Recombinant Staphylococcus aureus UPF0060 membrane protein SAUSA300_2286 (SAUSA300_2286)

What is the SAUSA300_2286 protein and why is it significant in S. aureus research?

SAUSA300_2286 is a UPF0060 family membrane protein found in Staphylococcus aureus strain USA300, with UniProt accession number Q2FEF7. This protein consists of 108 amino acids and is classified as a transmembrane protein with multiple membrane-spanning regions . Its significance stems from being part of a clinically relevant strain (USA300) that is frequently associated with community-acquired infections and antibiotic resistance. Research into membrane proteins like SAUSA300_2286 is crucial for understanding bacterial physiology, pathogenesis mechanisms, and developing novel antimicrobial strategies that target membrane components.

What is the amino acid sequence and predicted structural features of SAUSA300_2286?

The full amino acid sequence of SAUSA300_2286 is: mLYPIFIFILAGLCEIGGGYLIWLWLREGQSSLVGLIGGAILmLYGVIATFQSFPSFGRVYAAYGGVFIIMSLIFAMVVDKQMPDKYDVIGAIICIVGVLVmLLPSRA .

Structural prediction analysis suggests that SAUSA300_2286 is a highly hydrophobic protein with multiple transmembrane domains. The presence of numerous hydrophobic residues (particularly leucine, isoleucine, valine, and phenylalanine) supports its classification as an integral membrane protein. Based on sequence characteristics, researchers predict the protein contains multiple membrane-spanning alpha-helical regions with hydrophilic loops connecting them. These structural features are consistent with other UPF0060 family proteins that typically have roles in membrane integrity or transport functions.

What are the recommended protocols for expression and purification of recombinant SAUSA300_2286?

For recombinant expression and purification of SAUSA300_2286, researchers should consider:

Expression System Selection:

• E. coli-based systems with specialized strains designed for membrane protein expression (e.g., C41(DE3), C43(DE3))

• Cell-free expression systems for difficult-to-express membrane proteins

• S. aureus expression systems for native-like folding environment

Expression Protocol:

• Clone the SAUSA300_2286 gene (expression region 1-108) into an appropriate vector with a solubility or affinity tag

• Transform into expression hosts using electroporation techniques optimized for membrane proteins

• Induce expression at lower temperatures (16-25°C) to facilitate proper folding

• Supplement growth media with specific lipids if needed for stability

Purification Strategy:

• Cell disruption using specialized techniques for membrane proteins (French press or sonication)

• Membrane fraction isolation through ultracentrifugation

• Solubilization using appropriate detergents (DDM, LDAO, or other mild detergents)

• Affinity chromatography using the fusion tag

• Size exclusion chromatography for final purification

Storage Recommendations:

• Store in Tris-based buffer with 50% glycerol at -20°C for standard storage

• For extended storage, maintain at -80°C

• Avoid repeated freeze-thaw cycles

• Working aliquots can be kept at 4°C for up to one week

How can researchers effectively transform S. aureus with recombinant SAUSA300_2286 constructs?

Effective transformation of S. aureus with recombinant SAUSA300_2286 constructs requires specialized protocols due to the thick peptidoglycan layer of S. aureus:

Electroporation Method:

• Prepare electrocompetent S. aureus cells by growing to mid-logarithmic phase (OD600 of 0.5-0.8)

• Wash cells multiple times with ice-cold electroporation buffer (typically 10% glycerol with reduced salt concentration)

• Concentrate cells to high density (~10¹⁰ cells/mL)

• Mix plasmid DNA (purified using endotoxin-free kits) with cells

• Perform electroporation using optimized settings (typically 2.0-2.5 kV, 25 μF, 100 Ω)

• Immediately add recovery media and incubate at 30-37°C for 1-3 hours

• Plate on selective media with appropriate antibiotics

For NTML Library Strains:
When using Nebraska Transposon Mutant Library (NTML) strains or creating complementation constructs:

• Design complementation plasmids with appropriate promoters for expression

• Verify sequence integrity before transformation

• After transformation, confirm successful integration through PCR and sequencing

• Validate functional complementation through phenotypic assays

For optimal results, researchers should consider strain-specific modifications, as transformation efficiency can vary significantly between different S. aureus strains.

