Recombinant Pseudomonas fluorescens Cardiolipin synthase (cls)

What are the main cardiolipin synthase genes in Pseudomonas fluorescens?

Pseudomonas fluorescens contains at least two cardiolipin synthase genes: clsA and clsB. These genes encode enzymes that catalyze the formation of cardiolipin (CL) from two phosphatidylglycerol (PG) molecules. In P. fluorescens UM270, both clsA and clsB have been identified and characterized through deletion studies . Unlike some other bacterial species that may have additional cls homologs, these two appear to be the primary cardiolipin synthases in P. fluorescens.

What is the physiological role of cardiolipin in P. fluorescens?

Cardiolipin plays a fundamental role in the adaptation of P. fluorescens to various environmental conditions, particularly saline stress. Research with P. fluorescens UM270 demonstrates that cardiolipin is essential for normal bacterial physiology, including optimal growth under salt stress conditions (100-200 mM NaCl) . Beyond basic membrane structure, cardiolipin in P. fluorescens contributes to plant growth-promoting characteristics and root colonization capacity, suggesting its importance in bacteria-plant interactions .

How does cardiolipin synthesis affect bacterial growth phenotypes?

Deletion of cls genes in P. fluorescens UM270 (ΔclsA and ΔclsB mutants) results in significant reduction of cardiolipin synthesis (approximately 58% reduction in ΔclsA and 53% in ΔclsB) compared to wild-type . While growth rates of these mutants are not affected under normal conditions, they show impaired growth at elevated salt concentrations (100 and 200 mM NaCl) . This indicates that cardiolipin synthesis becomes particularly important for bacterial fitness under stress conditions.

What expression systems are recommended for recombinant P. fluorescens Cls?

Based on approaches used for related cardiolipin synthases, heterologous expression in E. coli is a viable strategy for P. fluorescens Cls. Previous studies with enterococcal Cls enzymes established successful expression and purification schemes that can be adapted for P. fluorescens Cls . When designing expression constructs, it's important to consider that Cls proteins contain transmembrane domains, particularly in the N-terminal region, which may affect solubility and proper folding.

What purification strategies maximize yield and activity of recombinant Cls?

Purification of active recombinant Cls has historically been challenging, as noted in previous characterization studies of bacterial cardiolipin synthases . Historical approaches relied on crude membrane or partially purified preparations. For recombinant P. fluorescens Cls, a multi-step purification protocol should include:

• Cell lysis in buffer containing appropriate detergents to solubilize membrane proteins

• Initial separation using affinity chromatography (if tagged constructs are used)

• Further purification by ion exchange and/or size exclusion chromatography

• Careful selection of buffer conditions to maintain enzyme stability

The goal should be to achieve highly purified and active protein suitable for biochemical and structural studies.

How do immobilization conditions affect recombinant enzyme activity?

While not specific to Cls, studies with P. fluorescens lipase demonstrate that immobilization conditions dramatically affect enzyme properties. For example, varying pH, ionic strength, buffer composition, and additives during immobilization can alter enzyme activity by 50-60% and stability by up to fourfold . For recombinant Cls, similar principles may apply when creating immobilized enzyme preparations for analytical or biotechnological applications.

What are the established assays for measuring Cls catalytic activity?

Cardiolipin synthase activity can be measured using several approaches:

• Radiometric assays: Use of radiolabeled substrates (e.g., [14C]acetate-labeled phospholipids) followed by thin-layer chromatography (TLC) separation and quantification of cardiolipin formation .

• Phosphate release assays: Monitoring the release of inorganic phosphate during the condensation reaction.

• HPLC or LC-MS based methods: For detailed product analysis and kinetic studies.

When using recombinant P. fluorescens Cls, it's essential to optimize reaction conditions including pH, temperature, divalent cation concentration (particularly Ca2+), and detergent/lipid composition to ensure maximal enzymatic activity.

How can the structure-function relationship of Cls be investigated?

Based on homology to phospholipase D enzymes, cardiolipin synthases likely have active sites composed of functional groups from two PLD domains (PLD1 and PLD2), with a conserved histidine serving as the putative active-site nucleophile . To investigate structure-function relationships:

• Site-directed mutagenesis targeting conserved residues in the catalytic domains

• Creation of truncated variants to identify minimal functional units

• Domain swapping between clsA and clsB to determine specificity determinants

• Crystallographic or cryo-EM studies for structural characterization

Studies with enterococcal Cls enzymes revealed that mutations near the catalytic site (e.g., H215R and R218Q) can significantly alter enzyme activity .

