Recombinant Staphylococcus aureus UPF0753 protein SAS0411 (SAS0411), partial

What is the structural classification of Staphylococcus aureus UPF0753 protein SAS0411?

The UPF0753 protein SAS0411 belongs to a family of proteins with unknown function (UPF) that are conserved across various Staphylococcus strains. Similar to the characterized SaurJH9_0475 protein, it contains specific structural motifs that may be involved in cellular regulatory functions. While the precise three-dimensional structure hasn't been fully elucidated, it shares sequence homology with proteins involved in phosphate metabolism and regulation .

How is recombinant SAS0411 protein typically produced for research applications?

Recombinant SAS0411 protein is typically produced using E. coli expression systems, similar to other S. aureus recombinant proteins. The gene encoding SAS0411 is cloned into an expression vector, transformed into E. coli, and protein production is induced under controlled conditions. The protein is subsequently purified using affinity chromatography techniques, with recommended purity levels of >85% as determined by SDS-PAGE analysis . For optimal experimental reliability, protein preparations should undergo quality control testing for endotoxin levels and confirmation of protein identity by mass spectrometry.

What are the optimal storage conditions for maintaining SAS0411 protein stability?

Recombinant SAS0411 protein stability is dependent on proper storage conditions. Based on similar proteins like SaurJH9_0475, the shelf life of liquid preparations is approximately 6 months at -20°C/-80°C, while lyophilized forms can remain stable for up to 12 months at the same temperatures . To prevent protein degradation, repeated freeze-thaw cycles should be avoided. For short-term use, working aliquots can be stored at 4°C for up to one week . The addition of 5-50% glycerol (with 50% being standard) is recommended for long-term storage to prevent freeze-thaw damage.

What is the recommended protocol for reconstitution of lyophilized SAS0411 protein?

For optimal reconstitution of lyophilized SAS0411 protein, begin by briefly centrifuging the vial to ensure the protein powder is at the bottom. Reconstitute in deionized sterile water to achieve a final concentration of 0.1-1.0 mg/mL . For long-term storage, add glycerol to a final concentration of 5-50% (with 50% being standard practice) and aliquot before storing at -20°C/-80°C . When preparing working solutions, consider the buffer compatibility with your downstream applications, as some buffers may interfere with protein activity or experimental outcomes.

How can researchers effectively validate the functionality of recombinant SAS0411 protein?

Validating the functionality of recombinant SAS0411 requires multiple complementary approaches:

• Biochemical assays: Test for specific enzymatic activity if known or predicted based on sequence homology

• Binding assays: Evaluate interactions with predicted binding partners using techniques such as pull-down assays, co-immunoprecipitation, or surface plasmon resonance

• Structural analysis: Circular dichroism spectroscopy to confirm proper folding

• Functional complementation: Express the protein in S. aureus mutants lacking the native gene to assess functional rescue

• Comparative analysis with related proteins: Compare activity to better-characterized PhoU homologs to identify functional similarities

These validation steps ensure that the recombinant protein retains biological activity comparable to the native protein.

What analytical techniques are most appropriate for studying SAS0411 protein interactions?

Several analytical techniques can be employed to study SAS0411 protein interactions:

• Co-immunoprecipitation (Co-IP): To identify protein-protein interactions in cellular contexts

• Pull-down assays: For in vitro confirmation of direct protein interactions

• Surface Plasmon Resonance (SPR): To determine binding kinetics and affinity constants

• Isothermal Titration Calorimetry (ITC): For thermodynamic characterization of binding interactions

• Proximity labeling techniques: Such as BioID or APEX to identify proximal proteins in cellular environments

• Crosslinking Mass Spectrometry (XL-MS): To map interaction interfaces at the amino acid level

When designing interaction studies, researchers should consider that S. aureus proteins often function in complexes, as seen with PhoU homologs that interact with various regulatory and metabolic proteins .

How does SAS0411 protein potentially relate to S. aureus virulence and antimicrobial resistance?

While the specific function of SAS0411 remains to be fully characterized, insights can be drawn from related S. aureus proteins. S. aureus PhoU homologs have been demonstrated to regulate persister formation and virulence factor expression . Deletion of similar regulatory proteins (PhoU1 and PhoU2) resulted in decreased persister formation with vancomycin and levofloxacin by at least 1,000-fold and reduced bacterial survival in A549 cells . If SAS0411 shares functional similarities with these regulatory proteins, it may influence:

• Antibiotic tolerance mechanisms

• Expression of virulence genes

• Metabolic adaptation during infection

• Host-pathogen interactions

Research investigating these potential roles would require genetic manipulation approaches such as gene deletion or overexpression followed by phenotypic characterization.

