Recombinant Salmonella typhimurium Inner membrane protein yejM (yejM)
Description
Biochemical Properties of Recombinant YejM
Recombinant YejM is typically expressed in E. coli with an N-terminal His tag for purification . Key specifications include:
-
Molecular weight: ~66 kDa (theoretical).
-
Stability: Lyophilized powder stored at -80°C; reconstitution in Tris/PBS buffer with 6% trehalose .
Enzymatic Activity:
-
Demonstrates magnesium-dependent phosphatase activity linked to OM lipid regulation .
-
Active site residues (e.g., F349) and the arginine-rich linker are essential for function .
Functional Roles and Mechanisms
YejM is indispensable for bacterial viability and virulence through two primary pathways:
Lipid Homeostasis
-
Binds cardiolipin and regulates its transport to the OM, enhancing bacterial survival under PhoPQ-activated conditions .
-
Modulates lipopolysaccharide (LPS) biosynthesis by stabilizing LpxC, a key enzyme in lipid A production .
Interaction with Proteolytic Complexes
-
Inhibits the YciM/FtsH protease complex, preventing excessive degradation of LpxC .
-
Deletion of YejM leads to hyperactivation of FtsH, causing lethal OM defects .
Research Applications
Recombinant YejM is utilized in:
-
Antibiotic resistance studies: YejM-deficient strains show hypersensitivity to vancomycin and detergents .
-
Structural biology: Crystallography studies to elucidate mechanisms of OM remodeling .
-
Drug discovery: Target validation for novel antimicrobials due to its essential role .
Outstanding Research Questions
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them during order placement. We will accommodate your needs whenever possible.
Note: All our proteins are shipped with standard blue ice packs by default. If dry ice shipping is required, 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
- This study presents the first crystal structure of the soluble periplasmic globular domain of PbgA. The analysis reveals that the globular domains of PbgA resemble the structures of the arylsulfatase protein family and contains a novel core hydrophobic pocket potentially responsible for binding and transporting cardiolipins. PMID: 27487745
KEGG: stm:STM2228
STRING: 99287.STM2228
Q&A
What is the essential biological role of YejM in Salmonella typhimurium?
YejM is an inner membrane (IM) metalloenzyme critical for maintaining outer membrane (OM) asymmetry by regulating lipopolysaccharide (LPS) biosynthesis. It senses periplasmic LPS levels through its periplasmic domain and modulates proteolytic degradation of LpxC, the rate-limiting enzyme in lipid A biosynthesis . Deletion of yejM leads to lethal OM permeability defects due to unbalanced LPS synthesis, as demonstrated by suppressor mutations in lpxC or ftsH that restore viability . Methodologically, essentiality is confirmed through conditional knockout strains and complementation assays using plasmid-borne yejM .
What experimental systems are used to express recombinant YejM for functional studies?
Recombinant YejM is typically expressed in E. coli or Salmonella systems with codon optimization for improved solubility. Key protocols include:
-
Membrane extraction: Detergent-based solubilization (e.g., 1% DDM) followed by Ni-NTA affinity chromatography .
-
Domain engineering: Truncation of the flexible periplasmic linker (residues 191–240) to enhance crystallizability, as shown in construct YejM241-586 .
-
Activity assays: Phosphatase activity is measured using p-nitrophenyl phosphate (pNPP) with magnesium dependence, confirming its metalloenzyme function .
How is YejM’s interaction with LPS or cardiolipin validated experimentally?
-
Lipid-binding assays: Surface plasmon resonance (SPR) or liposome co-flotation with purified periplasmic domains (e.g., YejM241-586) .
-
Genetic suppression: yejM mutants with OM permeability defects are rescued by lpxC mutations, linking YejM activity to LPS regulation .
-
Mass spectrometry: Quantitative lipidomics of OM vesicles from yejM mutants shows reduced cardiolipin under PhoPQ activation .
How do researchers reconcile contradictory findings about YejM’s role in cardiolipin transport versus LPS regulation?
The controversy arises from two observations:
-
yejM essentiality is independent of cardiolipin synthases, implicating LPS as the primary substrate .
Resolution strategies:
-
Conditional knockdowns: Test OM lipid composition in yejM depletion strains lacking cardiolipin synthases (e.g., clsABC mutants) .
-
Biochemical reconstitution: Measure YejM’s lipid transport activity in proteoliposomes with fluorescently tagged LPS/cardiolipin .
-
Structural analysis: Compare cardiolipin-binding pockets in YejM homologs (e.g., EptA) to identify functional motifs .
What structural biology approaches are used to study YejM’s mechanism?
Key advancements include:
Table 1: YejM Constructs for Crystallography
Methodological insights:
-
Lipidic cubic phase (LCP) crystallization: Enables membrane protein crystal growth in a lipid-rich environment .
-
Anomalous scattering: Identifies metal ions (Mg²⁺/Mn²⁺) in the active site using X-ray fluorescence .
How does YejM’s phosphatase activity influence OM remodeling?
YejM’s periplasmic domain shares structural homology with arylsulfatases and contains a conserved metalloenzyme active site (Fig. 1A in ). Functional studies reveal:
-
Kinetic parameters: Kₘ = 2.1 mM for pNPP, with optimal activity at pH 7.5 and 10 mM Mg²⁺ .
-
Physiological substrates: Likely dephosphorylates LPS precursors (e.g., lipid IVA) based on structural docking .
-
Genetic validation: Alanine substitutions in catalytic residues (H349A, D412A) abolish activity and cause OM defects .
How are yejM suppressor mutations analyzed to identify regulatory networks?
Suppressor screens in yejM mutants involve:
-
Random mutagenesis: Ethyl methanesulfonate (EMS) treatment to generate suppressors .
-
Whole-genome sequencing: Identify mutations in lpxC, ftsH, or lapB that restore OM integrity .
-
Transcriptional profiling: RNA-seq to assess PhoPQ regulon changes in suppressor strains .