How does Cls activity contribute to bacterial stress responses?

Cardiolipin synthesis is crucial for P. fluorescens adaptation to environmental stresses, particularly salt stress. ΔclsA and ΔclsB mutants show:

• Reduced growth at high salt concentrations (100-200 mM NaCl)

• Impaired root colonization capacity

• Altered production of plant growth-promoting factors

These findings indicate that cardiolipin synthesis is a key component of the bacterial stress response machinery, particularly for environmental adaptations relevant to plant-microbe interactions.

What is the relationship between Cls activity and plant growth promotion?

Cardiolipin synthesis in P. fluorescens UM270 plays an important role in promoting plant growth, especially under salt stress conditions. Research demonstrates that:

These data indicate that functional Cls enzymes in P. fluorescens contribute substantially to the bacterium's ability to promote plant growth, particularly under salt stress conditions .

How does Cls activity affect bacterial membrane properties?

Cardiolipin significantly affects membrane properties due to its unique structure with four acyl chains and small polar head group. In P. fluorescens, cls deletion results in:

• Altered membrane phospholipid composition

• Changes in membrane fluidity and permeability

• Modified protein-lipid interactions affecting membrane-associated functions

• Impaired physiological processes including indole acetic acid production and biofilm formation

These changes in membrane properties likely underlie the observed phenotypic effects of cls deletion.

How can Cls mutations be engineered to enhance stress tolerance?

Based on studies of Cls mutations in other bacteria such as enterococci, where certain mutations increased enzyme activity , targeted engineering approaches could include:

• Introducing point mutations near the catalytic site to modify enzyme kinetics

• Altering regulatory regions to modulate expression levels

• Engineering chimeric enzymes combining domains from different Cls homologs

• Screening for variants with enhanced activity under specific stress conditions

Mutations that increase Cls activity might enhance P. fluorescens stress tolerance and plant growth-promoting abilities, although this would require careful phenotypic characterization.

What techniques can determine how Cls interacts with other membrane proteins?

To investigate Cls interactions with other membrane components:

• Protein crosslinking followed by mass spectrometry

• Bacterial two-hybrid or split-protein complementation assays

• Co-immunoprecipitation with tagged Cls variants

• Fluorescence resonance energy transfer (FRET) between labeled proteins

• Lipidomic analysis of membrane microdomains in wild-type versus cls mutants

Studies of lipid transport proteins like MlaA, MlaZ, and the lipase MlaY in P. aeruginosa suggest that lipid transport machinery may interact with phospholipid synthesis enzymes .

How can recombinant Cls be used to study bacterial-plant interactions?

Recombinant P. fluorescens Cls can be employed in several experimental approaches:

• In vitro reconstitution of cardiolipin synthesis using purified enzymes and defined lipid substrates

• Creation of P. fluorescens strains with controlled Cls expression levels

• Supplementation of cls-deficient strains with purified recombinant enzyme to assess complementation

• Structural studies to understand how Cls activity is regulated during plant colonization

Such approaches could help elucidate the molecular mechanisms by which cardiolipin synthesis contributes to plant growth promotion.

How can low activity of recombinant Cls be addressed?

Low activity of recombinant Cls enzymes can result from several factors:

• Improper folding due to membranous nature of the protein

• Loss of essential cofactors during purification

• Suboptimal reaction conditions

Potential solutions include:

• Optimization of detergent type and concentration during purification

• Addition of specific lipids or phospholipids to reaction mixtures

• Testing different divalent cation concentrations, particularly Ca2+ which has been shown to enhance enzyme activity in P. fluorescens systems

• Use of liposome reconstitution to provide a more native-like membrane environment

What strategies help resolve contradictory results in Cls activity assays?

When facing contradictory results in Cls activity measurements:

• Standardize lipid substrate preparation methods and composition

• Control for product inhibition effects

• Verify enzyme stability throughout the assay period

• Consider the influence of detergents, which may either stimulate or inhibit activity

• Ensure proper negative controls using heat-inactivated enzyme or known inhibitors

Studies with P. aeruginosa phospholipases demonstrate that phospholipid metabolism can be affected by various inhibitors including AACOCF3, VU0364739, OBAA, FIPI, and darapladip , which could serve as useful controls in Cls activity assays.