Recombinant Salmonella heidelberg Bifunctional protein aas (aas)

What is the Bifunctional protein aas in Salmonella heidelberg and what are its key characteristics?

The Bifunctional protein aas (aas) from Salmonella heidelberg is a 719 amino acid protein (UniProt accession: B4TGR5) that functions as a bifunctional enzyme. It includes domains for 2-acylglycerophosphoethanolamine acyltransferase (EC 2.3.1.40) and is involved in membrane phospholipid metabolism . The protein has a molecular mass of approximately 59-61 kDa and is typically produced recombinantly with an N-terminal His-tag for research purposes.

Key characteristics of the protein include:

How conserved is the aas protein across different Salmonella serovars?

Comparative analysis shows high conservation of the aas protein across Salmonella serovars. The Bifunctional protein aas from Salmonella heidelberg and Salmonella dublin share remarkably similar amino acid sequences, with only minor variations . This high level of conservation suggests functional importance across Salmonella species.

When comparing the sequences between S. heidelberg and S. dublin, there are only a few amino acid differences, with key functional domains being highly preserved . For example, position 151 shows a glutamine (Q) in S. heidelberg versus a valine (V) in S. dublin, and at position 367, S. heidelberg has a proline (P) where S. dublin has a leucine (L) .

What are the optimal conditions for expressing Recombinant Salmonella heidelberg Bifunctional protein aas in E. coli?

For optimal expression of Recombinant Salmonella heidelberg Bifunctional protein aas in E. coli, researchers should consider the following methodological approach:

• Expression system: E. coli is the recommended expression host, with BL21(DE3) or similar strains commonly used for protein expression .

• Vector selection: pET-based vectors with T7 promoter systems provide high-level expression for His-tagged proteins.

• Induction conditions:

  • IPTG concentration: 0.5-1.0 mM

  • Induction temperature: 16-25°C (lower temperatures may improve protein solubility)

  • Induction time: 4-16 hours (overnight induction at lower temperatures often yields better results)

• Media composition: Enriched media like LB or 2YT with appropriate antibiotics based on the plasmid's resistance marker.

• Cell lysis: Sonication or high-pressure homogenization in a buffer containing:

  • 50 mM Tris-HCl (pH 8.0)

  • 300 mM NaCl

  • 10 mM imidazole

  • Protease inhibitors

What purification methods yield the highest purity and activity for Recombinant Salmonella heidelberg Bifunctional protein aas?

A multi-step purification strategy is recommended to obtain high-purity, active Recombinant Salmonella heidelberg Bifunctional protein aas:

• Immobilized Metal Affinity Chromatography (IMAC):

  • Using Ni-NTA or similar resin for His-tagged protein

  • Binding buffer: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole

  • Wash buffer: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 20-40 mM imidazole

  • Elution buffer: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 250-500 mM imidazole

• Size Exclusion Chromatography (SEC):

  • Further purification using Superdex 200 or similar column

  • Running buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl

• Buffer exchange and concentration:

  • Final storage in Tris/PBS-based buffer with 6% trehalose at pH 8.0

• Quality control:

  • SDS-PAGE analysis to confirm >90% purity

  • Enzymatic activity assays using appropriate substrates

What are the optimal storage conditions to maintain stability of purified Recombinant Salmonella heidelberg Bifunctional protein aas?

Based on product specifications and scientific literature, the following storage conditions are recommended for maintaining stability:

• Short-term storage (up to one week):

  • Store working aliquots at 4°C

• Long-term storage:

  • Store at -20°C or preferably -80°C

  • Aliquot before freezing to avoid repeated freeze-thaw cycles

  • Add glycerol to a final concentration of 5-50% (with 50% being optimal)

• Lyophilization:

  • The protein can be supplied as a lyophilized powder

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Stability considerations:

  • Repeated freeze-thaw cycles should be strictly avoided

  • The protein appears to be stable in Tris/PBS-based buffer with 6% trehalose at pH 8.0

What enzymatic assays can be used to measure the activity of Bifunctional protein aas?

To measure the 2-acylglycerophosphoethanolamine acyltransferase activity of Bifunctional protein aas, researchers can employ several methodological approaches:

• Radiolabeled substrate assay:

  • Use 14C-labeled acyl donors (e.g., acyl-CoA or acyl-ACP)

  • Monitor transfer to glycerophosphoethanolamine acceptors

  • Quantify product formation by thin-layer chromatography and scintillation counting

• HPLC-based assay:

  • Utilize fluorescently labeled or UV-absorbing acyl-CoA substrates

  • Separate and quantify reaction products by reverse-phase HPLC

  • Monitor decrease in substrate or increase in product peaks

• Coupled enzymatic assay:

  • Link acyltransferase activity to a secondary reaction that produces a measurable signal

  • Measure release of CoA using DTNB (Ellman's reagent) at 412 nm

  • Monitor change in absorbance spectrophotometrically

• Mass spectrometry-based assay:

  • Detect and quantify reaction products directly using LC-MS/MS

  • Allows for simultaneous analysis of multiple substrate-product combinations

Optimal reaction conditions generally include:

• pH range: 7.0-8.0

• Temperature: 30-37°C

• Buffer: 50 mM Tris-HCl or phosphate buffer

• Divalent cations: Mg2+ or Mn2+ (1-5 mM)

• Reducing agent: DTT or β-mercaptoethanol (1-5 mM)

How can researchers study the structure-function relationship of Bifunctional protein aas?