What advanced techniques are recommended for studying SAUSA300_2286 membrane topology and orientation?

Advanced Techniques for Topology Analysis:

• Cysteine Scanning Mutagenesis:

  • Systematically replace individual residues with cysteine

  • Use membrane-impermeable thiol-reactive reagents to determine exposure

  • Create a comprehensive topological map based on accessibility data

• Fluorescence-Based Approaches:

  • Generate GFP fusion constructs at various positions

  • Analyze cellular localization using confocal microscopy

  • Quantify fluorescence to determine membrane insertion efficiency

• Protease Protection Assays:

  • Express epitope-tagged versions of SAUSA300_2286

  • Subject intact cells, spheroplasts, and membrane vesicles to protease treatment

  • Analyze protection patterns to determine cytoplasmic vs. extracellular domains

• Advanced Biophysical Methods:

  • Solid-state NMR for structural determination in membrane environment

  • Cryo-electron microscopy for high-resolution structural analysis

  • Hydrogen-deuterium exchange mass spectrometry to probe dynamic regions

• Computational Prediction Validation:

  • Use algorithms like TMHMM, MEMSAT, and TOPCONS for initial predictions

  • Experimentally validate key predictions using techniques above

  • Refine computational models based on experimental data

These approaches provide complementary data that, when integrated, can generate a comprehensive understanding of SAUSA300_2286's membrane topology and orientation.

What methods are most effective for determining the function of SAUSA300_2286 in S. aureus?

Comprehensive Functional Analysis Approaches:

• Genetic Manipulation Strategies:

  • Generate clean deletion mutants using allelic replacement

  • Create conditional expression strains for essential functions

  • Use transposon mutagenesis libraries for high-throughput screening

  • Perform complementation studies to confirm phenotype specificity

• Phenotypic Characterization:

  • Assess growth kinetics under various conditions

  • Analyze membrane integrity using dye penetration assays

  • Evaluate stress responses (oxidative, osmotic, pH, temperature)

  • Measure biofilm formation capacity

  • Test antimicrobial susceptibility profiles

• Protein Interaction Studies:

  • Perform bacterial two-hybrid assays for protein-protein interactions

  • Use co-immunoprecipitation with tagged versions

  • Apply crosslinking approaches for transient interactions

  • Conduct pull-down assays with potential binding partners

• Transport Function Analysis:

  • Measure substrate uptake using radiolabeled compounds

  • Assess membrane potential changes upon substrate addition

  • Use fluorescent probes to monitor transport activity

  • Reconstitute purified protein in liposomes for transport assays

• Comparative Genomics Approach:

  • Analyze conservation across different strains and species

  • Identify co-occurrence with functionally related genes

  • Examine genomic context for functional clues

Integration of these approaches provides a robust framework for functional characterization, with each method providing complementary insights into SAUSA300_2286's biological role.

How does the SAUSA300_2286 protein potentially contribute to S. aureus pathogenesis and virulence?

While the exact function of SAUSA300_2286 in pathogenesis is not fully characterized, several methodological approaches can be employed to investigate its potential role in virulence:

• Infection Model Comparisons:

  • Compare wild-type and SAUSA300_2286 mutant strains in:

    • Cell culture infection models (adhesion, invasion, persistence)

    • Animal infection models (colonization, dissemination, mortality)

    • Ex vivo tissue models for organ-specific pathogenesis

• Virulence Factor Expression Analysis:

  • Examine how SAUSA300_2286 deletion affects expression of:

    • Toxins and hemolysins (particularly in UTI contexts)

    • Adhesins and biofilm components

    • Immune evasion factors

    • Stress response proteins

• Host-Pathogen Interaction Studies:

  • Assess SAUSA300_2286's impact on:

    • Immune cell recognition and activation

    • Survival within macrophages

    • Cytokine/chemokine induction profiles

    • Neutrophil recruitment and function

• Membrane Physiology and Adaptation:

  • Investigate SAUSA300_2286's contribution to:

    • Membrane stability during host-induced stress

    • Adaption to urinary tract conditions (especially in UTI strains)

    • pH tolerance and ionic stress responses

    • Antimicrobial peptide resistance

Research on other S. aureus strains suggests that membrane proteins can significantly impact virulence through altered adherence properties, biofilm formation, and stress responses, particularly in urinary tract infection contexts where S. aureus ST1 strains show distinct phenotypic adaptations .

