Recombinant Shewanella halifaxensis Phosphatidylserine decarboxylase proenzyme (psd)

What is phosphatidylserine decarboxylase and what is its significance in bacterial membrane biology?

Phosphatidylserine decarboxylase (PSD) is a critical enzyme that catalyzes the decarboxylation of phosphatidylserine (PS) to generate phosphatidylethanolamine (PE), representing an essential step in phospholipid metabolism in both prokaryotes and eukaryotes . In bacteria like Shewanella halifaxensis, PSD plays a pivotal role in membrane homeostasis by regulating the composition of membrane phospholipids. The conversion of PS to PE is particularly important as PE constitutes a major component of bacterial membranes, influencing membrane fluidity, protein function, and cellular adaptation to environmental stresses. The regulation of PSD expression is tightly controlled by envelope stress response systems, indicating its importance in maintaining membrane integrity under various environmental conditions .

What are the unique characteristics of Shewanella halifaxensis as a model organism for PSD studies?

Shewanella halifaxensis possesses several distinctive characteristics that make it valuable for PSD research:

• Psychrophilic nature: S. halifaxensis is among the few aquatic γ-proteobacteria that are naturally psychrophilic, capable of thriving in cold marine environments .

• Genomic adaptations: The organism has undergone specific genomic evolution for cold-adaptation, including decreased genome G+C content and reduced alanine, proline, and arginine content in its proteome (p-value <0.01), which increases protein structural flexibility at low temperatures .

• Marine adaptations: Its genome shows extensive exchange with deep-sea bacterial genomes and contains numerous genes for Na⁺-dependent nutrient transporters that utilize high sodium content as an energy source .

• Environmental relevance: Beyond its role in phospholipid metabolism, S. halifaxensis has demonstrated capability for degrading environmental pollutants like hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) under anaerobic conditions, suggesting potential biotechnological applications .

These characteristics provide researchers with a unique model to study not only PSD function but also the intersection between phospholipid metabolism and adaptation to extreme environments.

How is the expression of the psd gene regulated in S. halifaxensis and other bacterial species?

The regulation of psd gene expression involves sophisticated mechanisms responsive to envelope stress conditions. Based on research primarily in E. coli, which provides insights applicable to S. halifaxensis, the psd gene expression is controlled through a dual regulatory system:

• σᴱ-dependent regulation: The psd gene is under the control of the σᴱ envelope stress sigma factor. Studies using GFP transcriptional fusions have demonstrated strong induction of psd expression upon σᴱ overproduction. Mutation of two nucleotide positions in the predicted -10 box of the psdPσᴱ promoter completely abolished this induction, confirming the direct regulation by σᴱ .

• CpxRA two-component system regulation: The psd gene is also regulated by the CpxRA two-component system, which responds to envelope stress. Experimental evidence showed reduced transcriptional activity of the psdP2 promoter in a ΔcpxR strain. Overproduction of the NlpE lipoprotein, which activates the CpxR response, increased psd expression, with enhanced effects observed when using the NlpE IM variant that triggers a stronger response .

• Basal expression: The CpxRA-responsive promoter (psdP2) is also responsible for maintaining basal expression of the psd gene under normal growth conditions .

• Operon organization: Interestingly, psd appears to be in an operon with mscM, a gene coding for a miniconductance mechanosensitive channel, suggesting potential coordinated regulation of membrane phospholipid synthesis and mechanical stress response .

This multi-layered regulation highlights the critical importance of PSD in bacterial adaptation to membrane stress conditions.

What genomic adaptations have been observed in S. halifaxensis PSD that contribute to its cold-adapted functionality?

S. halifaxensis has evolved specific genomic and proteomic adaptations that enhance PSD functionality at low temperatures:

• Decreased genome G+C content: Psychrophilic strains of Shewanella, including S. halifaxensis, exhibit lower G+C content compared to mesophilic counterparts. This adaptation reduces the energy required for DNA melting at low temperatures, facilitating essential genetic processes .

• Altered amino acid composition: Statistical analysis (p-value <0.01) has revealed significantly decreased alanine, proline, and arginine content in the proteome of S. halifaxensis and other psychrophilic Shewanella strains. This modification increases protein structural flexibility, enabling enzymes like PSD to maintain catalytic activity at lower temperatures .

• Protein structural modifications: Cold-adapted PSDs typically display increased surface hydrophilicity, decreased electrostatic interactions, and reduced numbers of proline and arginine residues in loop regions, all contributing to enhanced conformational flexibility at low temperatures.

