Recombinant Pseudomonas entomophila Cardiolipin synthase (cls)

What is the role of cardiolipin synthase in Pseudomonas entomophila membrane homeostasis?

Cardiolipin synthase (cls) plays a critical role in the homeoviscous adaptation of bacterial membranes. As demonstrated in related Pseudomonas species, cardiolipin is synthesized via the condensation of two phosphatidylglycerol (PG) molecules by phospholipase D-type cardiolipin synthases (PLD-type Cls) . In Pseudomonas species, cardiolipin contributes to membrane rigidity, cellular size regulation, and response to environmental stressors . Studies in P. putida have shown that cls mutants exhibit increased membrane rigidity and decreased cell size compared to wild-type strains . The enzyme likely serves similar functions in P. entomophila, contributing to membrane integrity and adaptation to changing environmental conditions. Membrane composition analysis using thin-layer chromatography and mass spectrometry would be recommended for researchers investigating the specific membrane modifications in P. entomophila.

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

Based on successful approaches with related bacterial cardiolipin synthases, E. coli expression systems represent the primary choice for recombinant production. Heterologous expression of cardiolipin synthase genes from Agrobacterium tumefaciens in E. coli has been demonstrated to successfully produce functional enzyme, resulting in accumulation of cardiolipin in the host membranes . For P. entomophila cls, researchers should consider using expression vectors with inducible promoters (such as pET series vectors with T7 promoters) and E. coli strains optimized for membrane protein expression (such as C41(DE3) or C43(DE3)).

The methodology should include:

• Amplification of the cls gene with appropriate restriction sites

• Cloning into a suitable expression vector with an affinity tag (His6 is commonly used)

• Transformation into an E. coli expression strain

• Induction of protein expression under optimized conditions (temperature, IPTG concentration, and duration)

• Membrane fraction isolation followed by detergent solubilization

• Purification via affinity chromatography

How do mutations in P. entomophila cardiolipin synthase affect bacterial pathogenicity?

The relationship between cardiolipin synthesis and pathogenicity in P. entomophila represents a sophisticated research question with significant implications. Based on studies in P. putida, disruption of the cls gene results in compromised membrane function and increased sensitivity to antibiotics, suggesting altered efflux pump activity . In P. entomophila, which exhibits bactericidal effects against certain bacterial species like Xanthomonas citri , cls mutations may significantly impact pathogenicity mechanisms.

Research methodology to investigate this relationship should include:

• Generation of cls knockout mutants using transposon mutagenesis or CRISPR-Cas9 techniques

• Comparative virulence assays using Drosophila infection models

• Analysis of membrane permeability and antibiotic resistance profiles

• Evaluation of bactericidal compound production against target bacteria

• Transcriptomic analysis to identify compensatory mechanisms

Studies in Drosophila infection models have demonstrated that adaptation to P. entomophila is route-specific , suggesting that membrane composition changes may differentially affect various infection mechanisms. Researchers should consider both oral and systemic infection routes when evaluating the impact of cls mutations on pathogenicity.

What are the optimal conditions for assaying recombinant P. entomophila cardiolipin synthase activity in vitro?

Recombinant cardiolipin synthase activity can be measured through various biochemical approaches. The following methodology is recommended based on successful approaches with related enzymes:

Standard in vitro assay protocol:

• Enzyme preparation: Purify recombinant cls using affinity chromatography followed by size exclusion chromatography

• Substrate preparation: Prepare phosphatidylglycerol (PG) liposomes of defined composition

• Reaction conditions: 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl₂, 0.1 mg/ml PG

• Incubation: 30°C for 30 minutes

• Product analysis: Extract lipids using Bligh-Dyer method and analyze by thin-layer chromatography or LC-MS

Researchers should validate these conditions specifically for P. entomophila cls, as optimal conditions may vary between species.

How does cardiolipin content affect antibiotic resistance in P. entomophila?

Based on studies in P. putida, cardiolipin content significantly impacts antibiotic resistance through multiple mechanisms. In P. putida, cls mutants with reduced cardiolipin show increased sensitivity to various antibiotics, suggesting compromised function of resistance-nodulation-division (RND) efflux pumps . These efflux systems are critical for extruding antibiotics and toxic compounds from bacterial cells.

To investigate this relationship in P. entomophila, researchers should:

• Generate cls mutants with varying cardiolipin levels

• Perform comprehensive antibiotic susceptibility testing (MIC determination)

• Assess accumulation of efflux pump substrates (like ethidium bromide)

• Analyze membrane fluidity using fluorescence polarization with DPH probe

• Quantify expression levels of key efflux pump components

The experimental design should include positive controls (wild-type strains) and comparative analyses with other Pseudomonas species. Changes in membrane permeability can be assessed using fluorescent dyes like NPN (1-N-phenylnaphthylamine), which penetrates damaged outer membranes.

What are the major challenges in purifying active recombinant P. entomophila cardiolipin synthase?

Purification of functional cardiolipin synthase presents several technical challenges due to its nature as a membrane-associated enzyme. Key challenges and recommended solutions include:

For activity retention, researchers should consider incorporating the enzyme directly into nanodiscs or liposomes immediately after purification, which better mimics the native membrane environment and preserves enzymatic function.

How can researchers accurately quantify cardiolipin in bacterial membranes following recombinant cls expression?

