Recombinant Salmonella typhimurium UPF0208 membrane protein YfbV (yfbV)

What is YfbV protein and where is it localized in Salmonella typhimurium?

YfbV (gene designation: STM2336) is classified as an UPF0208 family membrane protein in Salmonella typhimurium, consisting of 151 amino acids. Cellular fractionation studies indicate that YfbV is predominantly localized to the inner membrane . The protein contains transmembrane domains that facilitate its integration into the lipid bilayer, similar to its E. coli homolog which is also found in the inner membrane .

How conserved is YfbV across different bacterial species?

YfbV is highly conserved across Enterobacteriaceae, particularly among Salmonella serovars and E. coli strains. Sequence alignment analyses reveal >90% amino acid identity between Salmonella typhimurium YfbV and its homologs in other Salmonella species (S. paratyphi, S. enteritidis, S. heidelberg) . The E. coli YfbV homolog shares approximately 85-90% sequence identity with the Salmonella versions, suggesting functional conservation . This high level of conservation indicates evolutionary importance and potential functional significance across enteric bacteria.

What structural domains or motifs are present in YfbV?

YfbV contains a characteristic DUF412 (Domain of Unknown Function 412) motif which is the defining feature of the UPF0208 protein family . Structural predictions indicate that YfbV possesses multiple transmembrane helices that likely anchor the protein to the inner membrane. Secondary structure analysis suggests approximately 60-65% alpha-helical content with the remaining regions comprised of loops and few beta-strands . The N-terminal region appears more conserved across species and may contain functionally important residues, while the C-terminal region shows higher variability.

What expression systems are most effective for producing recombinant YfbV?

E. coli expression systems have proven most efficient for recombinant YfbV production. The most commonly used approach employs BL21(DE3) strains with pET-based vectors containing an N-terminal His-tag for purification . Expression in E. coli typically yields 3-5 mg of protein per liter of culture. For improved folding of this membrane protein, expression at lower temperatures (16-18°C) after IPTG induction (0.5 mM) is recommended .

Alternative expression systems include:

• Yeast expressions systems (P. pastoris) - useful for post-translational modifications

• Baculovirus expression in insect cells - for projects requiring eukaryotic processing

• Cell-free expression systems - when rapid production is needed for initial screening

What purification strategy should be employed for recombinant YfbV?

The recommended purification protocol for His-tagged recombinant YfbV involves:

• Cell lysis under native conditions using mild detergents (0.5-1% n-dodecyl β-D-maltoside or CHAPS)

• Initial purification via IMAC (Immobilized Metal Affinity Chromatography) using Ni-NTA resin

• Buffer exchange to remove imidazole

• Secondary purification step using size exclusion chromatography

• Final concentration and storage in detergent-containing buffer with 6% trehalose at pH 8.0

This approach typically yields protein with >90% purity as determined by SDS-PAGE . For membrane protein studies requiring higher purity, additional ion exchange chromatography may be employed as a polishing step.

How can researchers assess the proper folding and activity of purified YfbV?

Assessing proper folding of YfbV can be challenging due to its membrane protein nature. Recommended approaches include:

• Circular Dichroism (CD) spectroscopy to confirm predicted secondary structure content

• Size exclusion chromatography to verify monodispersity

• Thermal shift assays to assess protein stability

• Limited proteolysis to evaluate compact folding

Since the specific biochemical activity of YfbV remains uncharacterized, functional assays must rely on indirect measurements or biological assays such as complementation of yfbV deletion strains .

What is the putative function of YfbV in Salmonella typhimurium?

While the precise function of YfbV remains uncharacterized, several lines of evidence suggest potential roles:

• Based on homology with E. coli YfbV, it may be involved in regulation of chromosome structure

• Its membrane localization suggests potential roles in:

  • Membrane integrity or organization

  • Transport or signaling across the inner membrane

  • Protein-protein interactions at the membrane interface

How does YfbV deletion affect Salmonella phenotype and virulence?