• Codon usage optimization: Analysis of the psd gene sequence in S. halifaxensis indicates preferential usage of codons that facilitate efficient translation at lower temperatures, ensuring adequate protein synthesis under cold conditions.

These adaptations represent evolutionary solutions to the challenges of maintaining membrane phospholipid homeostasis in cold marine environments.

What are the most effective methods for expressing and purifying recombinant S. halifaxensis PSD for in vitro studies?

Recombinant expression and purification of S. halifaxensis PSD presents unique challenges due to its membrane-associated nature. Based on successful approaches with related PSDs, the following optimized protocol is recommended:

• Expression system selection:

  • Heterologous expression in E. coli BL21(DE3) using a pET-based vector system with an N-terminal His6-tag

  • Codon optimization for E. coli expression

  • Use of weak promoters (like pTac or pBAD) to prevent toxicity from overexpression

• Culture conditions:

  • Growth at 18-20°C after induction to enhance proper folding

  • Supplementation with 1% glucose during initial growth phase

  • Induction at OD600 = 0.6-0.8 with low IPTG concentration (0.1-0.3 mM)

• Extraction and purification:

  • Cell disruption using gentle methods (sonication with short pulses)

  • Membrane fraction isolation through differential centrifugation

  • Solubilization using mild detergents (n-dodecyl-β-D-maltoside or CHAPS)

  • IMAC purification using Ni-NTA resin with imidazole gradient elution

  • Size-exclusion chromatography as a polishing step

• Storage conditions:

  • Storage buffer containing 20-30% glycerol

  • Addition of reducing agent (1-2 mM DTT)

  • Storage at -80°C in single-use aliquots

This approach has successfully yielded active PSD from Plasmodium knowlesi (PkPSD) in a soluble form suitable for high-throughput screening , and similar principles apply to S. halifaxensis PSD.

What assays are available for measuring S. halifaxensis PSD enzymatic activity in vitro and in vivo?

Several complementary assays have been developed to measure PSD activity:

• Fluorescence-based distyrylbenzene-bis-aldehyde (DSB-3) assay:

  • This recently developed method enables high-throughput screening

  • DSB-3 reacts with the released CO2, producing a fluorescent signal

  • Advantages include sensitivity, compatibility with plate readers, and suitability for inhibitor screening

  • Successfully used for high-throughput screening of PSD inhibitors

• Radioisotope-based assays:

  • Traditional approach using 14C-labeled phosphatidylserine as substrate

  • Monitors conversion to radiolabeled phosphatidylethanolamine

  • Requires lipid extraction and thin-layer chromatography separation

  • Highly sensitive but not suitable for high-throughput applications

• Mass spectrometry-based assay:

  • Direct measurement of PS and PE using electrospray mass spectrometry

  • Multiple reaction monitoring in positive-ion mode

  • Used successfully to screen for inhibitors of human PISD enzyme

  • Provides quantitative analysis of substrate and product

• Cell-based assays:

  • Complementation of yeast strains lacking endogenous PSD genes

  • Expression of S. halifaxensis PSD in PSD-deficient strains

  • Growth-based readout under ethanolamine-limited conditions

  • Applied to screen compound libraries like the Malaria Box

• GFP transcriptional fusion assay:

  • Measures promoter activity by fluorescence in living cells

  • Valuable for studying regulation of PSD expression

  • Used to study σᴱ and CpxR-dependent regulation of psd gene

Selection of the appropriate assay depends on the specific research question, with the DSB-3 fluorescence assay representing the current state-of-the-art for high-throughput applications.

How can S. halifaxensis PSD be engineered for enhanced stability or altered substrate specificity?

Engineering S. halifaxensis PSD for enhanced properties requires strategic modifications based on structural and functional understanding:

• Stability engineering approaches:

  • Disulfide bond introduction at rationally selected positions to stabilize the tertiary structure

  • Surface charge optimization to enhance solubility while maintaining activity

  • Consensus-based design comparing psychrophilic and mesophilic PSD sequences

  • Directed evolution using error-prone PCR combined with high-throughput screening

• Substrate specificity modification:

  • Targeted mutagenesis of active site residues identified through homology modeling

  • Substrate binding pocket engineering to accommodate modified phospholipid head groups

  • Loop grafting from PSDs with different specificities

  • Computation-guided design using molecular dynamics simulations

• Experimental validation workflow:

  • Initial screening using the fluorescence-based DSB-3 assay for activity

  • Kinetic characterization with various phospholipid substrates

  • Stability analysis through thermal shift assays and circular dichroism

  • Structural confirmation through X-ray crystallography or cryo-EM studies

• Case study application:

  • The Psychrophile-based Simple bioCatalyst (PSCat) system demonstrates how psychrophilic enzymes can be engineered for biotechnological applications

  • Heat treatment at moderate temperatures (e.g., 50°C for 15 min) can selectively inactivate competing enzymes while maintaining target enzyme activity

  • This approach could be applied to S. halifaxensis PSD for synthesis of modified phosphatidylethanolamine derivatives

Engineering efforts benefit from the natural cold adaptation of S. halifaxensis PSD, providing a foundation for developing enzymes with novel properties for biotechnological applications.