What computational approaches are most effective for predicting structural characteristics of SAUSA300_2286?

Advanced Computational Structure Prediction Workflow:

• Primary Sequence Analysis:

  • Apply hydropathy analysis (Kyte-Doolittle, Goldman-Engelman-Steitz)

  • Identify conserved motifs through multiple sequence alignments

  • Map evolutionary conservation patterns

• Secondary Structure Prediction:

  • Use membrane-specific algorithms (PSIPRED, JPred)

  • Apply consensus methods combining multiple algorithms

  • Identify transmembrane helices using specialized tools (TMHMM, MEMSAT)

• Tertiary Structure Modeling:

  • Implement homology modeling if structural homologs exist

  • Apply ab initio approaches for novel folds

  • Use specialized membrane protein modeling tools (ROSETTA Membrane)

  • Employ the recently developed computational workflow for membrane protein design

• Model Refinement and Validation:

  • Perform molecular dynamics simulations in membrane environments

  • Validate models using statistical potentials

  • Cross-reference with experimental data when available

• Functional Site Prediction:

  • Identify potential binding pockets

  • Predict protein-protein interaction interfaces

  • Map conservation onto structural models to identify functional residues

The novel computational workflow reported by Scripps Research for membrane protein design provides particularly promising approaches for structural characterization of challenging membrane proteins like SAUSA300_2286 .

How can researchers analyze the evolutionary conservation of SAUSA300_2286 across different S. aureus strains and related species?

Comprehensive Evolutionary Analysis Methodology:

• Sequence Retrieval and Database Mining:

  • Extract homologous sequences from:

    • Completed S. aureus genomes (various strains including clinical isolates)

    • Related staphylococcal species (S. epidermidis, S. haemolyticus, etc.)

    • Other genera in the Staphylococcaceae family

  • Use BLAST, HMMER, and specialized bacterial genome databases

• Multiple Sequence Alignment and Conservation Analysis:

  • Generate alignments using membrane protein-optimized algorithms

  • Calculate conservation scores for individual residues

  • Identify highly conserved motifs and variable regions

  • Generate sequence logos to visualize conservation patterns

• Phylogenetic Analysis:

  • Construct phylogenetic trees using:

    • Maximum likelihood methods

    • Bayesian inference approaches

    • Distance-based methods with appropriate substitution models

  • Calculate divergence times if molecular clock assumptions can be applied

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to identify sites under selection

  • Perform codon-based tests of selection

  • Identify lineage-specific selection patterns

• Structural Mapping of Conservation:

  • Map conservation scores onto predicted structural models

  • Identify structurally conserved regions despite sequence variation

  • Correlate conservation with predicted functional sites

• Strain-Specific Variation Analysis:

  • Compare sequences between pathogenic vs. commensal strains

  • Analyze variations between strains isolated from different infection sites

  • Examine specific variations in UTI-associated strains like ST1

This approach provides insights into SAUSA300_2286's evolutionary history, functional constraints, and potential adaptations in different ecological niches.

How can SAUSA300_2286 be evaluated as a potential drug target for anti-staphylococcal therapeutics?

Drug Target Validation Methodology:

• Target Essentiality Assessment:

  • Generate conditional knockout strains to test growth dependence

  • Use transposon sequencing (Tn-Seq) for high-throughput essentiality screening

  • Evaluate fitness costs in various growth conditions

  • Determine if SAUSA300_2286 is essential or associated with reduced virulence

• Druggability Analysis:

  • Identify potential binding pockets through computational analysis

  • Assess conservation across S. aureus strains to predict resistance emergence

  • Evaluate structural uniqueness compared to host proteins

  • Use computational workflow designed for membrane proteins

• Functional Assay Development:

  • Design high-throughput assays to measure protein activity

  • Develop reporter systems for target engagement

  • Establish clear structure-activity relationships

• In Silico Screening Approaches:

  • Perform virtual screening against predicted binding sites

  • Apply molecular dynamics for binding energy calculations

  • Use machine learning to prioritize candidates

• Experimental Screening and Validation:

  • Design competitive binding assays

  • Develop phenotypic screens based on knockout phenotypes

  • Test promising compounds for:

    • Target engagement using thermal shift assays

    • Antimicrobial activity (MIC determination)

    • Specificity and off-target effects

    • Resistance development frequency

This systematic approach follows the modern target-based drug discovery paradigm while incorporating specialized techniques for membrane protein targets.

