Recombinant Bacillus subtilis Phosphate acyltransferase (plsX)

What is the basic function of PlsX in Bacillus subtilis?

PlsX is a peripheral enzyme that plays a crucial role in phospholipid synthesis in Bacillus subtilis. Specifically, it catalyzes acyl transfer to orthophosphate in the phosphatidic acid (PA) pathway, serving as a phosphate acyl-acyl carrier protein (ACP) acyltransferase . Research has revealed that PlsX performs a dual function, acting both as a catalyst in the phosphatidic acid pathway and as a chaperone protein that facilitates substrate channeling into the pathway . This dual role emphasizes the importance of PlsX beyond its enzymatic activity, highlighting its significance in maintaining proper phospholipid synthesis flux.

How does PlsX associate with the cell membrane?

PlsX associates with the cell membrane through direct binding to lipid bilayers via a specific membrane anchoring moiety. This anchoring structure consists of a hydrophobic loop located at the tip of two amphipathic dimerization helices . Studies have demonstrated that recombinant PlsX directly binds to protein-free total lipids from B. subtilis, and mutations such as K257A can weaken this interaction . The membrane association is critical for PlsX's function, as it enables proper delivery of its product (acyl-phosphate) to PlsY, the next enzyme in the phosphatidic acid pathway . This association with regions of increased fluidity (RIFs) in the membrane appears to be essential for optimal enzymatic activity and cellular growth.

What are the conserved structural features of PlsX across bacterial species?

PlsX contains several strictly conserved residues that contribute significantly to its catalytic activity. The active site includes many residues that line a long, narrow gorge at the dimeric interface. Two positive residues form an ACP docking pad adjacent to this interfacial gorge . The catalytic mechanism specifically involves substrate activation and transition-state stabilization by two strictly conserved residues: Lys184 and Asn229 . Another notable structural feature is the release mechanism of the acyl phosphate product near the membrane, which may facilitate its membrane insertion. These conserved features distinguish PlsX's substrate recognition mode and catalytic mechanism from other enzymes like phosphotransacetylases that catalyze similar acyl transfer reactions.

What is the recommended protocol for expression and purification of recombinant PlsX?

For efficient expression and purification of recombinant PlsX with a C-terminal hexahistidine tag, researchers should follow this validated protocol:

• Amplify the plsX gene from B. subtilis genomic DNA using appropriate primers (such as PlsX-for and PlsX-rev).

• Integrate the amplified gene into the pET28a vector between NcoI and XhoI restriction sites.

• Verify the inserted gene sequence to ensure no mutations were introduced.

• Transform the recombinant plasmid into E. coli C43 (DE3) cells.

• Culture transformed cells overnight at 37°C on LB agar plates with 50 μg/ml kanamycin.

• Pick a single colony to inoculate 10 ml LB containing 50 μg/ml kanamycin and grow overnight at 37°C with shaking at 250 rpm.

• Use this starter culture to inoculate 4 L LB medium supplemented with 50 μg/ml kanamycin.

• Grow cells at 37°C with shaking at 250 rpm until OD600 reaches 0.4–0.6.

• Induce PlsX expression by adding isopropyl-β-d-thiogalactopyranoside to a final concentration of 0.2 mM.

• Continue expression at 18°C for 20 hours .

This protocol has been shown to yield functional protein suitable for subsequent biochemical and structural analyses. The use of E. coli C43 (DE3) is particularly important as this strain is optimized for the expression of membrane-associated proteins.

How can researchers create and express PlsX mutants for functional studies?

To create and express PlsX mutants for functional studies, researchers should employ site-directed mutagenesis on recombinant plasmids containing the wild-type gene. The QuickChange II XL site-directed mutagenesis kit has been successfully used for introducing point mutations into the amphipathic α-peptide of PlsX . For certain mutations (e.g., T253W, L254W, A261L, and A261W), alternative protocols may be required as reported in previous studies .

For expression in B. subtilis, researchers should:

• Amplify the wild-type or mutated plsX gene using primers such as MT-for and MT-rev.

• Insert the amplified gene into the vector pSG1151 between KpnI and XhoI restriction sites.

• Ensure all inserted genes have a stop codon at their 3′-end and verify sequences via full-length DNA sequencing.

• Transform B. subtilis strain 168 with the resulting plasmids.