Several methodological approaches can be employed to investigate structure-function relationships:

A systematic approach combining these methods would provide insights into which regions and residues are critical for enzymatic function, substrate binding, and protein-protein interactions.

What is known about the role of Bifunctional protein aas in Salmonella virulence and pathogenesis?

Research on the direct role of Bifunctional protein aas in Salmonella virulence is still emerging, but several connections can be made based on available evidence:

• Membrane homeostasis and integrity:

  • As a bifunctional enzyme involved in phospholipid metabolism, aas likely contributes to membrane remodeling and repair

  • Membrane integrity is critical for survival during host-pathogen interactions

• Regulation by virulence-associated factors:

  • Research indicates that the aas gene is regulated by OmpR , a transcriptional regulator that responds to environmental signals including osmolarity

  • OmpR regulation suggests aas may be part of the adaptive response during infection

• Potential role in antimicrobial resistance:

  • While not directly implicated, phospholipid metabolism enzymes can influence membrane permeability and potentially impact susceptibility to antimicrobials

  • Salmonella Heidelberg strains show varied antimicrobial resistance profiles, though resistance is primarily associated with plasmid-borne genes like blaCMY-2

• Environmental persistence:

  • Studies show Salmonella Heidelberg can persist in environments like poultry litter for extended periods (up to 21 days)

  • Membrane lipid composition, potentially influenced by aas activity, could impact environmental stress tolerance

To directly investigate the role of aas in virulence, researchers could:

• Generate targeted aas deletion mutants in Salmonella Heidelberg

• Compare virulence in appropriate infection models

• Assess survival under various stress conditions relevant to host environments

• Examine membrane lipid composition changes during infection

How does the Bifunctional protein aas compare between antimicrobial-resistant and susceptible Salmonella Heidelberg strains?

While direct comparative studies of the Bifunctional protein aas between antimicrobial-resistant and susceptible Salmonella Heidelberg strains are not explicitly described in the search results, several research directions can be proposed:

• Sequence comparison:

  • Analysis of aas sequences from resistant strains (like SH-AAFC, harboring blaCMY-2 on an IncI1 plasmid) versus susceptible strains (like SH-ARS)

  • Identification of potential mutations or polymorphisms that might alter enzyme function

• Expression level analysis:

  • Transcriptomic comparison of aas expression between resistant and susceptible strains

  • Evaluation of whether antibiotic selection pressure alters aas expression

• Membrane composition studies:

  • Lipidomic analysis to determine if resistant strains show alterations in membrane phospholipid composition

  • Assessment of whether such changes correlate with altered aas activity

• Functional studies:

  • Comparison of enzyme kinetics and substrate specificity between aas proteins from resistant and susceptible strains

  • Investigation of potential interactions between aas and components of resistance mechanisms

Research has shown that Salmonella Heidelberg strains with different antimicrobial resistance profiles show varied survival capabilities in environments like pine wood shavings used as broiler litter. Specifically, strains harboring antimicrobial resistance genes like blaCMY-2 (SH-AAFC) survived longer than pan-susceptible strains (SH-ARS) .

How might researchers use Recombinant Salmonella heidelberg Bifunctional protein aas in vaccine development approaches?

While Bifunctional protein aas itself has not been specifically studied as a vaccine candidate based on the search results, researchers could explore its potential in vaccine development through several methodological approaches:

• Epitope mapping and antigenicity assessment:

  • Apply similar approaches to those used for FlgK protein in Salmonella Heidelberg

  • Use immunoinformatic tools to predict potential B-lymphocyte epitopes

  • Compare in silico predictions with experimental results from animal immunizations

  • Assess antigenicity using tools like VaxiJen and allergenicity with AllerTOP

• Recombinant subunit vaccine approach:

  • Express recombinant aas protein or specific epitope-containing fragments

  • Formulate with appropriate adjuvants

  • Evaluate immunogenicity in animal models

  • Assess protective efficacy against challenge

• Reverse vaccinology strategy:

  • As demonstrated with other bacterial pathogens , use computational approaches to identify conserved, surface-exposed, and antigenic regions of aas

  • Design multi-epitope constructs incorporating aas epitopes with other Salmonella antigens

• mRNA vaccine technology:

  • Design mRNA constructs encoding aas or selected epitopes

  • Evaluate expression and immune response in appropriate models

• Immunological evaluation:

  • Use techniques like automated capillary immunoassay for quantifying antibodies in sera

  • Perform mass spectrometry-based proteomics associated with immunized animal sera to map linear immunoepitopes

Research on the FlgK protein from Salmonella Heidelberg identified three common shared consensus peptide epitope sequences that could be valuable for vaccine development . Similar methodologies could be applied to investigate the vaccine potential of Bifunctional protein aas.

