Recombinant Aliivibrio salmonicida Glycerol-3-phosphate acyltransferase (plsY)
Description
Introduction to Aliivibrio salmonicida and its Enzymatic Machinery
Aliivibrio salmonicida (formerly classified as Vibrio salmonicida) is a Gram-negative bacterium that functions as the causative agent of cold water vibriosis in sea-farmed Atlantic salmon and other commercially important fish species . This pathogen causes significant economic losses in aquaculture, though the disease is now largely controlled through vaccination programs . The bacterium exhibits a complex genome architecture consisting of two chromosomes and four plasmids, creating a six-partite structure that contributes to its evolutionary adaptability and pathogenic potential .
Glycerol-3-phosphate acyltransferase (plsY) represents a critical enzymatic component in the bacterial phospholipid biosynthesis pathway. This enzyme specifically catalyzes the transfer of acyl groups from acyl-phosphate to glycerol-3-phosphate, generating lysophosphatidic acid—a fundamental precursor molecule for membrane phospholipids . The recombinant form of this enzyme from Aliivibrio salmonicida has been expressed with an N-terminal histidine tag to facilitate laboratory purification and subsequent research applications.
Functional Role in Bacterial Metabolism
Glycerol-3-phosphate acyltransferase (plsY) from Aliivibrio salmonicida performs an essential function in bacterial membrane phospholipid biosynthesis. The enzyme is alternatively known by several descriptive synonyms that highlight its biochemical role: acyl-PO4 G3P acyltransferase, acyl-phosphate--glycerol-3-phosphate acyltransferase, G3P acyltransferase (GPAT), lysophosphatidic acid synthase, and LPA synthase .
The primary catalytic activity of plsY involves the transfer of an acyl group from acyl-phosphate to the sn-1 position of glycerol-3-phosphate, producing lysophosphatidic acid. This reaction constitutes the first committed step in phospholipid biosynthesis in most bacteria, making plsY crucial for membrane formation and cellular integrity. The enzyme's role in maintaining proper membrane composition may be particularly significant for Aliivibrio salmonicida, which must adapt to cold marine environments where membrane fluidity presents specific challenges.
Considering that Aliivibrio salmonicida causes tissue degradation, hemolysis, and sepsis in infected fish, the proper functioning of membrane-associated processes is likely essential for pathogenicity . While direct evidence linking plsY to virulence is limited in the available literature, its fundamental role in bacterial membrane formation suggests potential indirect contributions to the pathogen's survival and persistence in host environments.
Production and Purification Methodologies
The recombinant Aliivibrio salmonicida Glycerol-3-phosphate acyltransferase is produced through heterologous expression in Escherichia coli, taking advantage of established bacterial expression systems that facilitate high-yield protein production . The expression construct incorporates an N-terminal histidine tag sequence, which enables efficient purification through metal affinity chromatography while minimizing interference with the protein's native structure and function.
The purification process typically achieves greater than 90% purity as verified by SDS-PAGE analysis . Following purification, the protein undergoes lyophilization to create a stable powder form that can be stored long-term. This preparation method creates a product suitable for various downstream research applications, from structural studies to functional assays.
For optimal storage and handling, the following protocols are recommended:
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Store lyophilized protein at -20°C/-80°C upon receipt
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Perform brief centrifugation before opening to bring contents to the bottom of the vial
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Reconstitute in deionized sterile water to 0.1-1.0 mg/mL
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Add glycerol (5-50% final concentration) for long-term storage stability
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Aliquot to avoid repeated freeze-thaw cycles
These procedures ensure the maintenance of protein integrity and enzymatic activity for experimental applications.
Comparative Analysis with Related Acyltransferases
Comparing the Aliivibrio salmonicida plsY with homologous enzymes from other bacterial species provides valuable insights into evolutionary conservation and functional specialization. One relevant comparison can be made with the plsY from Prochlorococcus marinus, another marine microorganism but one adapted to warm, oligotrophic ocean environments rather than cold waters.
Table 2 presents a comparative analysis of key features between the plsY enzymes from these two marine bacteria:
| Feature | A. salmonicida plsY | P. marinus plsY |
|---|---|---|
| Amino acid length | 193 residues | 206 residues |
| UniProt ID | B6EM14 | A9BBV0 |
| Expression host | E. coli | E. coli |
| N-terminal sequence | MTPLALIMIIIAYLLGS... | MELTKIFLAFLCIGVS... |
| Ecological niche | Cold marine environments | Warm oligotrophic oceans |
| Bacterial lifestyle | Fish pathogen | Photosynthetic cyanobacterium |
The length difference (193 vs. 206 amino acids) and sequence variations likely reflect adaptations to their respective environmental niches and physiological requirements . The A. salmonicida plsY may incorporate structural features that maintain enzymatic activity at lower temperatures consistent with the bacterium's cold-water habitat, while the P. marinus enzyme would be optimized for function in warmer ocean waters.