Studies examining yfbV deletion mutants in Salmonella have shown:

• No significant growth defects under standard laboratory conditions in rich media

• Potential alterations in membrane composition or permeability

• Possible changes in stress response, particularly under iron-limited conditions similar to those observed with YdiU-mediated regulation

The effects of yfbV deletion on Salmonella virulence remain incompletely characterized but may involve altered host-pathogen interactions given the importance of membrane proteins in bacterial adaptation to host environments. More extensive phenotypic analyses under various stress conditions and in infection models would help clarify YfbV's role in Salmonella pathogenesis.

What are the optimal conditions for studying YfbV-protein interactions?

When designing experiments to identify YfbV-protein interactions, researchers should consider:

• Cross-linking approaches: Use membrane-permeable cross-linkers like DSP (dithiobis(succinimidyl propionate)) at 0.5-2 mM concentration in live cells to capture transient interactions

• Co-immunoprecipitation: Employ mild detergents (0.5% n-dodecyl β-D-maltoside) to solubilize membrane complexes while preserving protein-protein interactions

• Bacterial two-hybrid systems: Modified for membrane proteins, such as BACTH (Bacterial Adenylate Cyclase Two-Hybrid) system for screening potential interaction partners

• Proximity-based labeling: BioID or APEX2 fusion proteins to identify neighboring proteins in the native membrane environment

Control experiments should include:

• Non-specific binding controls (unrelated membrane proteins)

• Negative controls without cross-linking

• Validation of interactions using multiple complementary techniques

How can researchers design experiments to elucidate YfbV function?

A comprehensive experimental approach to determine YfbV function should include:

• Transcriptomic analysis: Compare gene expression profiles between wild-type and yfbV deletion strains under various conditions (rich media, minimal media, stress conditions)

• Phenotypic microarrays: Screen for growth differences across hundreds of conditions (carbon sources, nitrogen sources, pH, osmolarity, antibiotics)

• Genetic interaction mapping: Construct double mutants combining yfbV deletion with known pathway mutations to identify synthetic phenotypes, following approaches used for other Salmonella genes

• Localization studies: Fluorescent protein fusions to determine subcellular distribution patterns during different growth phases and stress responses

• In vivo infection models: Evaluate colonization, persistence, and virulence of yfbV mutants in appropriate animal models

This multi-faceted approach provides complementary data to triangulate YfbV's functional role.

What controls should be included when analyzing YfbV expression levels?

Proper experimental design for YfbV expression studies should include:

• Positive controls:

  • Housekeeping genes (rpoD, gyrB) for normalization in qRT-PCR

  • Known regulated genes under test conditions

• Negative controls:

  • Non-template controls for PCR contamination

  • Strains with deletion of yfbV to validate antibody specificity

• Experimental controls:

  • Growth curve synchronization to account for growth phase effects

  • Multiple biological and technical replicates (minimum n=3)

  • Comparison of multiple detection methods (qRT-PCR, Western blot, reporter fusions)

• Statistical analysis:

  • Appropriate statistical tests with correction for multiple comparisons

  • Transparent reporting of all data points and outliers

How might YfbV interact with host cellular components during infection?

While direct evidence for YfbV interactions with host factors is lacking, researchers investigating this question should consider:

• Potential parallels with YtfB, which has been shown to play a role in eukaryotic cell invasion processes in E. coli

• Experimental approaches to test host interactions:

  • Yeast two-hybrid screening against human protein libraries

  • Pull-down assays using purified YfbV against host cell lysates

  • Infection models comparing wild-type and ΔyfbV Salmonella

  • Transcriptomic analysis of host cells exposed to purified YfbV

• Special considerations when designing these experiments:

  • Use of membrane fractions rather than whole cell lysates

  • Careful negative controls with unrelated bacterial membrane proteins

  • Validation with multiple cell types relevant to Salmonella infection

How does YfbV expression change in response to environmental signals encountered during infection?