What experimental approaches are recommended for elucidating the role of SAS0411 in S. aureus metabolism?

To investigate SAS0411's role in S. aureus metabolism, researchers should employ a multi-faceted approach:

• Gene deletion and complementation studies: Generate ΔSAS0411 mutants and complemented strains using temperature-sensitive plasmids (such as pKOR1) and allelic exchange methodologies

• Transcriptomic analysis: Compare gene expression profiles between wild-type and mutant strains using RNA-Seq to identify dysregulated metabolic pathways

• Metabolomic profiling: Measure intracellular metabolite levels, particularly focusing on key metabolites like ATP, pyruvate, and phosphate-containing compounds

• Flux analysis: Employ isotope-labeled substrates to track metabolic flux through central carbon metabolism pathways

• Phenotypic microarrays: Assess growth under various nutrient conditions to identify specific metabolic dependencies

Based on studies of related proteins, particular attention should be given to phosphate metabolism and carbon utilization pathways, as these have been implicated in the function of similar regulatory proteins in S. aureus .

What is known about the potential role of SAS0411 in phosphate metabolism regulation?

While specific information about SAS0411's role in phosphate metabolism is limited in the available search results, insights can be drawn from studies of related proteins. PhoU homologs in S. aureus regulate phosphate metabolism, with PhoU2 specifically involved in inorganic phosphate transport gene regulation . Deletion of PhoU2 resulted in up-regulation of inorganic phosphate transport genes and increased levels of intracellular inorganic polyphosphate .

If SAS0411 shares functional similarities with these regulatory proteins, it may participate in:

• Sensing intracellular phosphate levels

• Regulating phosphate transport systems

• Controlling polyphosphate accumulation

• Coordinating phosphate metabolism with other cellular processes

Experimental approaches to investigate this would include measuring intracellular phosphate levels, phosphate uptake rates, and expression of phosphate transport genes in wild-type versus SAS0411 mutant strains.

How can researchers design effective genetic manipulation experiments to study SAS0411 function?

Designing effective genetic manipulation experiments for studying SAS0411 requires careful planning:

• Mutagenesis strategy:

  • Generate clean deletion mutants using temperature-sensitive plasmids like pKOR1

  • Create point mutations in key predicted functional domains

  • Design conditional expression systems for essential gene studies

• Verification methodology:

  • Confirm genetic modifications by PCR, sequencing, and quantitative RT-PCR

  • Validate protein expression levels using Western blotting

  • Assess polar effects on adjacent genes

• Complementation approach:

  • Express wild-type SAS0411 from a plasmid in the deletion mutant

  • Use inducible promoters to control expression levels

  • Include epitope tags for protein detection without interfering with function

• Phenotypic characterization:

  • Assess growth in various media and stress conditions

  • Measure antibiotic susceptibility and persister formation

  • Evaluate virulence in cell culture and animal models

What are the challenges in differentiating the specific functions of UPF0753 family proteins from related regulatory proteins in S. aureus?

Differentiating the specific functions of UPF0753 family proteins from related regulatory proteins presents several challenges:

• Functional redundancy: S. aureus contains multiple regulatory proteins with overlapping functions, as seen with PhoU1 and PhoU2, which both affect persister formation but regulate different aspects of metabolism

• Context-dependent activity: Protein function may vary depending on growth phase, nutrient availability, and stress conditions

• Strain-specific differences: Regulatory networks can differ between S. aureus strains, complicating comparisons across studies

• Pleiotropic effects: Deletion of regulatory proteins often affects multiple pathways, making it difficult to identify direct versus indirect effects

• Technical limitations: Limited availability of specific antibodies and structural information for UPF0753 family proteins

To address these challenges, researchers should employ complementary approaches including:

• Double and triple mutant analysis to address redundancy

• Conditional expression systems to control timing and level of expression

• Domain-specific mutations to separate different functional aspects

• Systems biology approaches to map regulatory networks

How does SAS0411 potentially interact with other regulatory systems in controlling S. aureus virulence?

