Recombinant Enterococcus faecalis UPF0374 protein EF_0697 (EF_0697)

What is the UPF0374 protein EF_0697 and what is its significance in Enterococcus faecalis?

UPF0374 protein EF_0697 belongs to a family of uncharacterized proteins found in Gram-positive bacteria. In E. faecalis, this protein is particularly significant as it may play a role in the bacterium's remarkable ability to adapt to hostile environments such as the urinary tract. While its exact function remains to be fully elucidated, proteomic studies suggest it could be involved in cellular adaptation mechanisms similar to other uncharacterized proteins identified in E. faecalis secretome analyses . The designation "UPF" (uncharacterized protein family) indicates that while the protein has been identified and sequenced, its biological function has not been fully characterized experimentally.

What expression systems are recommended for recombinant UPF0374 protein EF_0697 production?

Recombinant UPF0374 protein EF_0697 can be effectively expressed in multiple host systems, with E. coli and yeast offering optimal yields and shorter turnaround times for most research applications . For studies requiring post-translational modifications that might be critical for the protein's function, insect cell expression systems using baculovirus or mammalian cell systems may be preferable, as these can provide many of the modifications necessary for correct protein folding and activity maintenance . The choice of expression system should be guided by specific research objectives, whether prioritizing quantity, speed of production, or structural integrity.

What is known about the structural features of UPF0374 proteins?

Based on structural models of related UPF0374 proteins such as SEQ_1718 from Streptococcus equi, these proteins exhibit highly confident structural predictions with global pLDDT (predicted Local Distance Difference Test) scores above 90, indicating a well-defined three-dimensional structure . The models suggest that UPF0374 proteins may have structural similarities to components of bacterial phosphotransferase systems or receiver domains found in two-component signal transduction systems, though with notable differences in key functional residues . These structural insights, while not directly verified for EF_0697, provide a framework for understanding potential functional mechanisms.

How can researchers distinguish between the different functions of UPF0374 protein EF_0697 and its homologs in various Gram-positive bacteria?

Distinguishing functional differences requires a multi-faceted experimental approach combining:

• Comparative sequence analysis across bacterial species

• Gene knockout studies examining phenotypic changes

• Transcriptomic profiling under varying environmental conditions

• Protein-protein interaction studies to identify binding partners

For E. faecalis specifically, the adaptation to different environments (e.g., urine vs. standard media) significantly alters the protein expression profile . While examining homologs like UPF0374 protein SEQ_1718 from S. equi (which shares structural characteristics), researchers should note that despite structural similarities, specific insertions between residues (such as those observed in OG1RF_10478) may serve unique functions like dimerization interfaces . These differences highlight the importance of species-specific characterization rather than broad functional assumptions.

What are the most effective methods for studying the role of UPF0374 protein EF_0697 in E. faecalis adaptation to different environments?

The most effective methodological approach involves:

• Comparative proteomic analysis: Utilize label-free quantitative proteomics based on MaxQuant LFQ algorithms to identify differential protein expression across environmental conditions .

• Transcriptional regulation studies: Employ quantitative RT-PCR assays using the comparative CT method with appropriate reference genes (e.g., recA) to validate expression changes .

• Promoter mapping: Implement primer extension assays to identify transcription start sites and relevant promoter elements controlling expression .

• DNA-protein interaction analysis: Apply DNase I footprinting to identify potential regulatory protein binding sites influencing expression .

This multi-tiered approach allows researchers to develop a comprehensive understanding of how UPF0374 protein EF_0697 expression is regulated and contributes to environmental adaptation.

How reliable are computational structure predictions for UPF0374 proteins, and what experimental validations should be performed?

While computational structure predictions for UPF0374 proteins show high confidence scores (e.g., pLDDT global score of 96.91 for the related SEQ_1718 protein) , these remain theoretical models requiring experimental validation. Researchers should consider:

These experimental validations become especially important when predicted structures suggest functional similarities to known protein families but with critical differences in conserved residues, as observed in similar proteins from E. faecalis .

What are the optimal conditions for high-yield expression of recombinant UPF0374 protein EF_0697?

