Recombinant Saccharomyces cerevisiae Bypass of stop codon protein 2 (BSC2)

What is BSC2 and what is its primary function in Saccharomyces cerevisiae?

BSC2 (Bypass of Stop Codon protein 2) is a protein in Saccharomyces cerevisiae that plays multiple roles in cellular function. Originally identified through screening of gene deletion strains that affect premature stop codon bypass, BSC2 has been found to be important for cytoplasmic protein biosynthesis . More recent research has revealed its significant role in antioxidation processes and drug resistance mechanisms. The protein consists of 235 amino acids and has been successfully expressed as a recombinant protein with N-terminal His-tag fusion in E. coli expression systems .

How does BSC2 relate to stop codon bypass mechanisms?

BSC2 was identified through systematic screening of gene deletion strains that affect premature stop codon bypass in yeast. Researchers used expression plasmids (pUKC817, pUKC818, and pUKC819) containing different premature stop codons (UAA, UGA, and UAG, respectively) within a LacZ expression cassette to identify genes affecting translation termination. The deletion of BSC2 altered the efficiency of stop codon bypass, suggesting its involvement in translation fidelity maintenance. This identification process utilized large-scale β-galactosidase assays to evaluate the production of β-galactosidase in each gene deletion strain, with pUKC815 carrying the native LacZ gene as a control .

What expression systems are commonly used for recombinant BSC2 production?

Recombinant BSC2 is typically produced using E. coli expression systems for in vitro studies. For functional characterization within yeast cells, researchers employ both plasmid-based expression vectors and chromosomal integration approaches in S. cerevisiae. When expressing BSC2 in S. cerevisiae, the full-length protein (amino acids 1-235) can be fused with tags such as His-tag for purification and detection purposes . For chromosomal integration, researchers often select high-expression loci to ensure consistent expression levels. Comparative studies have shown that multicopy BSC2-expression vectors typically yield higher protein levels compared to single-copy chromosomal integrations, resulting in more pronounced phenotypic effects .

What are the recommended methods for manipulating BSC2 expression levels in yeast?

For BSC2 expression manipulation in S. cerevisiae, researchers can employ multiple strategies:

Plasmid-based overexpression:

• Use multicopy plasmids with constitutive promoters (e.g., TEF1, TDH3) for high-level expression

• Use centromeric plasmids (CEN plasmids) for moderate expression levels

• Employ inducible promoters (GAL1, CUP1) for controlled expression timing

Genomic integration:

• Single-copy integration at high-expression loci for stable expression

• CRISPR-Cas9 mediated gene editing for precise modifications

Gene deletion:

• Create knockout strains using homologous recombination techniques

• Use marker-based selection systems (e.g., URA3, LEU2) for screening

Comparative analysis has shown that multicopy BSC2 expression vectors in CEN.PK strain (CEN.PK-btrC) produce significantly higher protein levels than single-copy genomic integrations, with approximately 11-fold higher expression levels as measured by specific protein detection methods .

How can researchers assess BSC2's role in multidrug resistance?

To assess BSC2's role in multidrug resistance, implement the following experimental approach:

• Strain preparation:

  • Generate BSC2 deletion strains (bsc2Δ)

  • Create BSC2 overexpression strains using multicopy plasmids

  • Develop complementation strains with controlled BSC2 expression

• Drug susceptibility testing:

  • Perform spot assays on solid media containing various antifungal agents:

    • Amphotericin B (AMB)

    • Fluconazole (FLC)

    • Chlorhexidine (CHX)

    • 5-Fluorocytosine (5-FC)

    • Caspofungin (CAS)

• Growth inhibition measurement:

  • Conduct liquid culture growth assays with varying drug concentrations

  • Calculate IC50 values to quantify resistance levels

• Genetic interaction studies:

  • Test BSC2 function in various genetic backgrounds

  • Pay particular attention to FLO pathway mutants (FLO11Δ, FLO1Δ, FLO8Δ, TUP1Δ)

Research has demonstrated that BSC2 overexpression confers resistance to multiple antifungal compounds, while bsc2Δ strains show increased sensitivity compared to wild-type. The degree of resistance varies between drugs, with particularly strong effects observed for AMB and CAS .

What techniques can be used to study BSC2's impact on biofilm formation?