Accurate quantification of cardiolipin in bacterial membranes is essential for characterizing the activity of recombinant cardiolipin synthase. Multiple complementary approaches should be employed:

• Thin-layer chromatography (TLC):

  • Extract total lipids using Bligh-Dyer method

  • Separate on silica gel using chloroform/methanol/water (65:25:4)

  • Visualize with molybdenum blue or primuline spray

  • Quantify using densitometry against standards

• Mass spectrometry approaches:

  • Employ LC-MS/MS for accurate molecular species identification

  • Use internal standards with known concentrations

  • Multiple reaction monitoring (MRM) for specific cardiolipin species

  • Software analysis using integration of characteristic ions

• Fluorescent probes:

  • 10-N-nonyl acridine orange (NAO) binds specifically to cardiolipin

  • Measure fluorescence intensity in intact cells or isolated membranes

  • Calibrate using membranes with known cardiolipin content

For comparative studies, researchers should standardize extraction methods and analysis protocols across all experimental conditions to minimize technical variability.

How does the structure of P. entomophila cardiolipin synthase compare to cls enzymes from other bacteria?

While specific structural data for P. entomophila cardiolipin synthase is currently limited, insights can be derived from related bacterial cls enzymes. Cardiolipin synthases in most bacteria, including Pseudomonas species, belong to the phospholipase D (PLD) superfamily, characterized by conserved HKD motifs essential for catalytic activity .

Key structural features likely include:

• Transmembrane domains that anchor the enzyme in the cytoplasmic membrane

• HKD catalytic motifs that facilitate the condensation of phosphatidylglycerol molecules

• Substrate binding regions that recognize phosphatidylglycerol

Researchers investigating structural aspects should consider:

• Homology modeling based on related bacterial cls structures

• Site-directed mutagenesis of predicted catalytic residues

• Expression of truncated variants to identify essential domains

• Protein-lipid interaction studies using fluorescence techniques

The predicted structural elements can be validated through functional studies of recombinant enzyme with specific mutations in key domains.

What functional domains are critical for P. entomophila cardiolipin synthase activity?

Based on characterization of related bacterial cardiolipin synthases, several functional domains are likely critical for P. entomophila cls activity:

To investigate these domains, researchers should consider a methodical approach:

• Bioinformatic analysis to identify conserved domains

• Generation of targeted mutations in predicted functional regions

• Expression and purification of mutant proteins

• In vitro activity assays comparing wild-type and mutant enzymes

• Membrane association studies to determine localization effects

How does cardiolipin synthesis contribute to P. entomophila environmental adaptation?

Cardiolipin synthesis likely plays a crucial role in P. entomophila's adaptation to various environmental conditions. Research in P. putida has demonstrated that cardiolipin is involved in "homeoviscous adaptation," allowing bacterial membranes to modify their composition in response to environmental stressors . This adaptation mechanism enables Pseudomonas species to thrive in diverse ecological niches.

Key adaptation mechanisms likely include:

• Modulation of membrane fluidity in response to temperature changes

• Protection against organic solvent exposure (as seen with toluene in P. putida)

• Resistance to antimicrobial compounds present in natural habitats

• Adaptation to varying osmotic conditions

Researchers investigating these adaptive mechanisms should:

• Compare growth and survival of wild-type and cls mutants under various stress conditions

• Analyze membrane composition changes in response to environmental shifts

• Evaluate gene expression patterns of cls under different growth conditions

• Assess competitive fitness in mixed microbial communities

Unlike Agrobacterium tumefaciens, which can tolerate large changes in cardiolipin content , P. entomophila may be more sensitive to cls disruption based on observations in related Pseudomonas species, suggesting different evolutionary strategies for membrane adaptation.

How does cardiolipin content affect P. entomophila interactions with host organisms?

P. entomophila's interactions with host organisms, particularly insects like Drosophila, may be significantly influenced by cardiolipin content. Studies have shown that P. entomophila can establish infections through different routes, and host adaptation is contingent upon these infection routes . Membrane composition likely plays a crucial role in these host-pathogen interactions.

Researchers investigating this relationship should consider:

• Comparing virulence of wild-type and cls mutant strains in Drosophila infection models

• Evaluating both oral and systemic infection routes

• Assessing host immune responses to strains with varying cardiolipin content

• Analyzing bacterial persistence within host tissues

• Investigating potential trade-offs between virulence and environmental persistence

The specificity of host responses may be linked to membrane characteristics, as studies have shown that adaptation to P. entomophila through one infection route does not affect susceptibility to the same pathogen infecting from a different route . This suggests complex interactions between bacterial membrane components and host recognition systems.

How might recombinant P. entomophila cardiolipin synthase be utilized in synthetic biology applications?

Recombinant cardiolipin synthase from P. entomophila presents several promising applications in synthetic biology:

• Engineered membrane systems:

  • Development of bacterial chassis with enhanced stress resistance

  • Creation of membrane-based biocatalysts with improved stability

  • Design of bacteria with modified antibiotic resistance profiles

• Bioremediation applications:

  • Engineering strains with enhanced organic solvent tolerance

  • Development of bacteria capable of surviving in contaminated environments

  • Modification of membrane properties to facilitate pollutant uptake

• Methodological approaches:

  • Clone cls genes under controllable promoters

  • Introduce recombinant cls into heterologous hosts

  • Combine with other membrane-modifying enzymes

  • Quantify effects on membrane properties and stress resistance

The bactericidal properties of P. entomophila against plant pathogens like Xanthomonas suggest potential agricultural applications for recombinant strains with modified membrane compositions.

What are the latest methodological advances for studying cardiolipin dynamics in live bacterial cells?

Recent methodological advances have enhanced our ability to study cardiolipin dynamics in live bacterial cells:

Researchers studying P. entomophila cardiolipin dynamics should combine multiple techniques for comprehensive analysis. For example, fluorescent imaging approaches can provide spatial information, while mass spectrometry offers detailed compositional analysis. Time-resolved studies during environmental transitions can reveal the dynamics of cardiolipin remodeling in response to stress conditions.