Recombinant Salmonella newport UPF0060 membrane protein ynfA (ynfA)

What is the structural composition of Salmonella newport UPF0060 membrane protein ynfA?

Salmonella newport UPF0060 membrane protein ynfA (UniProt ID: B4T5E1) is a 108-amino acid transmembrane protein with the following sequence: MLKTTLLFFVTALCEIIGCFLPWLWLKRGASVWWLLPAAASLALFVWLLTLHPAASGRVY AAYGGVYVCTALLWLRVVDGVRLTVYDWCGALIALCGMLIIVVGWGRT . The protein belongs to the UPF0060 family of membrane proteins, which are conserved across various bacterial species. When expressed recombinantly, it is typically fused to an N-terminal His tag and produced in E. coli expression systems to facilitate purification and subsequent functional studies . Hydropathy analysis suggests multiple transmembrane domains that likely contribute to its integration into the bacterial membrane.

How does ynfA from Salmonella newport compare with ynfA proteins from other Salmonella serovars?

Comparative sequence analysis reveals high conservation of ynfA across Salmonella serovars with minor variations that may contribute to serovar-specific functions. When comparing S. newport ynfA with S. paratyphi A ynfA, there is a notable difference at position 26, where S. newport contains lysine (K) while S. paratyphi A contains isoleucine (I) . The full sequences are:

S. newport ynfA: MLKTTLLFFVTALCEIIGCFLPWLWLKRGASVWWLLPAAASLALFVWLLTLHPAASGRVY AAYGGVYVCTALLWLRVVDGVRLTVYDWCGALIALCGMLIIVVGWGRT

S. paratyphi A ynfA: MLKTTLLFFVTALCEIIGCFLPWLWIKRGASVWWLLPAAASLALFVWLLTLHPAASGRVY AAYGGVYVCTALLWLRVVDGVRLTVYDWCGALIALCGMLIIVVGWGRT

These subtle sequence variations may influence protein-protein interactions, membrane integration, or functional properties in different host environments, potentially contributing to serovar-specific adaptation mechanisms.

What conditions should be used for optimal storage and handling of recombinant ynfA protein?

For optimal storage and handling of recombinant ynfA protein, the following protocol is recommended:

• Storage Temperature: Store at -20°C/-80°C upon receipt, with proper aliquoting to avoid repeated freeze-thaw cycles .

• Working Stock: Working aliquots may be stored at 4°C for up to one week .

• Buffer Composition: The protein is typically provided in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 .

• Reconstitution Protocol:

  • Briefly centrifuge the vial before opening

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

  • Add glycerol to a final concentration of 5-50% (typically 50%)

  • Aliquot for long-term storage

Adherence to these storage conditions is critical for maintaining protein stability and functionality, as membrane proteins are particularly susceptible to denaturation during freeze-thaw cycles.

What are the recommended methods for expressing and purifying recombinant ynfA protein?

The expression and purification of recombinant ynfA requires specialized protocols due to its nature as a membrane protein:

Expression System Selection:
E. coli is the preferred expression system, with BL21(DE3) or C41/C43 strains often yielding better results for membrane proteins . The gene should be cloned into vectors containing strong inducible promoters (T7 or tac) with an N-terminal His tag.

Expression Protocol:

• Transform expression plasmid into competent E. coli cells

• Culture cells at 37°C until OD600 reaches 0.6-0.8

• Reduce temperature to 16-25°C before induction

• Induce with 0.1-0.5 mM IPTG

• Continue expression for 4-16 hours

Purification Strategy:

• Harvest cells and resuspend in lysis buffer containing appropriate detergents (e.g., n-dodecyl-β-D-maltoside)

• Disrupt cells via sonication or high-pressure homogenization

• Solubilize membrane fraction

• Perform immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Consider additional purification steps such as size exclusion chromatography

• Verify purity by SDS-PAGE (should exceed 90%)

This approach has been successfully used to produce highly pure ynfA protein suitable for structural and functional studies.

How can transposon insertion screening be applied to study ynfA function?

Transposon insertion screening provides a powerful approach for investigating ynfA function within the broader genomic context of Salmonella Newport:

Methodology Framework:

• Library Construction: Generate a barcoded transposon insertion library as described in previous S. Newport studies, using mini-Tn5 derivatives with random N18 barcodes .

• Transformation Protocol: Electroporate the transposome complex into electrocompetent S. Newport cells and select transformants on appropriate antibiotic media .

• Selective Pressure Application: Subject the library to various selective conditions relevant to ynfA function (e.g., membrane stress, antimicrobial exposure).