To investigate BSC2's effects on biofilm formation, employ the following methodological approaches:

• Flocculation assessment:

  • Measure sedimentation rates in culture tubes

  • Quantify flocculation using spectrophotometric methods

  • Compare wild-type, bsc2Δ, and BSC2-overexpression strains

• Cell surface hydrophobicity (CSH) analysis:

  • Implement hydrocarbon adherence assays

  • Measure water contact angles on cell lawns

  • Calculate hydrophobicity indices

• Biofilm formation quantification:

  • Grow biofilms on appropriate surfaces (polystyrene, glass)

  • Stain with crystal violet for biomass quantification

  • Use confocal microscopy for structural analysis

  • Implement fluorescent labeling for viability assessment

• Invasive growth evaluation:

  • Plate cultures on appropriate agar media

  • Wash plate surface and photograph remaining colonies

  • Quantify invasion depth using microscopy

  • Test in conjunction with FLO pathway mutants

Experimental data has shown that BSC2-overexpression strains exhibit enhanced flocculation, increased cell surface hydrophobicity, and more robust biofilm formation compared to control strains. These phenotypic changes are dependent on functional FLO pathway components, as BSC2 overexpression fails to induce these changes in FLO pathway mutants .

How does BSC2 interact with the FLO pathway to regulate biofilm formation and drug resistance?

BSC2 regulates biofilm formation and subsequent drug resistance through complex interactions with the FLO pathway. Mechanistically, BSC2 overexpression significantly increases the mRNA expression of key FLO genes, including FLO11, FLO1, FLO8, and TUP1 . This transcriptional upregulation leads to enhanced cellular flocculation, increased cell surface hydrophobicity, and robust biofilm development.

The relationship between BSC2 and the FLO pathway is hierarchical, with BSC2 functioning upstream as a regulator. This is evidenced by the inability of BSC2 overexpression to induce caspofungin (CAS) resistance or affect invasive growth in FLO pathway mutants (FLO11Δ, FLO1Δ, FLO8Δ, and TUP1Δ) . Different FLO genes contribute distinctly to the resistance phenotype, with FLO8Δ and FLO11Δ strains exhibiting CAS resistance, while FLO1Δ and TUP1Δ strains show CAS sensitivity, indicating that biofilm formation alone is insufficient for multidrug resistance (MDR) .

The regulatory mechanism appears to involve cell wall remodeling, as BSC2 overexpression increases mannose content in the cell wall and compensates for chitin synthesis defects, contributing to cell wall integrity maintenance. This cell wall modification likely contributes to the drug resistance phenotype by limiting drug penetration and activating stress response pathways .

What is the relationship between BSC2's functions in stop codon bypass and multidrug resistance?

The dual functionality of BSC2 in both translation fidelity (stop codon bypass) and multidrug resistance presents an intriguing research question. While direct experimental evidence linking these two functions is limited, several hypothetical mechanisms can be proposed:

• Stress response integration: Both translation fidelity and drug resistance mechanisms may be interconnected components of the cellular stress response. BSC2 might serve as a regulatory node that coordinates these responses.

• Proteome remodeling: Alterations in stop codon bypass efficiency could modify the cellular proteome, potentially including proteins involved in drug resistance pathways. This could include increased readthrough of premature stop codons in genes relevant to drug efflux or detoxification.

• Translational control of resistance factors: BSC2 might specifically regulate the translation of mRNAs encoding proteins involved in drug resistance, biofilm formation, or cell wall biosynthesis.

The genetic screening that identified BSC2 as affecting stop codon bypass utilized plasmids containing premature stop codons within a LacZ expression cassette, allowing quantitative assessment of bypass efficiency through β-galactosidase activity . Complementary approaches examining translation fidelity in strains with altered drug resistance profiles could help elucidate the connection between these functions.

How does BSC2 contribute to cell wall integrity and morphology maintenance?

BSC2 plays a crucial role in maintaining cell wall integrity and normal morphology in S. cerevisiae through several mechanisms:

• Chitin synthesis modulation: BSC2 overexpression compensates for chitin synthesis defects, helping maintain cell wall structural integrity. This was demonstrated by the ability of BSC2 overexpression to significantly reduce cell morphology abnormalities induced by caspofungin (CAS), an echinocandin antifungal that inhibits β-1,3-glucan synthesis .

• Mannose content regulation: BSC2 overexpression increases mannose levels in the cell wall, potentially modifying cell wall architecture and permeability. This effect appears mechanistically linked to DPM1 (dolichol phosphate mannose synthase), as DPM1 overexpression in both wild-type and bsc2Δ backgrounds conferred resistance to CAS and AMB .

• FLO pathway-dependent regulation: BSC2's effects on cell wall integrity are dependent on a functional FLO pathway, as BSC2 overexpression failed to repair cell wall damage caused by CAS in FLO mutant strains. This suggests that BSC2 regulates cell wall integrity at least partially through the FLO pathway components .

