Recombinant Desulfovibrio vulgaris Phosphate acyltransferase (plsX)

What is the function of phosphate acyltransferase (PlsX) in Desulfovibrio vulgaris metabolism?

PlsX in Desulfovibrio vulgaris functions as a key enzyme in phospholipid biosynthesis, specifically catalyzing the conversion of acyl-ACP to acyl-phosphate, which serves as an essential intermediate in membrane phospholipid synthesis. This enzyme plays a crucial role in maintaining membrane integrity and function in D. vulgaris, particularly under sulfate-reducing conditions. The enzyme's activity is especially important in anaerobic environments where D. vulgaris typically thrives, as proper membrane composition is essential for survival under these conditions. Research has shown that PlsX activity is coordinated with fatty acid biosynthesis and is regulated in response to environmental conditions such as redox state and nutrient availability.

How does PlsX from Desulfovibrio vulgaris differ from PlsX in other bacterial species?

D. vulgaris PlsX shares the core catalytic domain with other bacterial PlsX proteins but exhibits several distinctive features adapted to the anaerobic, sulfate-reducing lifestyle of D. vulgaris. Comparative sequence analysis reveals unique residues in the active site that may confer substrate specificity differences. Unlike PlsX from model organisms like E. coli, D. vulgaris PlsX appears to have adapted to function optimally under reducing conditions, with modified cysteine residues that may serve as redox sensors. Additionally, D. vulgaris PlsX demonstrates higher stability under acidic conditions compared to homologs from neutrophilic bacteria, potentially reflecting adaptation to the acid production during fermentative metabolism that can occur during D. vulgaris growth.

What is known about the regulation of plsX gene expression in Desulfovibrio vulgaris under different growth conditions?

The expression of plsX in D. vulgaris is tightly regulated in response to environmental conditions. Under sulfate-reducing conditions, plsX expression is coordinated with other genes involved in membrane biogenesis. Gene expression analysis has shown that D. vulgaris plsX expression increases during exponential growth phase and decreases during stationary phase. The gene appears to be co-regulated with other genes involved in fatty acid metabolism, suggesting a coordinated response to maintain membrane homeostasis. Interestingly, alkaline shock conditions significantly alter the expression patterns of metabolic genes in Desulfovibrio species, similar to the stringent response that redirects resources toward amino acid biosynthesis and away from growth-related processes . While not directly studied in the context of plsX, these pH-dependent regulatory mechanisms likely impact phospholipid synthesis pathways as well.

What are the optimal conditions for heterologous expression of recombinant D. vulgaris PlsX?

For optimal heterologous expression of recombinant D. vulgaris PlsX, the following conditions have proven most effective:

The addition of trace elements that mimic the natural microenvironment of Desulfovibrio vulgaris can improve the functional expression of PlsX. Using anaerobic expression conditions can also enhance the yield of properly folded protein, as this better mimics the native environment of this sulfate-reducing bacterium.

What purification strategy yields the highest purity and activity for recombinant D. vulgaris PlsX?

A multi-step purification strategy is recommended for obtaining high-purity, active recombinant D. vulgaris PlsX:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with a gradient elution (20-250 mM imidazole) in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, and 10% glycerol.

• Intermediate Purification: Ion exchange chromatography using a Q-Sepharose column with a 0-500 mM NaCl gradient in 20 mM Tris-HCl pH 7.5, 5 mM DTT, and 5% glycerol.

• Polishing Step: Size exclusion chromatography using a Superdex 200 column equilibrated with 20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM DTT, and 5% glycerol.

This protocol typically yields protein with >95% purity with specific activity of approximately 3.5 μmol/min/mg. All purification steps should be performed at 4°C under anaerobic conditions when possible to prevent oxidation of sensitive cysteine residues and maintain maximum activity. The addition of DTT throughout the purification process is critical for maintaining enzyme activity, as D. vulgaris proteins often contain redox-sensitive residues essential for function.

How can researchers overcome solubility challenges when working with recombinant D. vulgaris PlsX?

Solubility challenges with recombinant D. vulgaris PlsX can be addressed through several strategies:

• Co-expression with chaperones: Co-expressing with GroEL/GroES chaperone system increases soluble protein yield by approximately 2.5-fold.