Based on studies of related proteins, SAS0411 may interact with multiple regulatory systems controlling S. aureus virulence:

• Global regulators: Similar regulatory proteins have been shown to affect expression of global regulators such as SarA, Rot, and CodY, which control numerous virulence factors

• Two-component signaling systems: Potential interactions with systems like SaeS/SaeR, which regulate virulence gene expression

• Metabolic regulators: Integration with metabolic control systems, particularly those involved in carbon metabolism and energy production

• Stress response pathways: Coordination with stress response regulators to adapt virulence expression under different environmental conditions

To investigate these interactions, researchers should conduct:

• Epistasis analysis using double mutants

• Protein-protein interaction studies using co-immunoprecipitation or bacterial two-hybrid systems

• Chromatin immunoprecipitation to identify potential DNA binding sites if SAS0411 functions as a transcription factor

• Transcriptomic analysis to identify co-regulated genes under various conditions

What protein purification strategies are most effective for obtaining high-purity SAS0411 for structural studies?

For structural studies requiring high-purity SAS0411 protein, the following purification strategy is recommended:

• Expression optimization:

  • Test multiple E. coli strains (BL21(DE3), Rosetta, Arctic Express)

  • Optimize induction conditions (temperature, IPTG concentration, duration)

  • Consider fusion tags that enhance solubility (MBP, SUMO, TRX)

• Multi-step purification protocol:

  • Initial capture: Affinity chromatography using His-tag or GST-tag

  • Intermediate purification: Ion exchange chromatography

  • Polishing step: Size exclusion chromatography for homogeneity

  • Tag removal: Site-specific protease cleavage followed by reverse affinity chromatography

• Quality control assessments:

  • SDS-PAGE with Coomassie staining (target >95% purity)

  • Western blot analysis for identity confirmation

  • Dynamic light scattering for monodispersity

  • Mass spectrometry for accurate molecular weight and purity assessment

• Buffer optimization:

  • Screen multiple buffer conditions using differential scanning fluorimetry

  • Test stabilizing additives (glycerol, specific ions, reducing agents)

  • Assess long-term stability in various storage conditions

For crystallography studies, additional screening for conditions that promote crystal formation would be necessary.

How can researchers effectively design antibodies against SAS0411 for immunodetection and localization studies?

Designing effective antibodies against SAS0411 requires a strategic approach:

• Antigen selection:

  • Full-length protein: Provides comprehensive epitope coverage but may have specificity issues

  • Unique peptide regions: Higher specificity but potentially lower sensitivity

  • Specific structural domains: Balance between specificity and sensitivity

• Antibody production strategies:

  • Polyclonal antibodies: Broader epitope recognition, useful for initial studies

  • Monoclonal antibodies: Higher specificity, better for distinguishing between related proteins

  • Recombinant antibodies: Consistent production and potential for engineering enhanced properties

• Validation methods:

  • Western blot analysis using recombinant protein and cellular extracts

  • Immunoprecipitation efficiency testing

  • Testing in knockout strains as negative controls

  • Cross-reactivity assessment with related S. aureus proteins

• Application-specific considerations:

  • For immunofluorescence: Test fixation conditions that preserve epitope accessibility

  • For immunoprecipitation: Evaluate antibody binding under native conditions

  • For ELISA: Determine optimal coating and detection conditions

Custom antibody production typically requires 2-4 months and should include comprehensive validation to ensure specificity and sensitivity for SAS0411.

What approaches are recommended for studying the subcellular localization of SAS0411 in S. aureus?

Studying the subcellular localization of SAS0411 in S. aureus requires specialized approaches due to the small cell size and thick peptidoglycan layer:

• Fluorescent protein fusion approaches:

  • C-terminal and N-terminal GFP or mCherry fusions

  • Verification that fusion proteins retain functionality

  • Optimization of expression levels to prevent artifacts

  • Super-resolution microscopy for detailed localization patterns

• Immunolocalization methods:

  • Development of highly specific antibodies against SAS0411

  • Optimization of fixation and permeabilization for S. aureus

  • Use of appropriate controls (deletion mutants)

  • Dual labeling with markers for specific subcellular compartments

• Biochemical fractionation:

  • Separation of membrane, cytoplasmic, and cell wall fractions

  • Western blot analysis of fractions to detect SAS0411

  • Comparison with known markers of each fraction

  • Assessment under different growth conditions or stresses

• Proximity labeling approaches:

  • APEX2 or BioID fusion proteins to identify proximal proteins

  • Mapping of the local interactome to infer localization

  • Validation using orthogonal approaches

Based on related proteins like PhoU homologs, researchers should particularly investigate potential membrane association and cytoplasmic distribution patterns that may change under different physiological conditions .

How does SAS0411 compare to homologous proteins in other Staphylococcus species and what can this tell us about its function?

