Recombinant Enterococcus faecalis Phosphate acyltransferase (plsX)

What is the role of phosphate acyltransferase (PlsX) in Enterococcus faecalis?

PlsX in Enterococcus faecalis functions as an acyl-acyl carrier protein (ACP) phosphate acyltransferase that plays a critical role in both phospholipid synthesis and exogenous fatty acid incorporation into membrane lipids. This enzyme serves as a key intermediary in the process of converting fatty acids into membrane phospholipids, making it essential for proper cell membrane formation and function. PlsX is located adjacent to the acpB gene, which encodes an auxiliary ACP involved in regulating fatty acid synthesis, highlighting its integration within the fatty acid synthesis gene cluster .

How does deletion of the plsX gene affect E. faecalis growth and membrane composition?

Deletion of the plsX gene almost completely blocks growth of E. faecalis by severely decreasing de novo phospholipid synthesis. This growth inhibition is manifested as extremely weak growth in laboratory conditions. Microscopically, the deletion leads to abnormally long-chain acyl groups in the cell membrane phospholipids, indicating disruption of normal phospholipid synthesis pathways. The ΔplsX strain fails to grow without supplementation with appropriate exogenous fatty acids, demonstrating that PlsX is essential for normal membrane phospholipid formation and consequently cellular viability .

What is the relationship between PlsX and the fatty acid synthesis pathway in E. faecalis?

PlsX interfaces directly with the fatty acid synthesis pathway by participating in the conversion of acyl-ACP (products of the fatty acid synthesis pathway) to acyl-phosphates, which are then incorporated into phospholipids. In the fatty acid synthesis gene cluster, plsX is positioned adjacent to acpB, indicating coordinated regulation of these processes. The relationship is bidirectional, as disruptions in PlsX function affect fatty acid chain length in membrane phospholipids. This suggests PlsX provides feedback to the fatty acid synthesis pathway about membrane phospholipid needs, creating an integrated system for maintaining membrane homeostasis .

What techniques are used to generate E. faecalis ΔplsX strains?

The generation of E. faecalis ΔplsX strains typically employs a temperature-sensitive plasmid-based gene replacement strategy. The methodology involves:

• Construction of a plsX gene knockout cassette cloned into a temperature-sensitive plasmid (e.g., pQZ149)

• Transformation of E. faecalis cells with the recombinant plasmid

• Selection of transformants on medium containing erythromycin (5 mg/L) and X-Gal (100 mg/L) at 30°C

• Identification of blue colonies containing the integrated plasmid

• Temperature shift to 42°C to select for plasmid excision and gene replacement

• PCR verification of the plsX deletion

• Supplementation with palmitate (0.1 mM) to support growth of the ΔplsX strain

This process must include fatty acid supplementation (typically palmitate) to ensure survival of the mutant strain .

How can researchers construct E. faecalis plsX overexpression systems?

Construction of E. faecalis plsX overexpression systems involves the following methodological steps:

• PCR amplification of the plsX gene using high-fidelity DNA polymerase

• Ligation of the amplified plsX gene into an appropriate shuttle vector (e.g., pQZ28 derived from pZL277)

• Replacement of the original chloramphenicol resistance gene with an erythromycin resistance gene

• Transformation of the recombinant plasmid into E. faecalis cells

• Selection of transformants on media containing appropriate antibiotics

• Verification of overexpression through quantitative PCR or Western blotting

This approach allows for controlled expression of PlsX for functional studies and complementation experiments. Similar methodologies can be applied for constructing overexpression plasmids for other related genes such as fabK, fabI, and tesE .

What genetic compensatory mechanisms can restore growth in E. faecalis ΔplsX strains?

Several genetic compensatory mechanisms can restore growth in E. faecalis ΔplsX strains:

These mechanisms demonstrate the metabolic plasticity of E. faecalis in adapting to the loss of PlsX through alternative pathways for phospholipid synthesis .

How does exogenous fatty acid supplementation affect the growth of ΔplsX strains?

Exogenous fatty acid supplementation is critical for the survival of ΔplsX strains, with different fatty acids producing varying effects:

• Palmitic acid (saturated) supplementation leads to faster growth rates compared to oleic acid (unsaturated) supplementation

• Improved phospholipid acyl chain synthesis occurs with palmitic acid supplementation

• The ΔplsX strain cannot grow without appropriate exogenous fatty acid supplementation

• Saturated fatty acids are preferentially incorporated at the sn1-position of phospholipids

• Exogenous fatty acids are likely processed through the FakAB system for conversion to acyl-phosphates

The differential effects of fatty acid types indicate a preference in the alternative phospholipid synthesis pathway that becomes essential in the absence of PlsX .

What is the role of TesE thioesterase in relation to PlsX function in E. faecalis?

TesE thioesterase plays a crucial compensatory role in the absence of PlsX by:

• Cleaving acyl-ACPs to provide free fatty acids for phospholipid synthesis

• Showing substrate preference for unsaturated acyl-ACPs over saturated acyl-ACPs

• Enabling the FakAB system to convert released free fatty acids to acyl-phosphates

• Facilitating incorporation of acyl-phosphates into position sn1 of phospholipids via PlsY

How does the FakAB system interact with the PlsX pathway in phospholipid synthesis?