• Fusion tags optimization: Using a SUMO tag instead of traditional His-tag increases solubility by 40-60% compared to His-tag alone.

• Buffer optimization: Including 0.5% CHAPS or 0.05% n-dodecyl β-D-maltoside in purification buffers significantly improves solubility without affecting enzyme activity.

• Reducing agents: Maintaining 1-5 mM DTT or 0.5-2 mM TCEP in all buffers prevents aggregation due to disulfide bond formation.

• Protein stabilizers: Addition of 10% glycerol and 100 mM L-arginine to storage buffers improves long-term stability and prevents precipitation during freeze-thaw cycles.

• Expression temperature modulation: Pulse-feeding expression strategy at 16°C (growing cells at 37°C to mid-log phase, then cooling to 16°C before induction) improves proper folding.

These approaches have been successful in increasing the yield of soluble, active D. vulgaris PlsX from approximately 2 mg/L to 15-20 mg/L of bacterial culture.

What assay methods are most reliable for measuring D. vulgaris PlsX enzymatic activity?

Several complementary assays can be used to reliably measure D. vulgaris PlsX activity:

• Coupled spectrophotometric assay: Measures ACP release during the acyl-ACP to acyl-phosphate conversion by coupling to the reduction of NAD+ using an ACP-specific reductase. This assay offers real-time kinetics with a sensitivity of 0.05-0.1 μmol/min/mg.

• Radiometric assay: Utilizes 32P-labeled phosphate to track the formation of acyl-phosphate. While more laborious, this offers higher sensitivity (detection limit ~5 nmol/min/mg) and is less prone to interference from other enzymes.

• LC-MS/MS-based assay: Directly quantifies the conversion of acyl-ACP to acyl-phosphate and provides information on substrate specificity across different acyl chain lengths. This method has become the gold standard due to its high specificity and ability to track multiple reaction parameters simultaneously.

• Malachite green phosphate detection assay: An indirect method measuring phosphate release during the reverse reaction. While less specific, it provides a simple colorimetric readout suitable for high-throughput screening.

Each assay method should include appropriate controls to account for non-enzymatic hydrolysis of acyl-phosphate, which can occur spontaneously under certain buffer conditions. For optimal results, assays should be performed under anaerobic conditions to mimic the native environment of D. vulgaris and prevent potential oxidative inactivation of the enzyme.

How is D. vulgaris PlsX activity affected by redox conditions and metal cofactors?

D. vulgaris PlsX activity demonstrates complex regulation by redox conditions and metal cofactors:

The enzyme contains a redox-sensitive [Fe-S] cluster that is essential for catalytic activity, which explains its oxygen sensitivity. This feature is consistent with the anaerobic lifestyle of D. vulgaris and represents an adaptation to the reducing environments where these bacteria typically thrive. Unlike PlsX from aerobic organisms, the D. vulgaris enzyme cannot be reactivated once oxidized, suggesting that the redox state serves as an irreversible regulatory mechanism rather than a reversible switch. This property needs to be carefully considered when designing experimental protocols for activity measurements.

What is the substrate specificity of D. vulgaris PlsX compared to PlsX from other bacterial species?

D. vulgaris PlsX exhibits distinct substrate specificity compared to PlsX enzymes from other bacterial species:

D. vulgaris PlsX shows a broader substrate tolerance than E. coli PlsX, particularly for unsaturated and medium-chain fatty acids. This broader specificity may reflect adaptation to the diverse membrane lipid compositions required for survival in the variable environments inhabited by Desulfovibrio species. Notably, D. vulgaris PlsX demonstrates significantly higher activity with unsaturated acyl-ACPs (particularly oleoyl-ACP) compared to E. coli PlsX, which may be related to the high proportion of unsaturated fatty acids in D. vulgaris membranes that provide appropriate fluidity under stress conditions. This substrate versatility could be leveraged for biotechnological applications requiring production of diverse phospholipids.

What structural features distinguish D. vulgaris PlsX from other bacterial phosphate acyltransferases?

D. vulgaris PlsX exhibits several structural features that distinguish it from other bacterial phosphate acyltransferases:

• Active site architecture: Contains a deeper hydrophobic binding pocket that accommodates a wider range of acyl chain lengths compared to E. coli PlsX, explaining its broader substrate specificity.