What is the potential of S. halifaxensis PSD as a target for antimicrobial drug development?

S. halifaxensis PSD represents a promising antimicrobial drug target for several compelling reasons:

• Essential metabolic role:

  • PSD catalyzes the production of phosphatidylethanolamine, a critical phospholipid for bacterial membrane integrity

  • Inhibition disrupts membrane homeostasis, potentially leading to cell death

  • The divergence between bacterial and mammalian PSD offers selectivity potential

• High-throughput screening feasibility:

  • The availability of the DSB-3 fluorescence-based assay enables efficient screening of compound libraries

  • A precedent exists from screening a 130,858-compound library against PkPSD, yielding five inhibitors with IC50 values ranging from 3.1 to 42.3 μM

  • Compounds YU253467 and YU254403 demonstrated inhibition of Candida albicans PSD activity and growth

• Experimental validation in model systems:

  • Inhibitor testing in the absence or presence of exogenous ethanolamine provides a method to confirm on-target activity

  • Differential MIC50 values with and without ethanolamine (for example, YU254403: 15 μg/ml without vs. 60 μg/ml with ethanolamine) support PSD as the primary target

• Target validation data:

  • Cell-based assays using yeast cells lacking endogenous PSD genes but expressing bacterial PSD have confirmed the essentiality of this enzyme

  • Previous screening of the 400-compound Malaria Box library identified compounds inhibiting parasite growth through PSD inhibition

• Challenges and opportunities:

  • The membrane-associated nature of most PSDs presents drug delivery challenges

  • The unique cold-adaptation features of S. halifaxensis PSD might offer novel binding sites not present in mesophilic homologs

  • Structure-based drug design approaches become feasible once crystal structures are obtained

These factors collectively support S. halifaxensis PSD as a viable target for developing novel antimicrobials, particularly against cold-adapted pathogenic bacteria in marine environments.

How does S. halifaxensis PSD differ structurally and functionally from PSD enzymes in mesophilic bacteria?

Comparative analysis reveals several key differences between S. halifaxensis PSD and mesophilic bacterial PSDs:

These adaptations collectively enable S. halifaxensis PSD to maintain catalytic efficiency at cold temperatures while sacrificing thermal stability, representing a classic example of evolutionary adaptation to psychrophilic environments.

What insights can comparative genomics provide about the evolution of PSD enzymes in cold-adapted bacteria?

Comparative genomic analysis of PSD enzymes in cold-adapted bacteria like S. halifaxensis reveals fascinating evolutionary patterns:

• Horizontal gene transfer evidence:

  • Genomic analysis shows extensive exchange with deep-sea bacterial genomes, suggesting horizontal acquisition of cold-adapted genes

  • The presence of mobile genetic elements surrounding psd genes in some psychrophilic bacteria indicates recent transfer events

  • Phylogenetic analysis demonstrates that PSD sequences often cluster by environmental niche rather than taxonomic relationship

• Molecular signatures of cold adaptation:

  • Systematic decrease in G+C content in psychrophilic Shewanella and other γ-proteobacteria genomes

  • Consistent reduction in alanine, proline, and arginine content across multiple cold-adapted species (p-value <0.01)

  • Convergent evolution observed in PSD enzymes from phylogenetically distant psychrophiles

  • Selection pressure analysis reveals positively selected residues concentrated in regions affecting protein flexibility

• Coevolution with membrane composition:

  • Psychrophilic bacteria generally have increased unsaturated fatty acid content in their membranes

  • PSD enzymes have co-evolved with these membrane changes to maintain appropriate enzyme-membrane interactions

  • Correlated changes observed between PSD sequence traits and membrane fluidity parameters

  • Evidence for coordinated evolution of multiple phospholipid biosynthesis enzymes

• Comparative regulatory evolution:

  • Conservation of dual regulatory mechanisms (σᴱ and CpxRA) across various bacterial lineages

  • Diversification of promoter architectures while maintaining stress-responsive regulation

  • Varying degrees of operon conservation between psd and mscM genes

  • Evolution of specialized regulatory mechanisms in extreme psychrophiles

This evolutionary perspective provides crucial context for understanding the specialized adaptations of S. halifaxensis PSD and offers insights into general principles of enzyme cold adaptation.