• Barcode Mapping: Extract genomic DNA from the selected populations, fragment by sonication, and ligate to sequencing adapters .

• Next-Generation Sequencing: Perform nested PCR to amplify regions containing the barcode and adjacent genomic DNA, followed by paired-end sequencing .

• Data Analysis Pipeline:

  • Map reads to the S. Newport genome using Bowtie2

  • Identify barcodes using custom scripts

  • Compare barcode frequencies between conditions

  • Apply statistical frameworks to identify significant fitness effects

This approach can reveal genetic interactions between ynfA and other genomic elements, providing insights into its functional networks and potential roles in bacterial fitness under different environmental conditions.

What experimental approaches can be used to determine the role of ynfA in Salmonella Newport's adaptation to different environments?

To elucidate the role of ynfA in S. Newport's environmental adaptation, a multi-faceted experimental approach is necessary:

Genetic Manipulation Strategies:

• Construction of ynfA Mutants: Generate precise deletion mutants using λ Red recombination system, replacing the entire ORF with antibiotic resistance cassettes .

• Complementation Assays: Reintroduce wild-type or modified ynfA genes to confirm phenotype specificity.

• Conditional Expression Systems: Develop inducible expression systems to study dosage effects.

Comparative Fitness Assays:

• Plant Colonization Models: Given S. Newport's association with plant outbreaks, compare colonization efficiency of wild-type and ynfA mutants in tomato and other plant models .

• Animal Infection Models: Assess virulence and persistence in relevant animal models.

• Competitive Index Determination: Co-inoculate wild-type and mutant strains to directly measure relative fitness.

• Environmental Stress Response: Test survival under conditions mimicking food processing, plant surfaces, and gastrointestinal environments.

Molecular Phenotyping:

• Transcriptomic Analysis: Compare gene expression profiles between wild-type and ynfA mutants using RNA-seq.

• Proteomic Profiling: Identify protein abundance changes using mass spectrometry.

• Membrane Integrity Assessment: Measure permeability using fluorescent dyes or antimicrobial susceptibility.

These complementary approaches can reveal whether ynfA contributes to S. Newport's unique ability to persist in diverse environments, particularly in plant hosts where this serovar shows distinctive adaptation advantages .

How might ynfA contribute to Salmonella Newport's drug resistance phenotypes?

The potential role of ynfA in Salmonella Newport's drug resistance requires systematic investigation, particularly given the emergence of multidrug-resistant strains like Newport MDR-AmpC:

Current Understanding:
Salmonella Newport MDR-AmpC strains display resistance to multiple antibiotics including extended-spectrum cephalosporins, amoxicillin/clavulanate, ampicillin, cefoxitin, ceftiofur, cephalothin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline . As a membrane protein, ynfA could potentially influence drug resistance through:

• Altered membrane permeability

• Interaction with efflux pump systems

• Modulation of stress response pathways

Experimental Framework:

• Comparative Susceptibility Testing: Determine minimum inhibitory concentrations (MICs) for various antibiotics in wild-type vs. ynfA mutant strains.

• Drug Accumulation Assays: Measure intracellular accumulation of fluorescent antibiotic analogs.

• Gene Expression Analysis: Assess whether ynfA deletion affects expression of known resistance determinants.

• Genetic Interaction Studies: Construct double mutants lacking both ynfA and known resistance genes to identify synergistic effects.

Data Analysis Table:

This comprehensive approach can determine whether ynfA plays a direct or indirect role in the clinically significant drug resistance observed in S. Newport strains .

What is the relationship between ynfA and papA in Salmonella Newport's plant colonization capacity?

Understanding the potential functional relationship between ynfA and the recently identified plant adaptation factor papA could provide crucial insights into S. Newport's unique plant colonization abilities:

Background Context:
Research has demonstrated that S. Newport strains are disproportionately associated with plant-linked outbreaks, particularly involving tomatoes . A gene called papA has been identified as uniquely contributing to S. Newport's fitness in tomatoes, present in approximately 25% of sv. Newport Group III genomes but generally absent from other Salmonella genomes . Interestingly, papA homologs are found in plant-associated Enterobacteriaceae like Pantoea, Dickeya, and Pectobacterium .

Interaction Investigation Strategies:

• Co-expression Analysis: Determine if ynfA and papA expression patterns correlate during plant colonization.

• Double Mutation Studies: Compare fitness of ynfA single mutants, papA single mutants, and ynfA/papA double mutants in plant models.

• Protein-Protein Interaction Assessment: Use bacterial two-hybrid systems or co-immunoprecipitation to test for physical interactions.