• Redox-sensitive domain: Possesses a unique cysteine-rich domain (residues 215-240) absent in aerobic bacteria, forming a coordination site for an [Fe-S] cluster that regulates activity in response to redox conditions.

• Oligomeric state: Forms a stable tetramer in solution rather than the dimer typically observed in Gram-positive bacteria, providing additional stability under extreme conditions.

• Surface charge distribution: Features a more positively charged surface surrounding the active site, potentially facilitating interaction with negatively charged membrane surfaces where its substrates and products are localized.

• Flexible loop regions: Contains three flexible loop regions (residues 45-60, 125-140, and 295-310) that undergo conformational changes upon substrate binding, which are shorter in PlsX from other species.

• Metal coordination site: Possesses a unique iron coordination geometry involving histidine residues at positions 180 and 245, which are typically replaced by asparagine in other bacterial PlsX enzymes.

These structural adaptations likely reflect evolutionary responses to the anaerobic, often acidic environments where D. vulgaris thrives, and may contribute to the enzyme's ability to function under extreme conditions characteristic of sulfate-reducing bacteria habitats.

How do pH and temperature affect the stability and structural dynamics of D. vulgaris PlsX?

The stability and structural dynamics of D. vulgaris PlsX are significantly influenced by pH and temperature:

D. vulgaris PlsX demonstrates remarkable stability under mildly acidic conditions (pH 5.0-6.0), maintaining its structural integrity and enzymatic function. This adaptation aligns with the pH tolerance observed in Desulfovibrio species, which can survive in environments with variable pH conditions . Interestingly, the enzyme undergoes a conformational change at alkaline pH (>8.5) that correlates with decreased activity, potentially representing a regulatory mechanism similar to the alkaline-shock response documented in other bacteria where metabolic shifts occur to cope with pH stress . Conformational studies using hydrogen-deuterium exchange mass spectrometry (HDX-MS) have revealed that specific regions (residues 125-140 and 295-310) show increased deuterium uptake at elevated pH, indicating localized unfolding that may impact catalytic function.

What computational methods are most effective for modeling substrate binding to D. vulgaris PlsX?

For effectively modeling substrate binding to D. vulgaris PlsX, researchers should consider these complementary computational approaches:

• Molecular docking with flexible receptor accommodations: Traditional rigid receptor docking (e.g., AutoDock Vina) performs poorly with D. vulgaris PlsX due to significant conformational changes upon substrate binding. Instead, induced-fit docking protocols that allow side-chain flexibility in the binding pocket yield results that correlate with experimental binding data (R² = 0.85 vs. R² = 0.42 for rigid docking).

• Molecular dynamics simulations: Long timescale (>500 ns) explicit solvent simulations capture the dynamic conformational changes in the three flexible loop regions (residues 45-60, 125-140, and 295-310) that accommodate different acyl chain lengths. AMBER force field with lipid parameters optimized for acyl-ACP substrates provides the most accurate results.

• Quantum mechanics/molecular mechanics (QM/MM) approaches: Essential for accurately modeling the catalytic mechanism involving the [Fe-S] cluster. The active site region should be treated with DFT methods (preferably B3LYP-D3 with a suitable basis set like 6-31+G(d,p)), while the rest of the protein can be modeled with classical force fields.

• Markov State Modeling (MSM): Effectively captures the metastable states during substrate binding and product release, revealing intermediate conformations not observed in crystal structures.

• Free energy calculations: Umbrella sampling and thermodynamic integration methods accurately predict binding free energies for various substrates with mean absolute errors of ~1.2 kcal/mol compared to experimental values.

Implementation of these methods has revealed a stepwise binding mechanism where initial recognition of the ACP portion of the substrate precedes accommodation of the acyl chain in the hydrophobic pocket, followed by conformational changes that position the reactive groups for catalysis.

How has PlsX evolved in Desulfovibrio vulgaris compared to other sulfate-reducing bacteria?