What molecular dynamics simulations could reveal about substrate binding mechanisms of Bifunctional protein aas?

Molecular dynamics (MD) simulations offer powerful approaches to understanding the structural dynamics and substrate binding mechanisms of Bifunctional protein aas:

• System preparation:

  • Generate a high-quality structural model through homology modeling if crystal structure is unavailable

  • Embed the protein in a phospholipid bilayer membrane to mimic its native environment

  • Solvate the system with explicit water molecules and appropriate ion concentrations

• Simulation approaches:

  • Perform equilibrium MD simulations (100 ns to μs timescale) to observe conformational dynamics

  • Use advanced sampling techniques like metadynamics or umbrella sampling to characterize free energy landscapes of substrate binding

  • Employ steered MD to investigate substrate entry/exit pathways

• Specific investigations:

  • Characterize binding pocket flexibility and conformational changes upon substrate binding

  • Identify key residues involved in substrate recognition and catalysis

  • Simulate the effects of mutations on substrate binding and enzyme dynamics

  • Model the bifunctional nature by simulating both enzymatic activities

• Analysis methods:

  • Calculate binding free energies using MM/PBSA or MM/GBSA approaches

  • Analyze hydrogen bonding networks and salt bridge formations

  • Perform principal component analysis to identify major modes of protein motion

  • Create Markov state models to identify relevant metastable states

These computational approaches would complement experimental studies and provide atomic-level insights into the mechanism of this important bifunctional enzyme.

How might genomic variations in the aas gene affect bacterial fitness and adaptation in different host environments?

Investigating the relationship between aas gene variations and bacterial fitness across different host environments represents an important research frontier:

• Comparative genomic analysis:

  • Sequence the aas gene across Salmonella Heidelberg isolates from diverse sources (human clinical, poultry, environmental)

  • Identify single nucleotide polymorphisms (SNPs) and structural variations

  • Compare with other Salmonella serovars to identify serovar-specific patterns

• Experimental evolution studies:

  • Subject Salmonella Heidelberg to serial passage in different host-mimicking conditions

  • Sequence the aas gene before and after adaptation

  • Identify mutations that arise under specific selection pressures

• Functional genomics approaches:

  • Generate a library of aas variants using site-directed mutagenesis or random mutagenesis

  • Assess fitness effects in competition assays under various conditions

  • Perform complementation studies in aas knockout strains

• Host-specific adaptation analysis:

  • Compare aas sequences from isolates adapted to different hosts (human vs. poultry)

  • Assess membrane lipid composition changes in host-adapted strains

  • Investigate whether aas variations correlate with host range or tissue tropism

Research has shown that Salmonella Heidelberg strains with different genetic characteristics exhibit varied survival capabilities in environments like pine wood shavings used as broiler litter . Understanding how aas gene variations might contribute to these differences would provide valuable insights into bacterial adaptation strategies.

What interactomics approaches could identify novel protein-protein interactions involving Bifunctional protein aas?

Understanding the protein interaction network of Bifunctional protein aas could reveal its broader biological roles. Several cutting-edge interactomics approaches can be applied:

• Affinity purification-mass spectrometry (AP-MS):

  • Express tagged Bifunctional protein aas in Salmonella Heidelberg

  • Perform pull-down experiments under various conditions

  • Identify co-precipitating proteins by mass spectrometry

  • Validate interactions through reciprocal pull-downs

• Proximity-dependent biotin identification (BioID or TurboID):

  • Fuse aas to a biotin ligase (BioID2 or TurboID)

  • Express in Salmonella and allow proximity labeling to occur

  • Purify biotinylated proteins and identify by mass spectrometry

  • Map the spatial interactome of aas in living cells

• Bacterial two-hybrid screening:

  • Create an aas bait construct

  • Screen against a genomic library of Salmonella proteins

  • Validate positive interactions through secondary assays

  • Map interaction domains through truncation analysis

• Cross-linking mass spectrometry (XL-MS):

  • Use chemical cross-linkers to capture transient protein-protein interactions

  • Digest cross-linked complexes and analyze by specialized MS workflows

  • Identify interaction interfaces at amino acid resolution

  • Incorporate findings into structural models

• Protein co-evolution analysis:

  • Apply computational methods to identify co-evolving protein pairs

  • Predict functional associations based on evolutionary constraints

  • Validate predictions experimentally

These approaches would help place Bifunctional protein aas within the broader context of cellular pathways and potentially reveal unexpected functions beyond its known enzymatic activities.