What role might SAUSA300_2286 play in antibiotic resistance mechanisms in S. aureus?

Investigation Framework for Resistance Mechanisms:

• Expression Analysis Under Antibiotic Stress:

  • Measure SAUSA300_2286 expression changes upon exposure to:

    • Different antibiotic classes

    • Sub-inhibitory concentrations

    • Various exposure durations

  • Use qRT-PCR, RNA-Seq, and proteomics approaches

• Knockout/Overexpression Impact on Susceptibility:

  • Create deletion mutants and overexpression strains

  • Determine minimum inhibitory concentrations (MICs) for various antibiotics

  • Assess changes in susceptibility patterns

  • Measure killing kinetics and persistence

• Membrane Integrity and Permeability Assessment:

  • Evaluate membrane potential changes using fluorescent dyes

  • Measure antibiotic accumulation in wild-type vs. mutant strains

  • Analyze membrane fluidity and composition alterations

• Transporter Function Analysis:

  • Determine if SAUSA300_2286 affects:

    • Antibiotic influx/efflux rates

    • Transport of specific compounds

    • Interactions with known resistance transporters

• Genetic Context Examination:

  • Analyze genomic neighborhood for resistance-associated genes

  • Look for co-regulation with known resistance factors

  • Identify potential regulatory elements

• Clinical Isolate Comparisons:

  • Sequence SAUSA300_2286 in resistant clinical isolates

  • Correlate sequence variations with resistance phenotypes

  • Examine expression levels in resistant vs. susceptible isolates

The Nebraska Transposon Mutant Library (NTML) approach has been valuable for identifying membrane transporters involved in antibiotic uptake in S. aureus, providing a methodological framework applicable to SAUSA300_2286 research .

What are the major challenges in working with recombinant SAUSA300_2286 and how can they be addressed?

Common Challenges and Solutions:

• Low Expression Yields:

  • Challenge: Membrane proteins often express poorly in heterologous systems

  • Solutions:

    • Optimize codon usage for expression host

    • Use specialized strains (C41/C43 for E. coli)

    • Test multiple fusion tags and their positions

    • Reduce expression temperature and inducer concentration

    • Consider cell-free expression systems

• Protein Misfolding and Aggregation:

  • Challenge: Improper folding in non-native membrane environments

  • Solutions:

    • Co-express molecular chaperones

    • Add specific lipids during expression

    • Screen multiple detergents during purification

    • Include stabilizing agents (glycerol, specific salts)

    • Use mild solubilization conditions

• Detergent Selection and Optimization:

  • Challenge: Finding detergents that maintain native structure and function

  • Solutions:

    • Screen detergent panels systematically

    • Test detergent mixtures and novel amphipathic polymers

    • Implement stability assays to monitor protein quality

    • Consider nanodiscs or liposomes for functional studies

• Maintaining Stability During Storage:

  • Challenge: Preventing degradation and aggregation during storage

  • Solutions:

    • Use 50% glycerol in storage buffer

    • Store at -20°C for regular use or -80°C for long-term

    • Create small working aliquots to avoid freeze-thaw cycles

    • Keep working stocks at 4°C for only up to one week

• Functional Characterization Difficulties:

  • Challenge: Establishing reliable activity assays

  • Solutions:

    • Develop multiple complementary assay formats

    • Use liposome reconstitution for transport studies

    • Apply label-free techniques when possible

    • Compare activity in different membrane mimetics

These approaches address the specific challenges of membrane protein biochemistry while maximizing the chances of obtaining functional recombinant SAUSA300_2286.

What specialized techniques are required for studying interactions between SAUSA300_2286 and potential binding partners or substrates?