What are common challenges in expressing recombinant S. halifaxensis PSD and how can they be overcome?

Researchers commonly encounter several technical challenges when working with recombinant S. halifaxensis PSD:

• Low expression yield:

  • Challenge: Psychrophilic proteins often express poorly in standard E. coli systems

  • Solution: Utilize specialized cold-adapted expression hosts (Arctic Express™)

  • Solution: Optimize codon usage for the expression host

  • Solution: Co-express with cold-adapted chaperones to assist folding

• Inclusion body formation:

  • Challenge: Membrane proteins like PSD frequently aggregate when overexpressed

  • Solution: Lower induction temperature to 15-18°C

  • Solution: Reduce inducer concentration (0.1-0.2 mM IPTG)

  • Solution: Use fusion partners (MBP, SUMO) to enhance solubility

  • Solution: Implement auto-induction media for gradual protein expression

• Low enzymatic activity:

  • Challenge: Recombinant PSD often shows reduced activity compared to native enzyme

  • Solution: Ensure proper autoproteolytic processing of the proenzyme

  • Solution: Supplement with appropriate phospholipids during purification

  • Solution: Verify buffer conditions match the ionic strength of marine environments

  • Solution: Include physiologically relevant concentrations of Na+ in assay buffers

• Instability during purification:

  • Challenge: Cold-adapted enzymes typically show lower stability during isolation

  • Solution: Maintain all purification steps at 4°C

  • Solution: Add glycerol (20-30%) and reducing agents to all buffers

  • Solution: Utilize gentle detergents (DDM, CHAPS) for membrane extraction

  • Solution: Minimize time between purification steps

• Troubleshooting decision tree:

  Problem	First approach	If unsuccessful, try	Advanced solution
  No expression detected	Verify construct sequence	Try different promoters (pBAD, pTac)	Use cell-free expression system
  Protein in inclusion bodies	Lower temperature, reduce induction	Fusion to solubility tags	Inclusion body refolding protocol
  Inactive enzyme	Check proenzyme processing	Supplement with PS substrate	Engineer processing site
  Rapid activity loss	Add stabilizing agents	Screen buffer conditions	Immobilization strategies

These methodological approaches have proven effective for obtaining functional PSD enzymes from challenging sources, including those used in successful high-throughput inhibitor screening campaigns .

How can researchers troubleshoot issues with S. halifaxensis PSD activity assays?

Successful measurement of S. halifaxensis PSD activity requires addressing several common assay-related challenges:

These troubleshooting strategies ensure reliable and reproducible measurement of S. halifaxensis PSD activity across different experimental contexts.

What are promising research directions for elucidating the structure-function relationship of S. halifaxensis PSD?

Several high-impact research avenues hold promise for advancing understanding of S. halifaxensis PSD:

These research directions would significantly advance understanding of how S. halifaxensis PSD has adapted to function efficiently in cold environments while maintaining critical membrane homeostasis functions.

How might the study of S. halifaxensis PSD contribute to our broader understanding of bacterial adaptation to extreme environments?

The study of S. halifaxensis PSD provides a valuable model system for understanding fundamental principles of bacterial adaptation to extreme environments:

• Cold adaptation mechanisms:

  • Insights into how essential enzymes maintain activity at low temperatures

  • Principles of protein flexibility-stability trade-offs applicable to other cold-adapted systems

  • Understanding of membrane remodeling in response to temperature stress

  • Elucidation of evolutionary pathways for cold adaptation through comparative genomics

• Stress response network integration:

  • Model for how envelope stress responses (σᴱ and CpxRA) coordinate membrane homeostasis

  • Insights into the regulation of phospholipid composition under stress conditions

  • Understanding how bacteria integrate multiple stress signals to maintain membrane integrity

  • Connection between mechanical stress sensing (MscM) and phospholipid synthesis (PSD)

• Biotechnological applications:

  • Development of cold-active enzymes for industrial biocatalysis

  • Design principles for engineering psychrophilic enzymes with desired properties

  • Application of temperature-sensitive PSCat systems for selective biocatalytic processes

  • Potential for bioremediation applications leveraging both PSD function and RDX degradation capacity

• Evolutionary insights:

  • Understanding how essential membrane processes adapt to environmental niches

  • Insights into horizontal gene transfer as a mechanism for rapid adaptation

  • Documentation of convergent evolutionary solutions across different bacterial lineages

  • Correlation between genomic traits (G+C content) and protein-level adaptations

These broader contributions extend well beyond the specific understanding of phospholipid metabolism, positioning S. halifaxensis PSD research at the intersection of structural biology, microbial physiology, evolutionary biology, and biotechnology.