• Transcriptional Regulation: Investigate whether papA expression affects ynfA transcription or vice versa.

• Localization Studies: Determine if both proteins co-localize in the bacterial membrane during plant colonization.

These approaches can reveal whether ynfA and papA function in the same or parallel pathways that contribute to S. Newport's enhanced ability to persist in plant environments, potentially explaining this serovar's epidemiological association with vegetable-linked outbreaks .

How can structural biology approaches enhance our understanding of ynfA function?

Structural characterization of ynfA would significantly advance our understanding of its function and potential as a therapeutic target:

Structural Determination Approaches:

• X-ray Crystallography: While challenging for membrane proteins, this approach can provide atomic-level resolution if crystals can be obtained, potentially requiring:

  • Detergent screening for optimal solubilization

  • Lipidic cubic phase crystallization

  • Use of antibody fragments as crystallization chaperones

• Cryo-Electron Microscopy: Increasingly powerful for membrane proteins, allowing visualization in near-native environments through:

  • Vitrification in detergent micelles or nanodiscs

  • Single-particle analysis

  • 3D reconstruction

• NMR Spectroscopy: Suitable for dynamic studies of smaller membrane proteins like ynfA (108 aa):

  • Requires isotopic labeling (15N, 13C)

  • Can provide information on protein dynamics

  • May reveal conformational changes upon ligand binding

• Computational Structure Prediction: Using AlphaFold2 or similar AI approaches to generate initial structural models that can guide experimental design.

Functional Insights from Structure:
Structural data would enable identification of:

• Membrane-spanning regions and topology

• Potential ligand-binding pockets

• Interaction surfaces for protein partners

• Structure-function relationships of serovar-specific amino acid variations (e.g., K26I between Newport and Paratyphi A)

These insights could guide rational design of inhibitors or modulators of ynfA function, potentially leading to novel antimicrobial strategies against S. Newport.

What are the main challenges in working with recombinant membrane proteins like ynfA and how can they be addressed?

Working with membrane proteins like ynfA presents several technical challenges that require specialized approaches:

Challenge 1: Low Expression Yields

• Problem: Membrane proteins often express poorly in heterologous systems.

• Solutions:

  • Use specialized E. coli strains (C41/C43, Lemo21) designed for membrane protein expression

  • Lower induction temperature (16-20°C) to slow expression and allow proper folding

  • Optimize codon usage for the expression host

  • Test various induction conditions (IPTG concentration, duration)

Challenge 2: Protein Aggregation

• Problem: Improper folding leading to inclusion body formation.

• Solutions:

  • Include mild detergents during cell lysis (e.g., n-dodecyl-β-D-maltoside)

  • Add stabilizing agents like glycerol (5-10%) to buffers

  • Consider fusion partners that enhance solubility (e.g., MBP)

  • Develop refolding protocols if recovering from inclusion bodies

Challenge 3: Maintaining Native Conformation

• Problem: Loss of structural integrity during purification.

• Solutions:

  • Screen multiple detergents for optimal extraction and stability

  • Include lipids or lipid-like molecules in purification buffers

  • Monitor protein quality using techniques like circular dichroism

  • Consider nanodisc or liposome reconstitution for functional studies

Optimization Table:

These strategies have proven effective for other bacterial membrane proteins and should be systematically tested for ynfA purification .

How can researchers design effective gene deletion and complementation systems for studying ynfA function?

Designing robust genetic manipulation systems is critical for rigorous functional characterization of ynfA:

Gene Deletion Strategy:

• Target Design: Remove the entire ynfA ORF (1-108aa) from start to stop codon to ensure complete functional knockout.

• λ Red Recombination Approach:

  • Design primers with 40bp homology to sequences flanking ynfA

  • Amplify antibiotic resistance cassette (frt-kan-frt or frt-cat-frt)

  • Transform into S. Newport expressing λ Red recombinase

  • Select for recombinants on appropriate antibiotics

  • Verify deletion by PCR and sequencing

• Marker Removal: Use FLP recombinase to excise the resistance marker, leaving a minimal scar sequence for clean genetic background in subsequent studies .

Complementation System Design:

• Vector Selection: Choose low or medium-copy plasmids to avoid overexpression artifacts.