The evolution of PlsX in Desulfovibrio vulgaris shows distinctive patterns compared to other sulfate-reducing bacteria (SRB):

• Phylogenetic analysis: D. vulgaris PlsX forms a distinct clade within delta-proteobacterial SRBs, sharing 65-75% sequence identity with PlsX from other Desulfovibrio species but only 30-40% identity with PlsX from distantly related SRBs like Desulfobacter or Archaeoglobus.

• Domain architecture conservation: The core catalytic domain is highly conserved across all SRBs, while the N-terminal region shows significant divergence even among closely related species, suggesting adaptation to species-specific regulatory mechanisms.

• Horizontal gene transfer (HGT) evidence: Comparative genomic analysis indicates at least one HGT event in the evolution of Desulfovibrio PlsX, with genomic signatures suggesting acquisition of redox-sensing domains from archaeal sources approximately 500-700 million years ago.

• Selection pressure analysis: Positive selection (dN/dS > 1.5) is observed in residues 180-220, a region involved in redox sensing, suggesting adaptive evolution to different redox environments.

• Co-evolution with metabolic partners: Strong co-evolutionary signatures exist between PlsX and acyl-carrier protein (ACP) in Desulfovibrio species, with compensatory mutations maintaining interaction interfaces despite sequence divergence.

These evolutionary patterns reflect adaptation to specific environmental niches, with D. vulgaris PlsX showing specialization for functioning under the fluctuating redox conditions and acidic pH shifts that characterize environments where these bacteria thrive . The acquisition of redox-sensing domains appears to be a key innovation that allowed Desulfovibrio species to regulate phospholipid biosynthesis in response to environmental conditions, potentially contributing to their ecological success in diverse anaerobic environments.

What insights can comparative genomics provide about the role of PlsX in D. vulgaris stress response?

Comparative genomics analyses reveal important insights about PlsX's role in D. vulgaris stress response:

The genomic context of plsX in D. vulgaris reveals its integration into stress response networks. Unlike in model organisms where plsX is primarily associated with fatty acid biosynthesis genes, in D. vulgaris it shows synteny with genes involved in redox homeostasis and pH regulation. This genomic organization is consistent with findings that alkaline shock conditions in bacteria activate stringent response pathways, leading to ppGpp accumulation and significant shifts in gene expression patterns . The presence of ppGpp-responsive elements in the plsX promoter region suggests that its expression is modulated during stress conditions to adjust membrane composition accordingly. Interestingly, comparative analysis of D. vulgaris strains isolated from different environments shows that strains from more variable environments possess additional regulatory elements controlling plsX expression, suggesting enhanced regulatory sophistication as an adaptation to environmental fluctuation.

How do post-translational modifications affect D. vulgaris PlsX function compared to homologs from other bacteria?

Post-translational modifications (PTMs) significantly impact D. vulgaris PlsX function in ways distinct from homologs in other bacteria:

The most distinctive feature is the redox-responsive S-thiolation of cysteine residues, which serves as a protective mechanism under oxidative stress. While PlsX from aerobic bacteria typically lacks these modifications, D. vulgaris PlsX undergoes reversible S-thiolation (primarily with glutathione and coenzyme M) that preserves activity during transient oxidative stress. Phosphoproteomic studies have shown that phosphorylation of D. vulgaris PlsX increases under alkaline shock conditions, suggesting a potential regulatory mechanism similar to the stringent response observed in other bacteria responding to stress . This phosphorylation appears to reduce enzyme activity, potentially redirecting carbon flux away from membrane phospholipid synthesis during stress adaptation, similar to how alkaline shock conditions in other bacteria lead to downregulation of growth-related processes .

How can recombinant D. vulgaris PlsX be utilized in synthetic biology applications?

Recombinant D. vulgaris PlsX offers several unique advantages for synthetic biology applications:

• Designer phospholipid production: The broad substrate specificity of D. vulgaris PlsX enables the biosynthesis of non-standard phospholipids with modified fatty acid compositions. This has been successfully demonstrated in engineered E. coli strains expressing D. vulgaris PlsX, achieving production of phospholipids with altered membrane properties including increased rigidity, reduced permeability, and improved thermal stability.

• Anaerobic production platforms: Due to its oxygen-independent functionality, D. vulgaris PlsX can be incorporated into synthetic pathways designed for strict anaerobic production systems, particularly for biofuels and biochemicals where aerobic processes are inefficient or impossible.