Advanced Interaction Analysis Methods:

• Microscale Thermophoresis (MST):

  • Label protein with fluorescent dye

  • Measure thermophoretic movement changes upon ligand binding

  • Calculate binding affinities under near-native conditions

  • Advantage: Requires small sample amounts and works in detergent solutions

• Surface Plasmon Resonance (SPR) Optimization:

  • Immobilize purified SAUSA300_2286 on sensor chips

  • Use specialized capture approaches for membrane proteins

  • Measure real-time binding kinetics

  • Implementation challenges:

    • Detergent interference with baseline

    • Proper orientation on chip surface

    • Maintaining stability during experiment

• Isothermal Titration Calorimetry (ITC) for Membrane Proteins:

  • Measure heat changes during binding events

  • Determine thermodynamic parameters of interactions

  • Requires careful control experiments to account for detergent effects

  • Higher protein concentrations needed compared to other methods

• Native Mass Spectrometry Approaches:

  • Use specialized MS techniques compatible with membrane proteins

  • Identify binding partners from complex mixtures

  • Determine stoichiometry of complexes

  • Requires optimization of ionization conditions

• Förster Resonance Energy Transfer (FRET):

  • Label SAUSA300_2286 and potential partners with fluorophore pairs

  • Measure energy transfer as indicator of proximity

  • Can be performed in cellular contexts or with purified components

  • Allows for real-time monitoring of dynamic interactions

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Map regions involved in binding through protection from exchange

  • Identify conformational changes upon interaction

  • Requires specialized workflow for membrane proteins

  • Provides structural insights without need for crystals

These specialized approaches address the unique challenges of studying membrane protein interactions while providing robust data on SAUSA300_2286's potential binding partners and substrates.

How should researchers analyze and interpret experimental data related to SAUSA300_2286 function in the context of conflicting results?

Comprehensive Data Analysis Framework:

• Methodological Comparison Matrix:

  Method Aspect	Approach A	Approach B	Impact on Results
  Expression system	E. coli	Native S. aureus	Potential folding differences
  Membrane environment	Detergent micelles	Lipid bilayers	Functional state variation
  Assay conditions	In vitro reconstitution	Whole cell measurements	Context-dependent activity
  Strain background	Laboratory strain	Clinical isolate	Genetic context effects
  Growth conditions	Standard media	Host-mimicking conditions	Physiological relevance

• Systematic Resolution Strategies:

  • Repeat experiments with standardized protocols across laboratories

  • Test hypotheses explaining divergent results

  • Identify strain-specific or condition-dependent effects

  • Determine if contradictions reflect actual biological variability

• Meta-analysis Approach:

  • Weight evidence based on methodological robustness

  • Consider relevance of experimental conditions to physiological context

  • Integrate data across multiple experimental approaches

  • Identify consistent findings despite methodological differences

• Statistical Reanalysis:

  • Apply appropriate statistical tests for specific data types

  • Consider sample sizes and statistical power

  • Use Bayesian approaches to incorporate prior knowledge

  • Perform sensitivity analyses for key parameters

• Computational Modeling:

  • Develop models that can explain seemingly contradictory results

  • Test model predictions with targeted experiments

  • Use simulations to explore parameter spaces

  • Identify conditions where different outcomes would be expected

This structured approach helps researchers navigate conflicting data and develop a coherent understanding of SAUSA300_2286 function despite experimental variations.

What advanced quantitative methods should be applied when analyzing structure-function relationships of SAUSA300_2286?

Sophisticated Structure-Function Analysis Framework:

• Correlation Analysis Approaches:

  • Perform alanine scanning mutagenesis with quantitative functional readouts

  • Apply multivariate statistical methods to correlate sequence/structure with function

  • Use principal component analysis to identify key determinants

  • Implement machine learning for pattern recognition in structure-function data

• Network Analysis Methods:

  • Construct correlation networks between mutations and functional parameters

  • Identify clusters of residues with similar functional impacts

  • Apply graph theory to understand allosteric communication

  • Detect cooperative interactions between residues

• Energy Landscape Mapping:

  • Calculate energetic contributions of individual residues

  • Map energetic coupling between distant sites

  • Model conformational ensembles rather than single structures

  • Relate energy barriers to functional transitions

• Molecular Dynamics Integration:

