at atomic resolution . Such structural data would reveal the spatial arrangement of the active site, substrate binding pockets, and potential regulatory domains, providing crucial insights into the catalytic mechanism.

Cryo-electron microscopy (cryo-EM) offers complementary advantages for structural analysis, particularly for membrane-associated proteins like CLS that may be difficult to crystallize . This technique can potentially capture CLS in different conformational states during the catalytic cycle, yielding dynamic insights into the enzyme's mechanism that static crystallographic structures cannot provide.

Nuclear magnetic resonance (NMR) spectroscopy, while challenging for large proteins, could be applied to study specific domains or fragments of CLS to understand ligand binding, protein dynamics, and conformational changes . NMR is particularly valuable for characterizing protein-lipid interactions, which are central to CLS function within the bacterial membrane environment.

Computational approaches such as molecular dynamics simulations and homology modeling can complement experimental structural studies . Using the resolved structures of related enzymes as templates, researchers can generate preliminary structural models of P. syringae pv. tomato CLS to guide experimental design and interpretation. These models can predict how mutations might affect enzyme function and help identify potential inhibitor binding sites.

Integration of structural data with functional analyses through structure-guided mutagenesis experiments would be particularly valuable . By systematically mutating residues predicted to be involved in substrate binding or catalysis based on structural information, researchers can validate mechanistic hypotheses and establish structure-function relationships for this important enzyme.

What are the prospects for using CLS inhibitors as novel antimicrobials against P. syringae pv. tomato?

The development of CLS inhibitors as novel antimicrobials against P. syringae pv. tomato represents a promising approach with several potential advantages over conventional bactericides. Cardiolipin plays essential roles in bacterial membrane function and stress responses, making its synthesis an attractive target for disrupting pathogen viability and virulence . Unlike traditional antimicrobials that often target processes common to many bacteria, CLS inhibitors could potentially be designed with specificity toward plant pathogens, reducing impacts on beneficial microbiota.

Rational design of CLS inhibitors would benefit from structural information about the enzyme's active site and substrate binding pockets . Potential inhibitor classes include substrate analogs that competitively bind to the active site, allosteric inhibitors that alter enzyme conformation, and transition-state analogs that mimic the reaction intermediate. Each approach has distinct advantages and challenges for inhibitor development.

High-throughput screening methods can accelerate the discovery of CLS inhibitors. These methods typically involve measuring enzyme activity in the presence of compound libraries using fluorescence-based assays or mass spectrometry to detect changes in cardiolipin production . Promising hits from such screens would then undergo chemical optimization to improve potency, selectivity, and pharmacokinetic properties.

Evaluation of potential CLS inhibitors requires a multi-tiered approach. Initial in vitro enzyme assays should be followed by tests against whole bacteria to assess cell penetration and efficacy . Subsequently, plant infection models can evaluate the ability of compounds to reduce disease symptoms and bacterial populations in planta. Phytotoxicity and effects on non-target organisms must also be carefully assessed.

The potential for resistance development represents an important consideration for CLS inhibitors, as with any antimicrobial . Studies of phage resistance in P. syringae suggest that bacteria can modify membrane components with minimal fitness costs . Therefore, combination treatment strategies or multi-target approaches might be necessary to mitigate resistance development to CLS inhibitors.

How can systems biology approaches integrate CLS function into broader models of bacterial pathogenesis?

Systems biology approaches offer powerful frameworks for integrating CLS function into comprehensive models of bacterial pathogenesis through multi-scale data integration and network analysis. Genome-scale metabolic models incorporating detailed lipid metabolism pathways can predict how perturbations in CLS expression ripple through the bacterial metabolic network . These computational models can generate testable hypotheses about the systemic effects of altered cardiolipin synthesis on cellular energetics, membrane properties, and stress responses.

Multi-omics integration represents a core systems biology strategy for understanding CLS function in context. By simultaneously analyzing transcriptomics, proteomics, lipidomics, and metabolomics data from wild-type and cls mutant strains under various conditions, researchers can map the regulatory networks connecting CLS to other cellular processes . These integrated datasets reveal how bacteria compensate for altered cardiolipin levels and identify potential intervention points for disrupting pathogen adaptation.

Network analysis approaches can elucidate the position of CLS within the broader pathogenicity network of P. syringae pv. tomato. Protein-protein interaction networks, genetic interaction maps, and regulatory network reconstructions help place CLS in relation to virulence factors, stress response systems, and host interaction pathways . Such analyses may reveal unexpected connections between membrane lipid composition and specific virulence mechanisms.

Agent-based modeling and population-level simulations can bridge molecular-level understanding of CLS function to ecosystem-level dynamics of pathogen-host interactions . These computational approaches can predict how mutations affecting CLS might influence bacterial population dynamics in field settings, including spread patterns, persistence under stress, and evolution of resistance to bacteriophages or antimicrobials.

For practical application, systems biology approaches can guide experimental design by identifying high-value measurements that provide maximum information about system behavior. This iterative process of model development, prediction, experimental testing, and model refinement gradually builds a more comprehensive understanding of how CLS function contributes to P. syringae pv. tomato pathogenicity in complex agricultural ecosystems .