• Biofilm matrix contributions: The enhanced biofilm formation associated with BSC2 overexpression likely includes extracellular matrix components that contribute to both community structure and protection from environmental stressors, including antifungal drugs .

These functions collectively suggest that BSC2 serves as an important regulatory factor in cellular responses to cell wall stress, potentially coordinating transcriptional and translational responses to maintain cellular integrity under challenging conditions.

What are the key considerations when designing experiments to distinguish between BSC2's multiple cellular functions?

When investigating BSC2's diverse functions, researchers should implement targeted experimental designs that can isolate specific aspects of its activity:

• Domain mapping and mutational analysis:

  • Create truncated or point-mutated versions of BSC2

  • Test each variant for specific functions (translation, drug resistance, biofilm formation)

  • Identify domains critical for each function

  • Use complementation assays in bsc2Δ strains to validate domain functions

• Temporal control strategies:

  • Employ inducible expression systems (e.g., GAL1 promoter)

  • Implement rapid protein degradation systems (auxin-inducible degron)

  • Monitor different cellular processes during BSC2 expression/depletion time courses

  • Correlate BSC2 levels with phenotypic changes

• Pathway-specific reporters:

  • Develop reporters for translation fidelity (stop codon readthrough)

  • Create biofilm formation reporters linked to FLO gene expression

  • Implement cell wall integrity pathway reporters

  • Monitor multiple pathways simultaneously during BSC2 manipulation

• Genetic suppressor/enhancer screens:

  • Identify mutations that suppress or enhance specific BSC2-dependent phenotypes

  • Use genome-wide approaches (synthetic genetic array analysis)

  • Focus on genes with known functions in relevant pathways

These approaches can help distinguish between direct and indirect effects of BSC2 on various cellular processes, clarifying whether its multiple functions represent independent activities or interconnected aspects of a unified cellular role .

What analytical techniques are most effective for studying BSC2-mediated changes in cell wall composition?

To thoroughly characterize BSC2-mediated changes in cell wall composition, researchers should employ multiple complementary analytical approaches:

• Compositional analysis:

  • Quantify β-1,3-glucan content using aniline blue staining and fluorescence spectroscopy

  • Measure chitin levels using calcofluor white staining and flow cytometry

  • Determine mannose content through acid hydrolysis followed by HPLC analysis

  • Compare cell wall fractions between wild-type, bsc2Δ, and BSC2-overexpression strains

• Structural characterization:

  • Implement atomic force microscopy (AFM) for cell wall surface topography

  • Use transmission electron microscopy (TEM) to measure cell wall thickness and layering

  • Apply scanning electron microscopy (SEM) for surface feature analysis

  • Perform mechanical property testing using single-cell compression devices

• Functional assessments:

  • Test susceptibility to cell wall-perturbing agents (congo red, calcofluor white)

  • Measure osmotic stress tolerance

  • Evaluate resistance to cell wall-degrading enzymes (β-glucanase, chitinase)

  • Assess cell wall permeability using fluorescent dye uptake assays

• Molecular interactions:

  • Identify cell wall proteins with altered abundance or localization

  • Analyze protein glycosylation patterns

  • Study mannoproteins using lectin binding assays

  • Investigate protein-polysaccharide associations

Research has shown that BSC2 overexpression increases mannose content in the cell wall and compensates for chitin synthesis defects, suggesting a specific role in regulating these components of cell wall architecture . Comprehensive cell wall analysis will help elucidate the molecular mechanisms underlying these effects.

How can researchers effectively differentiate between direct and indirect effects of BSC2 on multidrug resistance?

Distinguishing direct from indirect effects of BSC2 on multidrug resistance requires sophisticated experimental approaches:

• Temporal analysis of gene expression:

  • Implement time-course RNA-seq after BSC2 induction

  • Identify immediate early response genes versus secondary effects

  • Compare expression profiles in different genetic backgrounds

  • Correlate gene expression changes with development of resistance

• Pathway dissection:

  • Test BSC2 effects in strains lacking specific resistance mechanisms

  • Analyze drug transport (efflux pump activity, drug uptake assays)

  • Measure reactive oxygen species (ROS) levels and oxidative stress responses

  • Evaluate cell wall permeability changes separately from other resistance mechanisms

• Protein interaction studies:

  • Perform co-immunoprecipitation to identify BSC2 binding partners

  • Use yeast two-hybrid or proximity labeling approaches

  • Conduct epistasis analysis with genes in relevant pathways

  • Implement ChIP-seq to identify potential transcriptional targets

• Direct measurement of drug interactions:

  • Quantify intracellular drug concentrations in different strains

  • Analyze drug binding to cellular targets

  • Implement fluorescently labeled drug analogs for localization studies

  • Compare biochemical drug targets in wild-type versus BSC2-modified strains

What statistical approaches are most appropriate for analyzing BSC2-related phenotypic data?