• Select for integration of the cloned gene at the plsX locus by plating on LB agar containing 5 μg/ml chloramphenicol.

• Incubate overnight at 37°C .

For functional studies comparing growth rates between wild-type and mutant strains, pick single colonies to inoculate 1 ml LB culture supplemented with 50 μg/ml tryptophan at 37°C. After overnight growth, dilute cultures to a low optical density (OD600 = 0.05) to inoculate fresh LB medium in separate shake bottles at 37°C. Monitor cell growth by measuring OD600 at different time points .

What techniques are effective for visualizing PlsX subcellular localization?

Green Fluorescent Protein (GFP) fusion is a highly effective technique for visualizing PlsX subcellular localization. The recommended approach is:

• Use the pSG1729 vector for N-terminal fusion of GFP to PlsX.

• Introduce the A206K mutation into the gfp gene to avoid dimerization, creating a monomeric fluorescent protein (GFPm).

• Amplify the plsX gene from B. subtilis genomic DNA using appropriate primers.

• Insert the amplified gene into the modified pSG1729 vector between BamHI and XhoI restriction sites.

• Verify the construct by sequencing before expression in B. subtilis .

For examining the localization of specific domains, such as the amphipathic α-peptide, insert the coding sequence for residues 250–262 to the 3′-end of the GFP gene in the vector. This approach has successfully demonstrated that PlsX localizes to regions of increased fluidity (RIFs) in the membrane, and mutations in the amphipathic α-peptide can disrupt this localization pattern .

Fluorescence microscopy can then be used to observe the subcellular distribution of the fusion proteins, providing insights into the relationship between protein structure and localization.

How do mutations in the amphipathic α-peptide affect PlsX membrane association and cellular function?

Mutations in the amphipathic α-peptide significantly impact PlsX membrane association and cellular function. Different point mutations have distinct effects, as evidenced by experimental data:

The K257A mutation substantially weakens PlsX interaction with lipid bilayers and causes the protein to become mostly delocalized to the cytosol . This mutation leads to significant growth impairment, with cells showing a two-hour delay in transition to the exponential phase and a lower growth plateau compared to wild-type cells .

The T255W mutation redistributes PlsX predominantly to non-RIFs membrane regions rather than completely delocalizing it to the cytosol. While this mutation also impairs growth, the effect is less severe than with K257A mutation, suggesting that association with the membrane, even if not specifically with RIFs, is partially functional .

The S256A mutation preserves normal RIFs localization and results in growth curves almost indistinguishable from wild-type, indicating that this residue is not critical for membrane association .

Importantly, these point mutations are located far from the suspected active site and do not affect the catalytic activity of the purified recombinant protein. This provides compelling evidence that the subcellular localization itself, rather than changes in enzymatic activity, is crucial for the physiological function of PlsX .

What is the relationship between PlsX membrane association and phospholipid synthesis?

The relationship between PlsX membrane association and phospholipid synthesis is direct and essential. Experimental evidence shows that phospholipid synthesis is severely hampered in cells where PlsX is detached from the membrane . Metabolic labeling studies have demonstrated that cells with membrane-dissociated PlsX accumulate free fatty acids, indicating a disruption in the phosphatidic acid synthesis pathway .

This relationship exists because membrane association is required for the proper delivery of PlsX's product (acyl-phosphate) to PlsY, the next enzyme in the phosphatidic acid pathway. Even though mutations that prevent membrane association do not affect PlsX's transacylase activity in vitro, they significantly disrupt the flux through the phosphatidic acid pathway in vivo .

These findings support the dual role of PlsX in phospholipid synthesis:

• As a catalyst performing the acyl transfer reaction

• As a chaperone protein mediating substrate channeling into the pathway

The membrane association therefore ensures efficient metabolic flux by facilitating the handoff of intermediates between pathway enzymes, a critical aspect of cellular metabolism that goes beyond simple catalysis.

How does PlsX interact with membrane lipids, and what techniques can measure these interactions?

PlsX interacts with membrane lipids through its hydrophobic loop located at the tip of two amphipathic dimerization helices. This interaction can be measured and characterized using several sophisticated techniques:

• Liposome Sedimentation Assays: This technique can determine the binding affinity of PlsX to artificial membrane vesicles of defined composition .