These comparative analyses could potentially inform research into temperature adaptation mechanisms in membrane-associated enzymes and provide insights into the evolution of phospholipid biosynthesis across diverse bacterial lineages.
Applications in Research and Biotechnology
Recombinant Aliivibrio salmonicida plsY has several significant applications in research and biotechnology fields:
Vaccine Development Research
Understanding the components of Aliivibrio salmonicida has direct implications for vaccine development against cold water vibriosis. While currently available vaccines effectively control the disease, the molecular mechanisms underlying protective immunity remain incompletely characterized . Investigation of membrane-associated proteins like plsY could potentially contribute to improved vaccine formulations or identification of novel antigenic targets.
Enzymatic Assays and Biochemical Tools
The recombinant enzyme serves as a valuable tool for studying phospholipid biosynthesis pathways. Researchers can utilize the purified protein in enzymatic assays to investigate substrate specificity, reaction kinetics, and regulatory mechanisms of bacterial membrane formation. Such studies could reveal unique aspects of membrane adaptation to cold environments.
Drug Discovery Applications
As bacterial membrane biosynthesis represents a potential target for antimicrobial development, recombinant plsY can facilitate screening assays for compounds that inhibit this essential pathway. The identification of molecules that selectively inhibit plsY function could lead to novel antibacterial agents with applications in aquaculture disease management.
Potential Role in Pathogenicity and Host Interaction
While direct evidence linking plsY to Aliivibrio salmonicida virulence is limited in the available literature, several lines of reasoning suggest potential contributions to pathogenicity:
Membrane Integrity and Environmental Adaptation
The phospholipid composition of bacterial membranes influences environmental adaptation, particularly in response to temperature fluctuations. As a cold-water pathogen, A. salmonicida must maintain proper membrane fluidity at lower temperatures to support essential cellular functions . The plsY enzyme, through its role in phospholipid biosynthesis, likely contributes to this adaptive capacity by influencing membrane composition.
Relation to Virulence Mechanisms
The genome of Aliivibrio salmonicida reveals evidence of gene acquisition related to pathogenicity, including iron-acquisition systems and protein secretion mechanisms . While plsY itself may not be directly classified as a virulence factor, proper membrane formation is prerequisite for the function of many virulence-associated systems, including protein secretion apparatus and surface adhesins that mediate host interaction.
Immune Recognition and Evasion
The cell envelope components of A. salmonicida play significant roles in immune recognition and evasion. Studies have identified outer membrane proteins and lipopolysaccharide complexes that function as immunodominant antigens . As phospholipids constitute a major component of bacterial membranes, the products of plsY activity may indirectly influence the presentation of surface antigens and subsequent host immune responses.
Future Research Directions
Several promising research directions could extend our understanding of Aliivibrio salmonicida plsY and its biological significance:
Structure-Function Relationship Studies
Detailed structural analysis through advanced techniques like X-ray crystallography could elucidate the catalytic mechanism of A. salmonicida plsY and identify specific adaptations to cold environments. Such studies might reveal unique structural features that distinguish this enzyme from homologs in mesophilic bacteria.
Role in Cold Adaptation
Investigating how plsY activity contributes to membrane composition and fluidity at low temperatures could provide insights into bacterial cold adaptation mechanisms. Comparative studies with homologous enzymes from bacteria adapted to different temperature ranges would be particularly informative.
Potential as a Therapeutic Target
Exploring plsY as a potential target for antimicrobial development could lead to novel treatments for cold water vibriosis. High-throughput screening of compound libraries against the recombinant enzyme might identify selective inhibitors with potential applications in aquaculture disease management.
Immunological Significance
Further investigation of the potential immunological significance of membrane components derived from plsY activity could enhance our understanding of host-pathogen interactions in cold water vibriosis and potentially inform improved vaccine formulations.
Product Specs
Note: We will prioritize shipping the format that we currently have in stock. However, if you have specific requirements for the format, please indicate them when placing your order, and we will prepare the product according to your request.