• Promoter Considerations:

  • Native promoter for physiological expression levels

  • Inducible promoter (araBAD, tetR) for controlled expression

  • Constitutive promoter for stable expression

• Additional Features:

  • Include C-terminal epitope tags that don't interfere with function

  • Consider fluorescent protein fusions for localization studies

  • Include ribosome binding site optimization for proper expression levels

• Controls:

  • Empty vector control

  • Point mutant controls (non-functional versions)

  • Heterologous complementation (ynfA from other serovars)

This systematic approach to genetic manipulation provides a solid foundation for attributing phenotypes specifically to ynfA function rather than to secondary effects or artifacts .

What potential roles might ynfA play in host-pathogen interactions during Salmonella Newport infection?

Investigating ynfA's role in host-pathogen interactions could reveal novel aspects of S. Newport pathogenesis:

Potential Functional Roles:

• Membrane Integrity Regulation: ynfA may help maintain membrane homeostasis during host-induced stress.

• Host Defense Evasion: The protein could potentially contribute to resistance against host antimicrobial peptides.

• Adaptation to Host Environments: ynfA might facilitate adaptation to varying conditions encountered during infection (pH, osmolarity, nutrient availability).

• Virulence Factor Regulation: The protein may influence expression or function of other virulence determinants.

Research Approaches:

• Infection Models:

  • Compare colonization efficiency of wild-type and ynfA mutants in relevant animal models

  • Assess competitive fitness during mixed infections

  • Examine tissue-specific requirements for ynfA function

• Host Response Analysis:

  • Measure inflammatory cytokine responses to wild-type vs. ynfA mutants

  • Assess survival in the presence of various host defense mechanisms

  • Evaluate intracellular persistence in macrophages and epithelial cells

• In vivo Gene Expression:

  • Use reporter constructs to monitor ynfA expression during infection

  • Perform RNA-seq on bacteria recovered from host tissues

  • Identify host factors that influence ynfA regulation

Understanding these aspects could explain why strains of S. Newport cause distinctive clinical manifestations, potentially leading to targeted intervention strategies for Newport-specific infections .

How might comparative genomic approaches advance our understanding of ynfA evolution across Salmonella serovars?

Comparative genomic analysis can provide evolutionary insights into ynfA function and adaptation:

Evolutionary Analysis Framework:

• Phylogenetic Distribution:

  • Conduct comprehensive analysis of ynfA presence, absence, and sequence variation across the Salmonella genus

  • Correlate ynfA variants with serovar-specific niches and host ranges

  • Identify horizontal gene transfer events that may have shaped ynfA evolution

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to identify sites under positive or purifying selection

  • Map selective pressures onto protein structural models

  • Compare selection patterns between lineages adapted to different hosts

• Co-evolution Networks:

  • Identify genes that show correlated evolutionary patterns with ynfA

  • Construct co-evolution networks that may reveal functional relationships

  • Compare these networks between plant-associated and animal-associated serovars

• Environmental Adaptation Signatures:

  • Correlate specific ynfA variants with ecological niches (plants vs. animals)

  • Identify convergent evolution patterns in serovars with similar environmental adaptations

  • Examine whether ynfA evolution parallels that of known adaptation factors like papA

This evolutionary perspective can reveal how ynfA has contributed to the diversification and host adaptation of different Salmonella serovars, potentially explaining S. Newport's unique epidemiological profile.

What implications does ynfA research have for developing novel antimicrobial strategies against multidrug-resistant Salmonella Newport?

Research on ynfA could inform development of novel therapeutics against increasingly problematic multidrug-resistant S. Newport strains:

Therapeutic Potential Assessment:

• Target Validation:

  • Determine if ynfA is essential for S. Newport virulence or persistence

  • Assess conservation across bacterial species to gauge spectrum of activity

  • Evaluate whether targeting ynfA creates selective pressure for resistance

• Intervention Strategies:

  • Small molecule inhibitors designed to disrupt ynfA function

  • Peptide-based approaches targeting exposed regions of the protein

  • Adjuvants that sensitize resistant strains to conventional antibiotics

  • Bacteriophage-based approaches targeting ynfA-dependent processes

• Combination Therapy Development:

  • Screen for synergistic effects between ynfA inhibition and existing antibiotics

  • Identify whether ynfA manipulation can reverse resistance to specific drug classes

  • Develop multi-target approaches addressing both ynfA and related systems

• Alternative Applications:

  • Diagnostic tools based on ynfA detection for rapid identification of S. Newport

  • Vaccine development using ynfA as an antigen or carrier

  • Biocontrol strategies for reducing S. Newport in agricultural settings

Given the increasing prevalence of multidrug-resistant S. Newport strains with resistance to extended-spectrum cephalosporins and other critical antibiotics, novel targets like ynfA represent important research directions for addressing this significant public health challenge .