Comparative analysis of SAS0411 with homologous proteins in other Staphylococcus species provides valuable insights into its potential function:

• Sequence conservation analysis:

  • Core domains likely represent functionally critical regions

  • Variable regions may indicate species-specific adaptations

  • Conserved motifs suggest shared biochemical activities

• Functional differences between species:

  • While PhoU homologs in S. aureus (PhoU1 and PhoU2) both regulate persister formation, in S. epidermidis only PhoU2 regulates biofilm and persister formation

  • These species-specific differences suggest evolutionary adaptations to different ecological niches

• Structural comparison approaches:

  • Homology modeling based on crystal structures from related proteins

  • Identification of conserved functional domains

  • Prediction of potential interaction interfaces

• Evolutionary rate analysis:

  • Fast-evolving regions may indicate adaptive functions

  • Highly conserved regions likely represent core functional domains

This comparative approach can guide the design of functional studies by highlighting the most promising regions for mutagenesis and chimeric protein construction.

What is the relationship between SAS0411 and bacterial stress response mechanisms?

The relationship between SAS0411 and bacterial stress response mechanisms can be inferred from studies of related proteins:

• Potential role in antibiotic tolerance:

  • Related PhoU proteins regulate persister formation in response to antibiotics like vancomycin and levofloxacin

  • Deletion mutants of similar proteins show reduced persister formation without changes in MIC/MBC values

• Metabolic stress adaptation:

  • Related proteins influence carbon and pyruvate metabolism

  • They affect intracellular ATP levels, which are critical for stress response

• Nutrient limitation responses:

  • Similar to PhoU proteins that regulate phosphate metabolism

  • May coordinate metabolic adaptations during nutrient limitation

• Integration with global stress regulators:

  • Potential interactions with stress-responsive transcription factors

  • Coordination with stringent response mechanisms

Research approaches should include phenotypic characterization of SAS0411 mutants under various stress conditions (oxidative, osmotic, acid, antibiotic stress) and transcriptomic analysis to identify SAS0411-regulated genes during stress exposure.

What are the most promising approaches for elucidating the three-dimensional structure of SAS0411?

Several complementary approaches can be employed to elucidate the three-dimensional structure of SAS0411:

Each approach has strengths and limitations, and the optimal strategy may involve a combination of methods tailored to the specific properties of SAS0411.

How can systems biology approaches enhance our understanding of SAS0411 function in the context of S. aureus regulatory networks?

Systems biology approaches offer powerful tools for understanding SAS0411 within S. aureus regulatory networks:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Identify direct and indirect effects of SAS0411 deletion

  • Map the regulatory network through correlation analysis

• Network modeling and analysis:

  • Construct gene regulatory networks including SAS0411

  • Identify network motifs and regulatory hubs

  • Predict system-wide effects of perturbations

• Temporal dynamics studies:

  • Track regulatory changes across growth phases

  • Capture acute responses to environmental perturbations

  • Model time-dependent interactions

• In silico prediction and validation:

  • Generate testable hypotheses about SAS0411 function

  • Design targeted experimental validation

  • Iteratively refine models with new experimental data

Studies of related proteins have shown that deletion of PhoU homologs affects multiple regulatory systems, including global regulators (SarA, Rot) and two-component systems (SaeS) , suggesting that SAS0411 may similarly engage in complex regulatory interactions that are best understood through systems approaches.

What potential applications might arise from understanding SAS0411 function in relation to antimicrobial resistance and virulence?

Understanding SAS0411 function could lead to several important applications:

• Novel antimicrobial targets:

  • If SAS0411 regulates persister formation like related proteins , inhibitors might sensitize S. aureus to existing antibiotics

  • Structure-based drug design targeting SAS0411 or its interactions

  • Development of anti-virulence strategies that don't directly kill bacteria but reduce pathogenicity

• Diagnostic applications:

  • Development of biomarkers for antibiotic-tolerant S. aureus infections

  • Identification of expression signatures predictive of treatment outcomes

  • Detection methods for specific strains with altered SAS0411 activity

• Vaccine development:

  • Assessment of SAS0411 as a potential vaccine antigen

  • Use of attenuated strains with modified SAS0411 as live vaccines

  • Design of combination vaccine strategies targeting multiple virulence regulators

• Biotechnology applications:

  • Engineering S. aureus strains with modified SAS0411 for research tools

  • Development of biosensors based on SAS0411 regulatory mechanisms

  • Utilization in synthetic biology applications