For optimal expression of recombinant UPF0374 protein EF_0697, the following considerations are critical:

• Host selection: E. coli BL21(DE3) or similar strains provide efficient expression for basic structural studies, while Pichia pastoris offers advantages for larger-scale production with eukaryotic folding machinery .

• Vector design: Incorporate a cleavable affinity tag (His6 or GST) for purification, preferably with a precision protease site to ensure native protein structure after tag removal.

• Induction conditions: For E. coli expression, IPTG concentration (typically 0.1-1.0 mM), induction temperature (16-37°C), and duration (3-24 hours) require optimization, with lower temperatures generally favoring proper folding.

• Media composition: Enriched media (2xYT or TB) generally yields higher biomass and protein production compared to minimal media .

• Co-expression considerations: For proteins requiring specific folding assistance, co-expression with chaperones (GroEL/ES, DnaK/J) may improve soluble yield.

Systematic optimization of these parameters through small-scale expression trials is recommended before scaling to production volumes.

What purification strategies maximize the recovery of correctly folded UPF0374 protein EF_0697?

A multi-step purification approach is recommended for obtaining high-purity, correctly folded protein:

• Initial capture: Immobilized metal affinity chromatography (IMAC) for His-tagged constructs or glutathione sepharose for GST-tagged proteins provides efficient initial purification .

• Tag removal: Site-specific protease cleavage (TEV or PreScission) followed by reverse affinity chromatography ensures removal of the tag and uncleaved protein.

• Intermediate purification: Ion exchange chromatography based on the protein's theoretical pI (anion exchange for pI < 7, cation exchange for pI > 7).

• Polishing: Size exclusion chromatography to separate monomeric protein from aggregates and to perform buffer exchange into the final storage buffer.

• Quality assessment: SDS-PAGE, dynamic light scattering, and circular dichroism should be employed to verify purity, homogeneity, and proper folding.

For EF_0697 specifically, monitoring protein stability during purification is critical, as proteins in this family may demonstrate conformational changes when shifting between different environmental conditions, similar to the adaptations observed in E. faecalis proteins responding to environmental changes .

How can researchers effectively investigate potential regulatory roles of UPF0374 protein EF_0697 in E. faecalis?

Investigating regulatory functions requires multiple complementary approaches:

• Transcriptomics: RNA-Seq or microarray analysis comparing wild-type and EF_0697 deletion mutants can identify genes with altered expression, revealing potential regulatory networks .

• Chromatin immunoprecipitation (ChIP): If EF_0697 functions as a DNA-binding protein, ChIP-seq can identify genomic binding sites, similar to methods used for characterizing the MafR transcriptional regulator in E. faecalis .

• Protein-protein interaction studies: Bacterial two-hybrid assays or co-immunoprecipitation followed by mass spectrometry can identify interaction partners potentially involved in regulatory complexes.

• Reporter gene assays: Fusing identified promoter regions to reporter genes (e.g., lacZ) allows quantitative assessment of transcriptional effects when EF_0697 levels are modulated .

• Epistasis analysis: Constructing double mutants with known regulators helps place EF_0697 within existing regulatory hierarchies.

These approaches have proven effective in characterizing other regulatory proteins in E. faecalis, such as the global transcriptional regulator MafR, which influences the expression of numerous genes including those encoding enzymes and transporters required for carbon source utilization .

What techniques are most suitable for determining the subcellular localization of UPF0374 protein EF_0697?

Determining subcellular localization requires multiple complementary methods:

• Fractionation studies: Differential centrifugation to separate cellular compartments (cytoplasm, membrane, cell wall) followed by Western blot analysis using antibodies against EF_0697.

• Fluorescent protein fusions: GFP-EF_0697 fusion constructs expressed in E. faecalis for live-cell fluorescence microscopy, bearing in mind potential effects on protein function.

• Immunogold electron microscopy: Ultra-high resolution localization using specific antibodies conjugated to gold particles.

• Computational prediction: Bioinformatic analysis for signal peptides, transmembrane domains, and localization signals using tools like PSORT and SignalP.

• Proteomic verification: Analysis of secretome fractions using methods similar to those in previous E. faecalis studies that identified secreted proteins in different environmental conditions .

Based on proteomic studies of E. faecalis, certain proteins show environment-specific localization, being differentially secreted when bacteria are cultured in urine versus standard laboratory media . This suggests environmental triggers may influence EF_0697 localization.