• Differential Scanning Calorimetry (DSC): DSC can measure thermodynamic parameters of PlsX-lipid interactions. The table below shows DSC measurements for wild-type PlsX and various mutants interacting with DMPG (dimyristoylphosphatidylglycerol) liposomes:

This data reveals that wild-type PlsX significantly affects membrane properties in a concentration-dependent manner, while mutations like L254E reduce this effect, confirming the role of specific residues in membrane interaction.

• Fluorescence Microscopy: Using GFP-PlsX fusion proteins to visualize subcellular localization and dynamics of membrane association in living cells .

• Direct Binding Assays: Recombinant PlsX has been shown to directly bind to protein-free total lipids from B. subtilis, and mutations can weaken this interaction, as demonstrated by supplementary assays in the literature .

These techniques collectively provide a comprehensive view of PlsX-membrane interactions and can help identify critical residues involved in these interactions.

What is the catalytic mechanism of PlsX and how does it differ from similar enzymes?

The catalytic mechanism of PlsX involves substrate activation and transition-state stabilization by two strictly conserved residues: Lys184 and Asn229 . This mechanism differs significantly from phosphotransacetylases that catalyze similar acyl transfer reactions.

The active site of PlsX includes numerous residues lining a long, narrow gorge at the dimeric interface. Additionally, two positive residues form an ACP docking pad next to this interfacial gorge, which facilitates substrate recognition .

A distinctive feature of PlsX catalysis is the release of the acyl phosphate product near the membrane, which likely facilitates its membrane insertion . This spatial positioning of the product release is critical for the subsequent steps in the phosphatidic acid pathway.

The catalytic cycle involves:

• Recognition and binding of acyl-ACP at the ACP docking pad

• Entry of the acyl chain into the narrow gorge

• Activation of the substrate by Lys184 and Asn229

• Transfer of the acyl group to orthophosphate

• Release of the acyl phosphate product near the membrane surface

• Release of the ACP

This mechanism optimizes the transfer of hydrophobic acyl groups from the water-soluble ACP carrier to membrane-embedded pathway components, highlighting the elegant design of the bacterial phospholipid synthesis machinery.

How does PlsX recognize and interact with acyl-ACP substrates?

PlsX recognizes acyl-ACP substrates through a specialized ACP docking pad composed of two positively charged residues located adjacent to the interfacial gorge of the enzyme . This recognition involves electrostatic interactions between the negatively charged surface of ACP and the positively charged docking pad of PlsX.

The interaction follows these steps:

• Initial recognition through electrostatic complementarity between ACP and the docking pad

• Positioning of the acyl chain for entry into the narrow gorge at the dimeric interface

• Transfer of the acyl group from ACP to the active site of PlsX

The specificity of PlsX for different acyl-ACP substrates depends on the length and saturation of the acyl chain. The narrow gorge at the dimeric interface accommodates the acyl chain and positions it for transfer to orthophosphate.

How can researchers accurately measure PlsX enzymatic activity in vitro?

Accurate measurement of PlsX enzymatic activity in vitro requires careful consideration of its dual role as both an acyltransferase and a membrane-associated protein. Here is a comprehensive protocol for assessing PlsX activity:

• Transacylase Activity Assay:

  • Prepare reaction mixtures containing purified recombinant PlsX, acyl-ACP substrate, and orthophosphate

  • Incubate at 37°C for defined time periods

  • Quantify acyl phosphate formation using colorimetric or radiometric methods

  • Include appropriate controls, particularly comparing wild-type PlsX with catalytic mutants

• Membrane Binding Assessment:

  • Perform liposome sedimentation assays with defined phospholipid compositions

  • Quantify bound versus unbound protein using SDS-PAGE and densitometry

  • Calculate binding parameters to determine affinity for different membrane compositions

• Integrated Assay System:

  • Develop reconstituted systems containing both PlsX and PlsY in liposomes

  • Measure the complete conversion from acyl-ACP to lysophosphatidic acid

  • This approach can assess both the catalytic activity and the substrate channeling function

When interpreting results, it's important to note that mutations affecting membrane association (such as K257A) do not necessarily impact the intrinsic catalytic activity of the enzyme in vitro . Supplementary assays have shown that the K257A mutant protein maintains catalytic activity comparable to non-mutated PlsX in purified enzyme assays, despite causing significant growth impairment in vivo . This highlights the importance of combining in vitro activity measurements with in vivo functional studies to fully understand the biological role of PlsX.