Note: All our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please inform us in advance, as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: vsa:VSAL_I2693
STRING: 316275.VSAL_I2693
Q&A
What is Aliivibrio salmonicida and what disease does it cause?
Aliivibrio salmonicida (previously known as Vibrio salmonicida) is a Gram-negative bacterium that causes cold-water vibriosis (CV), a hemorrhagic septicemia primarily affecting farmed Atlantic salmon (Salmo salar L.) . The disease mechanisms, host specificity, and adaptation processes of this pathogen remain largely unknown, although recent research has focused on identifying virulence factors and understanding host-pathogen interactions .
What is the function of Glycerol-3-phosphate acyltransferase in bacterial systems?
Glycerol-3-phosphate acyltransferase (GPAT) is a critical enzyme in bacterial phospholipid biosynthesis, catalyzing the first step in phospholipid synthesis by transferring an acyl group to glycerol-3-phosphate . In Aliivibrio salmonicida, this enzyme (EC 2.3.1.15) plays an essential role in membrane phospholipid formation, which is crucial for bacterial survival, adaptation to environmental conditions, and potentially pathogenicity .
How does plsB differ from plsY in Aliivibrio salmonicida?
Both plsB and plsY encode glycerol-3-phosphate acyltransferases but differ in substrate specificity and evolutionary origin. PlsB preferentially uses acyl-ACP or acyl-CoA as acyl donors, while plsY utilizes acyl-phosphate. Although the search results focus on plsB (UniProt No. B6ENU1), both enzymes perform the initial acylation of glycerol-3-phosphate in phospholipid biosynthesis . The differential expression of these enzymes may relate to adaptation strategies in different environmental conditions or during infection.
What expression systems are optimal for producing recombinant Aliivibrio salmonicida GPAT?
Based on current research, recombinant Aliivibrio salmonicida GPAT can be successfully expressed in both prokaryotic (E. coli) and eukaryotic (mammalian cell) systems . The E. coli system (product code CSB-EP478104AZM) typically yields higher protein quantities but may have limitations in post-translational modifications. The mammalian expression system (product code CSB-MP478104AZM) may provide more native-like modifications but generally with lower yields . The choice between these systems should depend on downstream applications and whether post-translational modifications are critical for the research question.
What purification strategies yield the highest activity for recombinant GPAT?
Successful purification of active recombinant GPAT requires careful consideration of buffer conditions and purification techniques. The available data indicates that purification to >85% homogeneity (as assessed by SDS-PAGE) can be achieved with both expression systems . Although specific purification protocols are not detailed in the search results, maintaining protein stability during purification likely requires including glycerol and avoiding repeated freeze-thaw cycles, as indicated by storage recommendations for the purified protein .
How should researchers validate the activity of purified recombinant GPAT?
Validation of enzymatic activity should include both spectrophotometric assays (measuring the formation of acylated glycerol-3-phosphate) and structural integrity assessment through circular dichroism or thermal shift assays. While specific activity data is not provided in the search results, researchers should establish baseline activity parameters for their purified preparations against known substrates before proceeding with experimental applications.
How can recombinant GPAT be used to study Aliivibrio salmonicida pathogenesis?
Research into Aliivibrio salmonicida pathogenesis has revealed that virulence gene expression is generally downregulated during in vivo infection compared to in vitro culture conditions . This suggests a potential strategy to evade host immune detection. Recombinant GPAT can serve as a tool to investigate phospholipid biosynthesis regulation during infection and how membrane composition changes contribute to virulence. Studies could involve comparing enzymatic activity under conditions that mimic the host environment versus standard laboratory conditions to understand adaptive responses.
What are the implications of GPAT in bacterial membrane adaptation during infection?
The ability of Aliivibrio salmonicida to cause cold-water vibriosis suggests specialized adaptations to low temperatures, potentially involving membrane fluidity regulation through phospholipid composition changes . GPAT plays a critical role in determining the initial acyl chain composition of phospholipids. Investigating substrate specificity and activity parameters of recombinant GPAT under various temperature conditions could provide insights into how this pathogen adapts its membrane composition during the infection process in cold-water environments.
How does GPAT activity correlate with immune evasion strategies of Aliivibrio salmonicida?
Studies on Atlantic salmon infected with Aliivibrio salmonicida have demonstrated a strong but short-lasting innate immune response, suggesting the bacterium possesses mechanisms to inhibit or resist host immunity . The expression profile of virulence factors shows general downregulation in vivo compared to in vitro conditions. While a direct link between GPAT activity and immune evasion remains unexplored, investigating how membrane phospholipid composition affects recognition by host pattern recognition receptors could provide valuable insights into bacterial persistence mechanisms.