  • Perform long-timescale simulations in membrane environments

  • Extract dynamic information not available from static structures

  • Calculate free energy profiles for proposed mechanisms

  • Validate computational predictions with experimental measurements

• Quantitative Structure-Activity Relationship (QSAR) Analysis:

  • Develop mathematical models relating structural parameters to function

  • Use regression techniques to identify key structural determinants

  • Apply dimensionality reduction to handle complex datasets

  • Generate predictive models for untested variants

This comprehensive analytical framework enables researchers to move beyond descriptive structure-function relationships toward quantitative predictive models of SAUSA300_2286 behavior.

What emerging technologies show promise for advancing our understanding of SAUSA300_2286 and similar membrane proteins?

Emerging Technologies with Transformative Potential:

• Cryo-EM Advances for Membrane Proteins:

  • Single-particle analysis at near-atomic resolution

  • Visualization of conformational ensembles

  • Reduced protein quantity requirements compared to crystallography

  • Capability to resolve structures in more native-like environments

• Integrative Structural Biology Approaches:

  • Combining complementary structural techniques (SAXS, NMR, EM)

  • Computational integration of sparse structural data

  • Mapping dynamics and conformational landscapes

  • Development of hybrid experimental-computational workflows

• In-Cell Structural Biology:

  • NMR methods for membrane protein structure in living cells

  • Advanced fluorescence techniques for in situ conformational studies

  • Cryo-electron tomography of intact cellular contexts

  • Correlative light and electron microscopy approaches

• Artificial Intelligence Applications:

  • Deep learning for improved structure prediction

  • Machine learning for function prediction from sequence/structure

  • Neural networks for identifying functional relationships

  • AI-assisted experimental design for efficient characterization

• Single-Molecule Techniques:

  • FRET spectroscopy for conformational dynamics

  • Force spectroscopy for mechanical properties

  • Single-molecule tracking in native membranes

  • Correlating structure, dynamics, and function at the single-molecule level

• Synthetic Biology Tools:

  • Designer membrane proteins with enhanced properties

  • Genetic code expansion for site-specific probes

  • Engineered cellular systems for functional testing

  • In vivo directed evolution for function discovery

The computational workflow developed by Scripps Research represents a particularly promising direction for custom design of proteins targeting membrane regions, directly applicable to SAUSA300_2286 research .

How might systems biology approaches contribute to understanding SAUSA300_2286's role in the broader context of S. aureus physiology?

Systems Biology Integration Framework:

• Multi-omics Data Integration:

  • Combine transcriptomics, proteomics, metabolomics, and fluxomics data

  • Generate condition-specific networks including SAUSA300_2286

  • Identify upstream regulators and downstream effectors

  • Map perturbation effects across biological scales

• Network Reconstruction and Analysis:

  • Position SAUSA300_2286 within protein-protein interaction networks

  • Construct regulatory networks governing expression

  • Develop metabolic models incorporating membrane functions

  • Apply graph theory to identify network motifs and hierarchies

• Genome-Scale Modeling:

  • Integrate SAUSA300_2286 into genome-scale metabolic models

  • Perform flux balance analysis with varying conditions

  • Model growth phenotypes of mutants

  • Predict emergent behaviors not obvious from reductionist approaches

• Comparative Systems Approaches:

  • Analyze system-level conservation across S. aureus strains

  • Compare network architectures between pathogenic and non-pathogenic strains

  • Identify condition-specific network rewiring

  • Examine particularly how UTI-associated strains differ at systems level

• Host-Pathogen Interface Modeling:

  • Model SAUSA300_2286's role during infection

  • Simulate interactions with host defense systems

  • Integrate temporal dynamics of infection process

  • Predict critical nodes for therapeutic intervention

• Experimental System Perturbations:

  • Design strategic perturbation experiments based on model predictions

  • Measure global responses to SAUSA300_2286 manipulation

  • Validate and refine network models iteratively

  • Identify emergent properties not predictable from individual components

This systems-level framework places SAUSA300_2286 research within the broader context of S. aureus biology, potentially revealing functions and interactions not apparent from reductionist approaches.