How does PlsX contribute to bacterial membrane homeostasis?

PlsX plays a critical role in bacterial membrane homeostasis through several mechanisms:

• Regulation of Phospholipid Synthesis: As a key enzyme in the phosphatidic acid pathway, PlsX controls the flow of acyl groups from fatty acid synthesis into phospholipid biosynthesis. This regulatory function helps maintain appropriate membrane phospholipid composition .

• Coordination with Fatty Acid Synthesis: PlsX couples fatty acid synthesis with phospholipid production, ensuring that newly synthesized fatty acids are efficiently incorporated into membrane phospholipids. This coordination is essential for balanced membrane biogenesis during cell growth .

• Localization to Regions of Increased Fluidity (RIFs): PlsX preferentially associates with RIFs in the bacterial membrane, where it colocalizes with other enzymes involved in lipid metabolism. This spatial organization creates a functional lipid synthesis complex that enhances metabolic efficiency .

• Substrate Channeling: Beyond its catalytic role, PlsX functions as a chaperone protein that facilitates the transfer of intermediates between enzymes in the phospholipid synthesis pathway. This substrate channeling prevents the loss of hydrophobic intermediates and ensures efficient metabolic flux .

The importance of PlsX in membrane homeostasis is evident from growth studies with PlsX mutants. Strains expressing PlsX variants with impaired membrane association (K257A or T255W) show significant growth defects, including delayed transition to exponential phase and lower growth plateaus . These defects occur despite normal catalytic activity of the mutant proteins, highlighting the critical importance of proper subcellular localization for PlsX function.

What is the relationship between PlsX and antibiotic resistance in Bacillus subtilis?

The relationship between PlsX and antibiotic resistance in Bacillus subtilis centers on the interaction between PlsX and the lipopeptide antibiotic daptomycin. Research has shown that:

• Daptomycin exerts its antibacterial effect partly by causing detachment of PlsX and other peripheral proteins from the Regions of Increased Fluidity (RIFs) in the bacterial membrane .

• This detachment of PlsX disrupts phospholipid synthesis and cell wall biosynthesis, contributing to the bactericidal activity of daptomycin .

• The membrane-disrupting effects of daptomycin are complex and include multiple mechanisms beyond simply affecting PlsX localization .

The study of PlsX mutants with altered membrane association provides insights into potential mechanisms of daptomycin resistance. Bacteria might develop resistance by modifying the membrane association properties of PlsX or altering membrane lipid composition to preserve PlsX localization in the presence of daptomycin.

Understanding the molecular details of how PlsX interacts with the membrane and how this interaction is disrupted by antibiotics could lead to the development of new antimicrobial strategies or methods to overcome resistance. Future research should investigate whether clinical isolates with daptomycin resistance show alterations in PlsX sequence, expression, or localization.

How is PlsX activity regulated in response to environmental stress?

The regulation of PlsX activity in response to environmental stress involves complex mechanisms that adjust phospholipid synthesis to changing conditions. While the search results don't provide explicit information on stress regulation of PlsX, we can infer several likely regulatory mechanisms based on available data:

Experimental approaches to investigate stress regulation of PlsX could include:

• Transcriptomic and proteomic analyses of B. subtilis under various stress conditions

• Analysis of PlsX membrane association in response to stresses using GFP-fusion proteins

• Measurement of PlsX enzymatic activity in membrane fractions isolated from stressed cells

• Investigation of potential regulatory protein-protein interactions that might modulate PlsX activity

Understanding these regulatory mechanisms could provide insights into bacterial adaptation strategies and potentially reveal new targets for antimicrobial development.

How can structural insights into PlsX contribute to antimicrobial drug development?

Structural insights into PlsX can contribute significantly to antimicrobial drug development through several approaches:

• Targeting the Active Site: The identification of the critical catalytic residues (Lys184 and Asn229) and the detailed structure of the active site gorge at the dimeric interface provides opportunities for designing inhibitors that block enzymatic activity. Small molecules that mimic transition states or substrate features could be developed as competitive inhibitors.

• Disrupting Membrane Association: The amphipathic α-peptide responsible for membrane association represents a novel target for antimicrobial development. Compounds that bind to this region could displace PlsX from the membrane, mimicking one of the mechanisms of daptomycin action . Since membrane association is critical for in vivo function despite not affecting in vitro activity, this approach targets a vulnerability distinct from simple enzyme inhibition.