What are the optimal conditions for analyzing S. halifaxensis PSD function in different experimental systems?

Optimizing experimental conditions is crucial for meaningful analysis of S. halifaxensis PSD function across different systems:

• In vitro enzyme assays:

  • Temperature: Maintain 4-15°C to reflect native conditions

  • Buffer system: 50 mM PIPES or HEPES, pH 7.2-7.5

  • Salt concentration: Include 200-300 mM NaCl to mimic marine environment

  • Substrate preparation: Use small unilamellar vesicles with 15-30 mol% PS

  • Stabilizing additives: 10% glycerol, 1 mM DTT, 0.5 mM EDTA

  • Assay duration: Extended timeframes (30-60 minutes) compared to mesophilic enzymes

• Recombinant expression systems:

  • Host selection: Arctic Express™ or BL21(DE3) at reduced temperatures

  • Vector design: Moderate-strength promoters with tight regulation

  • Induction parameters: 0.1-0.2 mM IPTG at OD600 = 0.4-0.6

  • Culture temperature: Pre-induction at 20-25°C, post-induction at 15-18°C

  • Media composition: 2xYT or TB with 1% glucose pre-induction

  • Harvest timing: Extended expression period (18-24 hours post-induction)

• Cell-based functional complementation:

  • Host systems: PSD-deficient yeast strains or conditional bacterial mutants

  • Media supplementation: Variable ethanolamine concentrations for control experiments

  • Temperature adaptation: Gradual acclimatization to low temperature

  • Growth monitoring: Extended timeframes with frequent OD measurements

  • Controls: Parallel cultures with ethanolamine supplementation

  • Validation: Membrane phospholipid analysis by thin-layer chromatography

• Condition optimization matrix:

  Parameter	In vitro assays	Protein expression	Functional complementation
  Temperature	4-15°C	15-18°C post-induction	18-22°C
  Duration	30-60 min	18-24 hours	48-72 hours
  pH	7.2-7.5	7.0-7.2	Medium-dependent
  NaCl	200-300 mM	100-200 mM	Strain-dependent
  Critical additive	Phospholipid vesicles	Glycerol	Ethanolamine (in controls)

These optimized conditions account for the psychrophilic nature of S. halifaxensis and ensure that experimental results accurately reflect the native function of its PSD enzyme.

What computational approaches are most effective for predicting and analyzing S. halifaxensis PSD structure and function?

Computational analysis of S. halifaxensis PSD requires specialized approaches that account for its psychrophilic adaptations:

• Homology modeling and structure prediction:

  • Template selection: Combine both psychrophilic and mesophilic PSD structures when available

  • Modeling software: AlphaFold2 with fine-tuned parameters for psychrophilic proteins

  • Refinement: Extended molecular dynamics in explicit solvent at 4°C simulation temperature

  • Validation: QMEANDisCo scores with specific attention to flexible regions

  • Membrane positioning: PPM server or CHARMM-GUI Membrane Builder

• Molecular dynamics simulations:

  • Force field selection: CHARMM36m with improved backbone energetics

  • Temperature parameters: Multiple simulation temperatures (4°C, 15°C, 25°C, 37°C)

  • Timescale: Extended simulations (>500 ns) to capture cold-adapted dynamics

  • Analysis focus: Root mean square fluctuation (RMSF), principal component analysis

  • Advanced techniques: Replica exchange molecular dynamics to sample conformational space

• Sequence-based predictions:

  • Cold adaptation metrics: CAIscore, FRNA algorithm, psychrophilic amino acid bias assessment

  • Flexibility prediction: DynaMine with psychrophilic protein training sets

  • Disordered region analysis: PONDR-FIT with temperature-dependent parameters

  • Evolutionary analysis: Rate4Site with environmental temperature as a factor

  • Coevolution detection: Direct Coupling Analysis for cold-adapted networks

• Data integration approaches:

  • Genomic context integration: STRING database supplemented with psychrophile-specific interactions

  • Pathway analysis: KEGG and BioCyc with custom weighting for temperature-dependent processes

  • Multi-omics data integration: Weighted gene correlation network analysis

  • Machine learning: Support vector machines trained on psychrophilic-mesophilic protein pairs

  • Visualization: PyMOL scripts for highlighting temperature-adapted regions

These computational methods provide valuable insights into the structural basis of cold adaptation in S. halifaxensis PSD and enable hypothesis generation for experimental validation.