What are the optimal storage conditions for maintaining recombinant GPAT stability?
For maximum stability and activity retention, recombinant GPAT should be stored at -20°C/-80°C with 50% glycerol as a cryoprotectant . Liquid formulations generally maintain stability for up to 6 months, while lyophilized preparations can remain stable for approximately 12 months under proper storage conditions . Working aliquots should be stored at 4°C and used within one week, while repeated freeze-thaw cycles should be strictly avoided to prevent activity loss through protein denaturation .
How should researchers design experiments to investigate GPAT substrate specificity?
Experimental designs for substrate specificity studies should incorporate:
| Parameter | Considerations | Measurement Approach |
|---|---|---|
| Acyl donor variety | Test range of acyl-CoA or acyl-ACP chain lengths | LC-MS quantification of products |
| Temperature range | 4°C to 20°C (relevant to cold-water vibriosis) | Activity assays at different temperatures |
| pH optimization | pH 6.5-8.0 range typical for enzymatic activity | Spectrophotometric activity assays |
| Divalent cation requirements | Mg²⁺, Mn²⁺, Ca²⁺ at various concentrations | Comparative activity measurements |
| Glycerol-3-phosphate concentration | Kinetic analysis with varying substrate | Michaelis-Menten parameters determination |
Researchers should also consider how host factors present during infection might influence substrate availability and enzyme activity.
How does GPAT expression correlate with bacterial proliferation during infection?
Analysis of Aliivibrio salmonicida gene expression during Atlantic salmon infection reveals a lag phase before day 4 post-infection, followed by exponential bacterial proliferation . While the search results don't specifically address GPAT expression patterns, studies on housekeeping genes (accD, gapA, and 16S rDNA) provide reference points for normalized expression analysis . Future research could investigate whether GPAT expression changes correlate with this biphasic growth pattern and how phospholipid synthesis rates relate to bacterial proliferation stages.
What immune factors specifically target bacterial membrane components during Aliivibrio salmonicida infection?
Atlantic salmon mount a rapid and strong innate immune response against Aliivibrio salmonicida, with significant upregulation of key cytokines (TNFα, IL-1β, and IL-6) and pattern recognition receptors like TLR5S within the first 8-48 hours post-infection . This response shows dramatic fold changes (122-fold for TNFα, 742-fold for IL-1β, and 817-fold for IL-6) but quickly diminishes despite continued bacterial proliferation . Understanding how these immune factors interact with bacterial membrane components, potentially modified by GPAT activity, could reveal mechanisms of bacterial persistence.
How can GPAT be utilized in developing new intervention strategies against cold-water vibriosis?
With the reported resurgence of cold-water vibriosis outbreaks despite vaccination , new intervention approaches are needed. GPAT, as an essential enzyme in bacterial membrane synthesis, presents a potential therapeutic target. Researchers could use the recombinant enzyme to screen for specific inhibitors that disrupt membrane biogenesis. Structure-based drug design approaches would benefit from crystallographic studies of the purified recombinant enzyme, while in vivo studies could evaluate whether GPAT inhibition affects bacterial virulence or survival within the host.
How do we reconcile the downregulation of virulence genes in vivo with the progressive disease pathology?
A significant contradiction in Aliivibrio salmonicida pathogenesis is that virulence genes appear downregulated in vivo compared to in vitro conditions, yet the infection progresses to cause severe disease . This suggests either that: (1) minimal expression of virulence factors is sufficient for pathogenesis, (2) currently unidentified virulence factors are upregulated during infection, or (3) the pathology depends more on host response than direct bacterial action. Researchers investigating GPAT should consider how phospholipid metabolism might contribute to this paradoxical virulence strategy and design experiments to test alternative hypotheses.
What methodological challenges exist in studying GPAT activity in infection-relevant conditions?
Recreating the in vivo environment for enzymatic studies presents significant challenges. The temperature (typically 10-15°C for cold-water vibriosis), host factors, and the low-nutrient environment during certain infection phases all potentially affect GPAT activity. Researchers should develop experimental systems that better mimic these conditions, possibly using ex vivo tissue models or microfluidic systems with controlled temperature and defined media compositions to bridge the gap between standard in vitro enzyme assays and the actual infection environment.