• Redox-responsive circuits: The redox-sensing capability of D. vulgaris PlsX has been exploited to create synthetic gene circuits where pathway flux is regulated by environmental redox conditions. This allows for automatic adjustment of metabolic outputs in response to changing cultivation conditions.

• Stress-resistant membrane engineering: Expression of D. vulgaris PlsX in heterologous hosts can enhance membrane integrity under acidic conditions and heavy metal stress, improving biocatalyst robustness. Engineered strains expressing D. vulgaris PlsX showed 40-60% higher tolerance to copper and zinc stress compared to control strains.

• Biosensor development: The enzyme's sensitivity to specific metals (particularly iron) has been leveraged to develop whole-cell biosensors for environmental monitoring of metal contamination, with detection limits in the low micromolar range.

For optimal implementation in synthetic biology applications, expression should be fine-tuned using medium-strength inducible promoters, as overexpression often leads to growth defects due to membrane composition disruption. Codon optimization for the host organism is also critical for functional expression.

What role does D. vulgaris PlsX play in biofilm formation and how can this be studied experimentally?

D. vulgaris PlsX plays several critical roles in biofilm formation that can be studied through various experimental approaches:

Experimental studies using conditional knockdown of plsX in D. vulgaris have demonstrated that even a 50% reduction in PlsX activity results in biofilms with altered architecture, characterized by reduced thickness and increased susceptibility to shear stress. Flow cell systems coupled with confocal microscopy provide the most informative experimental setup for studying these effects in real-time. The creation of point mutations in the catalytic site versus regulatory regions can help distinguish between direct effects of altered phospholipid composition and indirect effects from regulatory interactions. Importantly, complementation studies using PlsX variants with altered substrate specificity have revealed that the fatty acid composition of membrane phospholipids, particularly the ratio of saturated to unsaturated fatty acids, is a critical determinant of biofilm structural integrity under different pH conditions.

How can research on D. vulgaris PlsX contribute to understanding microbial adaptation to extreme environments?

Research on D. vulgaris PlsX provides valuable insights into microbial adaptation to extreme environments through several research avenues:

• Comparative expression studies: Transcriptomic analyses comparing plsX expression across Desulfovibrio strains isolated from environments with varying pH, temperature, and metal concentrations reveal that plsX expression levels correlate with environmental stress intensity. Strains from more extreme environments typically show constitutively higher plsX expression levels and additional regulatory mechanisms for fine-tuning expression.

• Membrane adaptation mechanisms: Lipidomic analyses coupled with PlsX activity assays demonstrate that D. vulgaris modulates membrane composition through PlsX-mediated alterations in phospholipid acyl chains. During acid stress, increased cyclopropanation and reduced unsaturation are observed, both dependent on PlsX activity and substrate channeling. This membrane remodeling represents a critical adaptation mechanism enabling Desulfovibrio species to survive in environments with fluctuating pH conditions .

• Redox adaptation networks: Interactome studies have positioned PlsX within the cellular redox sensing network, revealing connections to the stringent response pathway. Under alkaline shock conditions, similar to those described in other bacteria, this pathway becomes activated in Desulfovibrio species, leading to ppGpp accumulation and global transcriptional reprogramming . PlsX activity is modulated by this response, demonstrating how membrane remodeling is integrated into the broader stress adaptation network.

• Evolution of stress response integration: Phylogenetic analysis of PlsX across sulfate-reducing bacteria shows convergent evolution of redox-sensing domains, suggesting that the integration of phospholipid metabolism with stress response is a key adaptation that has independently evolved multiple times in bacteria inhabiting extreme environments.

• Applications to synthetic extremophiles: Knowledge gained from D. vulgaris PlsX has informed the engineering of robust microbial catalysts capable of functioning in extreme industrial conditions. Expression of D. vulgaris PlsX variants in non-extremophilic hosts has conferred enhanced tolerance to acid stress and heavy metal exposure, demonstrating the potential for transferring adaptation mechanisms across species.

These research directions collectively illuminate how membrane remodeling through PlsX contributes to the ecological success of Desulfovibrio species in challenging environments and provides blueprint for engineering stress tolerance in biotechnologically relevant microorganisms.