To investigate YfbV regulation during infection processes, researchers should design experiments that:

• Mimic host conditions: Examine YfbV expression under:

  • Acidic pH (pH 4.5-5.5, simulating phagosomes)

  • Nutrient limitation (particularly iron restriction)

  • Antimicrobial peptide exposure

  • Macrophage infection models

• Utilize reporter systems:

  • Transcriptional fusions (yfbV promoter-lacZ/GFP)

  • Translational fusions (if they don't disrupt function)

  • Ribosome profiling to examine translation efficiency

• Compare with known virulence regulators:

  • Examine dependence on PhoP/PhoQ, RpoS, and other stress-responsive regulators

  • Test for coordinated regulation with other virulence genes

Preliminary data suggests that YfbV expression may be altered under conditions similar to those that trigger UMPylation by YdiU, which affects flagellar biogenesis in Salmonella within host cells .

What approaches can be used to identify small molecule modulators of YfbV function?

For researchers interested in identifying small molecules that interact with or modulate YfbV:

• Initial screening approaches:

  • Thermal shift assays to identify compounds that alter protein stability

  • Surface plasmon resonance (SPR) or microscale thermophoresis (MST) for binding studies

  • In silico docking studies based on AlphaFold structural predictions

• Functional validation:

  • Growth inhibition or rescue of yfbV mutant phenotypes

  • Direct biochemical assays once a functional assay is established

  • Membrane permeability assays to detect changes in membrane properties

• Structure-activity relationship studies:

  • Systematic modification of hit compounds

  • Comparison across different bacterial species' YfbV homologs

  • Competition assays to determine binding sites

This research direction could provide tools for further functional characterization and potentially identify novel antimicrobial approaches targeting membrane protein functions.

How does Salmonella typhimurium YfbV compare with its E. coli homolog functionally?

While both proteins belong to the UPF0208 family and share high sequence similarity (85-90%), potential functional differences include:

Researchers investigating functional differences should consider complementation experiments (expressing E. coli YfbV in Salmonella yfbV deletion strains and vice versa) to determine functional conservation .

What experimental approaches can distinguish the roles of YfbV from other UPF0208 family proteins?

To differentiate the specific functions of YfbV from other related proteins:

• Domain swapping experiments:

  • Create chimeric proteins between YfbV and other UPF0208 family members

  • Test functionality in appropriate deletion backgrounds

  • Identify domains responsible for specific functions

• Differential phenotypic analysis:

  • Compare growth and stress responses of mutants lacking different UPF0208 family proteins

  • Use high-throughput phenotypic arrays to identify condition-specific roles

  • Examine genetic interactions with common pathways

• Co-expression network analysis:

  • Identify genes co-regulated with each UPF0208 family member

  • Compare transcriptomic responses to deletion of different family members

  • Build predictive models for functional relationships

These approaches can help delineate unique versus overlapping functions among UPF0208 family proteins.

How can YfbV be utilized in the development of attenuated Salmonella vaccine strains?

YfbV's potential application in vaccine development could include:

• As a mutation target for attenuation:

  • If yfbV deletion affects virulence without compromising immunogenicity

  • In combination with other attenuating mutations in well-established vaccine platforms

• As a delivery vehicle:

  • Fusion partner for antigenic epitopes (C-terminal fusions may be tolerated without disrupting membrane integration)

  • Surface display systems if portions are exposed to the periplasm or extracellular space

• Experimental design considerations:

  • Careful characterization of growth and survival in vivo

  • Assessment of immune responses to both vectored antigens and YfbV itself

  • Stability of expression in vivo without selective pressure

Any vaccine development application would require extensive safety and efficacy testing in appropriate animal models .

What considerations are important when using YfbV as a model for studying bacterial membrane protein structure and function?

YfbV presents both opportunities and challenges as a model membrane protein:

Advantages:

• Relatively small size (151 aa) facilitates expression and structural studies

• High conservation allows evolutionary and comparative analyses

• Presence in well-studied model organisms enables genetic manipulation

Challenges:

• Unknown native function complicates functional assays

• Membrane integration requires specialized handling for structural studies

• Potential toxicity when overexpressed

Recommended approaches:

• Use native membrane environment when possible (nanodiscs, liposomes)

• Consider detergent screening to identify optimal solubilization conditions

• Employ complementary structural techniques (X-ray crystallography, cryo-EM, NMR)

• Validate structural predictions with biochemical and biophysical experiments

YfbV's characteristics make it an interesting but challenging subject for membrane protein methodology development.