What are the key considerations for designing activity assays for UPF0374 protein EF_0697 when its function remains poorly characterized?

When designing activity assays for proteins with unknown functions like EF_0697, consider:

• Structural homology-based approaches: Based on structural similarities to EIIB components of phosphotransferase systems or receiver domains in two-component signal transduction systems , design phosphorylation assays using radioactive ATP or phosphate donors.

• Thermal shift assays: Screen for potential ligands or interaction partners by measuring protein thermostability shifts upon binding.

• Enzymatic activity screening: Test for common enzymatic activities (phosphatase, kinase, nuclease) using substrate libraries.

• Protein-protein interaction screening: Identify binding partners through pull-down assays coupled with mass spectrometry to provide functional clues.

• Phenotypic complementation: Express EF_0697 in heterologous systems with defined mutant phenotypes to observe functional rescue.

While designing these assays, consider that UPF0374 proteins may lack certain conserved residues typically required for specific functions (e.g., phosphorylation sites), necessitating modified assay conditions and careful interpretation of results .

How might UPF0374 protein EF_0697 contribute to E. faecalis pathogenicity and what experimental approaches can test this hypothesis?

UPF0374 protein EF_0697 may contribute to E. faecalis pathogenicity through mechanisms similar to other adaptation proteins:

• Infection models: Compare virulence of wild-type and EF_0697 deletion mutants in established in vivo models (urinary tract infection, endocarditis, systemic infection).

• Host cell interaction studies: Examine adhesion, invasion, and survival within host cells using tissue culture models.

• Stress response assessment: Evaluate the mutant's ability to withstand host defense mechanisms (oxidative stress, antimicrobial peptides, pH fluctuations).

• Transcriptome analysis: Compare gene expression profiles during infection to identify EF_0697-dependent virulence factor expression.

• Biofilm formation: Quantify biofilm development in clinical scenarios, as E. faecalis biofilms contribute significantly to pathogenicity.

E. faecalis is known for its adaptability to hostile environments such as the urinary tract and bladder , and uncharacterized proteins often play crucial roles in these adaptation processes. The connection between environmental adaptation and virulence is particularly relevant for opportunistic pathogens like E. faecalis.

What are the most promising approaches for identifying potential interaction partners of UPF0374 protein EF_0697?

To identify interaction partners, researchers should employ:

• Affinity purification-mass spectrometry (AP-MS): Express tagged EF_0697 in E. faecalis, perform pull-downs under native conditions, and identify co-purifying proteins by mass spectrometry.

• Bacterial two-hybrid screening: Test for interactions with a library of E. faecalis proteins using systems optimized for bacterial protein interactions.

• Surface plasmon resonance (SPR): Verify direct binding and determine kinetic parameters for candidate interactors.

• Crosslinking mass spectrometry: Use chemical crosslinking coupled with mass spectrometry to capture transient interactions in their native environment.

• Co-immunoprecipitation with specific candidates: Test interactions with proteins involved in pathways suggested by structural similarities, such as components of phosphotransferase systems or two-component signal transduction systems .

The potential dimerization interface identified in similar proteins suggests protein-protein interactions are likely critical to EF_0697 function, making interaction partner identification particularly valuable.

What comparative genomic approaches could help elucidate the evolutionary significance of UPF0374 proteins across bacterial species?

Comparative genomic approaches should include:

• Phylogenetic analysis: Construct evolutionary trees of UPF0374 proteins across bacterial species to identify conservation patterns and evolutionary relationships.

• Synteny analysis: Examine the genomic context of UPF0374 genes across species to identify consistently co-localized genes suggesting functional relationships.

• Selection pressure analysis: Calculate dN/dS ratios to determine whether UPF0374 proteins are under purifying or diversifying selection.

• Structural comparison: Compare AlphaFold or experimentally determined structures across species to identify conserved structural features despite sequence divergence.

• Horizontal gene transfer assessment: Evaluate evidence for horizontal acquisition that might explain distribution patterns across bacterial taxa.

These approaches can reveal whether UPF0374 proteins represent core bacterial functions or specialized adaptations, and whether their presence correlates with specific ecological niches or pathogenic potential across bacterial species.