• Interfering with Protein-Protein Interactions: The positive ACP docking pad identified near the interfacial gorge presents an opportunity to develop compounds that disrupt PlsX-ACP interaction, thereby preventing substrate recognition and processing.

• Structure-Based Design: The known three-dimensional structure of PlsX enables rational drug design approaches, including virtual screening of compound libraries, fragment-based drug discovery, and structure-activity relationship studies.

• Species Selectivity: Comparative analysis of PlsX structures from different bacterial species could reveal species-specific features that allow for the development of narrow-spectrum antibiotics targeting specific pathogens while sparing beneficial bacteria.

The dual role of PlsX as both a catalyst and a chaperone protein mediating substrate channeling offers multiple points of intervention, potentially reducing the likelihood of resistance development through mutations that alter only one aspect of its function.

What are the current challenges in expressing and purifying functionally active recombinant PlsX?

Expressing and purifying functionally active recombinant PlsX presents several technical challenges that researchers must address:

• Membrane Association Properties: PlsX's natural tendency to associate with membranes through its amphipathic α-peptide can cause aggregation or improper folding during heterologous expression. Researchers have addressed this by using E. coli C43 (DE3) strain, which is specifically engineered for expressing membrane-associated proteins .

• Maintaining Dimeric Structure: PlsX functions as a dimer with the active site located at the dimeric interface . Ensuring proper oligomerization during expression and purification is critical for obtaining functionally active protein.

• Preserving Catalytic Residues: The catalytic mechanism relies on specific residues like Lys184 and Asn229 . Purification conditions must avoid modifications of these residues (e.g., through oxidation or chemical adduction).

• Optimizing Expression Conditions: Successful protocols have employed lower expression temperatures (18°C for 20 hours) after induction, which likely promotes proper folding by slowing down protein synthesis .

• Assessing Functional Activity: Due to PlsX's dual role as both an enzyme and a membrane-associating protein, comprehensive activity assessment requires both enzymatic assays and membrane binding studies. This dual functionality complication necessitates multiple validation approaches.

Current successful strategies include:

• Using the pET28a vector system with C-terminal hexahistidine tags

• Expression in E. coli C43 (DE3) cells

• Induction with 0.2 mM IPTG at 18°C for extended periods

• Rigorous quality control through both activity assays and membrane binding studies

Future work might focus on developing improved expression systems or fusion tags that stabilize the protein while maintaining both catalytic activity and membrane association properties.

What emerging techniques could advance our understanding of PlsX function in vivo?

Several emerging techniques hold promise for advancing our understanding of PlsX function in vivo:

• Super-Resolution Microscopy: Techniques like PALM (Photoactivated Localization Microscopy) or STORM (Stochastic Optical Reconstruction Microscopy) could provide nanoscale visualization of PlsX localization in bacterial membranes, offering unprecedented insights into its dynamic association with RIFs and interaction with other proteins in the phospholipid synthesis pathway.

• Cryo-Electron Tomography: This technique could reveal the three-dimensional organization of PlsX in the native cellular context, potentially capturing its interaction with membrane structures and partner proteins.

• Proximity Labeling Proteomics: Methods like BioID or APEX2 could identify proteins that interact with PlsX in vivo by tagging proteins in close proximity to PlsX. This would help map the protein interaction network of PlsX and discover potential regulatory partners.

• Single-Molecule Tracking: By labeling individual PlsX molecules with photoactivatable fluorophores, researchers could track their movement and dynamics in living cells, providing insights into how they locate and associate with specific membrane regions.

• Metabolic Flux Analysis with Stable Isotopes: Combined with mass spectrometry, this approach could quantify how alterations in PlsX function affect the flow of metabolites through the phospholipid synthesis pathway under various conditions.

• CRISPR-Based Genetic Screens: Genome-wide screens using CRISPR-Cas9 could identify genes that synthetically interact with PlsX, revealing unexpected functional connections and regulatory mechanisms.

• In-Cell NMR: This emerging technique could potentially provide structural information about PlsX in its native cellular environment, offering insights into conformational changes associated with membrane binding and catalysis.

These advanced techniques, combined with established biochemical and genetic approaches, would provide a more comprehensive understanding of PlsX function in the complex cellular context, potentially revealing new regulatory mechanisms and functional interactions that are not apparent from in vitro studies.