The FakAB system functions as an alternative pathway that intersects with the PlsX-dependent phospholipid synthesis pathway:

• In normal conditions, PlsX converts acyl-ACPs to acyl-phosphates for phospholipid synthesis

• When PlsX is absent, the FakAB system can convert free fatty acids to acyl-phosphates

• These acyl-phosphates are then incorporated into position sn1 of phospholipids by PlsY

• Free fatty acids for the FakAB system can be derived from exogenous sources or from TesE-mediated cleavage of acyl-ACPs

• The FakAB pathway is less efficient than the PlsX pathway, explaining the growth defects in ΔplsX strains

This alternative pathway explains how ΔplsX strains can survive with appropriate fatty acid supplementation or with genetic adaptations that enhance fatty acid synthesis .

What analytical methods are used to determine phospholipid composition in E. faecalis strains?

The analysis of phospholipid composition in E. faecalis strains employs several complementary techniques:

• Lipid extraction using chloroform-methanol methods to isolate membrane phospholipids

• Thin-layer chromatography (TLC) for separation of phospholipid classes

• Gas chromatography-mass spectrometry (GC-MS) for analysis of fatty acid methyl esters derived from phospholipids

• Positional analysis to determine acyl chain distribution at the sn1 and sn2 positions

• Liquid chromatography-mass spectrometry (LC-MS) for more detailed phospholipid profiling

• Radiolabeling experiments using 14C-labeled acetic acid to track de novo fatty acid synthesis and incorporation

These techniques have revealed that saturated acyl chains dominate the sn1-position in E. faecalis phospholipids, indicating a preference for saturated fatty acids at this position .

What are the most effective protocols for constructing double mutants in E. faecalis?

Construction of double mutants in E. faecalis, such as the ΔplsX ΔfabT strain, requires specialized protocols:

• Sequential deletion approach: Delete one gene first, then use the resulting strain as the starting point for the second deletion

• Temperature-sensitive plasmids containing knockout cassettes (e.g., pQZ149 for plsX deletion)

• Selection on media containing appropriate antibiotics (e.g., erythromycin at 5 mg/L) and indicator compounds (e.g., X-Gal at 100 mg/L)

• Temperature shifts between 30°C and 42°C to control plasmid integration and excision

• Supplementation with appropriate fatty acids (e.g., 0.1 mM palmitate) if the mutations affect essential metabolic pathways

• PCR verification of both gene deletions

• Phenotypic characterization to confirm the expected combined effects of both mutations

This methodology has been successfully applied to generate the ΔplsX ΔfabT double mutant, which shows very weak growth compared to the plsX single mutant that cannot grow without fatty acid supplementation .

How does understanding PlsX function contribute to antibiotic development against E. faecalis?

Understanding PlsX function has significant implications for antibiotic development against E. faecalis:

• E. faecalis is an opportunistic pathogen with high levels of antibiotic resistance that causes hospital-acquired infections

• PlsX is essential for normal growth and membrane formation, making it a potential antibiotic target

• The PlsX pathway differs from mammalian phospholipid synthesis, allowing for selective targeting

• Inhibition of PlsX could disrupt membrane integrity, potentially increasing susceptibility to other antibiotics

• The alternative pathways that compensate for PlsX loss could inform combination therapy approaches

Given the clinical importance of E. faecalis infections and their growing resistance to current antibiotics, targeting essential phospholipid synthesis enzymes like PlsX represents a promising approach for developing new antimicrobial strategies .

What are the methodological approaches for evaluating potential PlsX inhibitors?

Methodological approaches for evaluating potential PlsX inhibitors include:

• In vitro enzymatic assays using purified recombinant PlsX to screen for direct inhibitors

• Growth inhibition assays comparing effects on wild-type versus PlsX-overexpressing strains

• Membrane integrity assays to assess the impact of PlsX inhibition on cell envelope function

• Phospholipid synthesis assays using radiolabeled precursors to measure specific inhibition of the PlsX pathway

• Synergy testing with existing antibiotics to identify combination effects

• Molecular modeling and structure-based drug design based on PlsX structure

• Assessment of inhibitor effects on ΔfabT and other compensatory mutants to evaluate escape mechanisms

These approaches would help identify compounds that specifically target PlsX function while considering potential resistance mechanisms and alternative pathways that might reduce inhibitor efficacy .

What are the remaining questions about PlsX regulation in E. faecalis?

Despite significant advances, several questions about PlsX regulation in E. faecalis remain:

• What transcriptional and post-translational mechanisms regulate PlsX expression and activity?

• How is PlsX activity coordinated with fatty acid synthesis and membrane needs during different growth phases?

• What environmental signals (pH, oxygen, nutrient availability) modulate PlsX function?

• How does PlsX interact with other membrane synthesis enzymes to maintain appropriate membrane composition?

• Are there additional compensatory mechanisms for PlsX function yet to be discovered?

• What is the three-dimensional structure of E. faecalis PlsX and how does it compare to orthologs in other bacteria?

Addressing these questions would provide deeper insights into membrane homeostasis in E. faecalis and potentially reveal new approaches for disrupting this essential process .

What experimental systems might advance our understanding of PlsX structure-function relationships?

Advanced experimental systems that could deepen our understanding of PlsX structure-function relationships include:

• CRISPR-Cas9 gene editing for precise modification of PlsX active sites

• Conditional expression systems to study PlsX essentiality under different conditions

• Fluorescently tagged PlsX to visualize subcellular localization and potential membrane associations

• Bacterial two-hybrid or co-immunoprecipitation approaches to identify PlsX interaction partners

• Cryo-electron microscopy to determine the three-dimensional structure of PlsX alone and in complex with substrates

• Molecular dynamics simulations to model substrate binding and catalytic mechanisms

• High-throughput suppressor mutation screening to identify additional genetic interactions

These approaches would integrate structural biology, genetics, and biochemistry to build a comprehensive model of how PlsX functions within the context of bacterial membrane synthesis .