When analyzing complex phenotypic data related to BSC2 function, appropriate statistical methods are crucial:

• For drug susceptibility testing:

  • Apply dose-response curve analysis with non-linear regression

  • Calculate and compare IC50 values with 95% confidence intervals

  • Implement two-way ANOVA to evaluate strain and drug interactions

  • Use repeated measures approaches for time-course experiments

• For biofilm quantification:

  • Apply appropriate data transformations to achieve normality

  • Implement mixed-effects models for multi-plate/batch experiments

  • Use non-parametric methods for non-normally distributed data

  • Apply multivariate analysis for multiple biofilm parameters

• For gene expression data:

  • Implement false discovery rate (FDR) correction for multiple testing

  • Use principal component analysis for pattern identification

  • Apply hierarchical clustering to identify co-regulated gene groups

  • Implement pathway enrichment analysis to identify biological processes

• For phenotypic correlations:

  • Calculate Spearman's rank correlation between different phenotypes

  • Apply multidimensional scaling to visualize relationships

  • Implement factor analysis to identify underlying variables

  • Use machine learning approaches for complex pattern recognition

When reporting results, researchers should include both statistical significance (p-values) and effect sizes, as the biological relevance of BSC2-mediated changes may not always correlate with statistical significance alone. Additionally, visualization methods that effectively communicate complex relationships between multiple variables should be employed .

How should researchers interpret contradictory findings regarding BSC2 function across different studies?

Contradictory findings regarding BSC2 function may arise from various experimental factors. Researchers should consider the following approaches for reconciliation:

• Strain background evaluation:

  • Compare genetic backgrounds used in different studies

  • Test BSC2 function across multiple strain backgrounds

  • Consider genome-wide variation and potential genetic interactions

  • Examine strain-specific phenotypes that might influence BSC2 function

• Expression level considerations:

  • Quantify BSC2 expression levels in different experimental systems

  • Create standardized expression constructs for cross-study comparisons

  • Develop dose-response relationships between BSC2 levels and phenotypes

  • Consider potential threshold effects or non-linear relationships

• Environmental condition analysis:

  • Compare growth media, temperature, pH, and other environmental factors

  • Test BSC2 function across a range of controlled conditions

  • Examine stress-specific responses versus general functions

  • Implement factorial experimental designs to identify condition interactions

• Methodological standardization:

  • Develop standard operating procedures for key assays

  • Implement both the original and new methodologies in parallel

  • Consider sensitivity and specificity of different detection methods

  • Use multiple complementary approaches to measure the same phenotype

For example, research has shown that BSC2's effects on drug resistance depend on a functional FLO pathway, which might explain discrepancies in resistance profiles observed in studies using different strain backgrounds with variations in FLO gene functionality. Additionally, the degree of BSC2 overexpression appears critical, with multicopy expression systems showing much stronger phenotypes than single-copy integrations .

What bioinformatic approaches can reveal insights about BSC2 function and evolutionary conservation?

To gain deeper insights into BSC2 function through bioinformatics:

• Structural analysis:

  • Predict protein structure using AlphaFold or similar tools

  • Identify functional domains and motifs

  • Perform molecular dynamics simulations to study conformational changes

  • Analyze potential binding sites for ligands or protein partners

• Comparative genomics:

  • Conduct comprehensive ortholog identification across fungal species

  • Examine synteny and genomic context conservation

  • Analyze selection pressure (dN/dS ratios) across domains

  • Investigate potential horizontal gene transfer events

• Interactome prediction:

  • Build protein-protein interaction networks from experimental and predicted data

  • Identify functional clusters and pathways

  • Predict genetic interactions using machine learning approaches

  • Integrate transcriptomic data to create condition-specific networks

• Evolutionary analysis:

  • Construct phylogenetic trees of BSC2 across fungal species

  • Identify lineage-specific functional adaptations

  • Correlate BSC2 sequence changes with phenotypic traits

  • Examine co-evolution with interacting partners

What emerging technologies could advance our understanding of BSC2's cellular functions?

Several cutting-edge technologies hold promise for elucidating BSC2's complex cellular roles:

• CRISPR-based approaches:

  • Implement CRISPRi/CRISPRa for precise temporal control of BSC2 expression

  • Use CRISPR screening to identify genetic interactions with BSC2

  • Apply base editing for targeted mutation of specific BSC2 residues

  • Implement CRISPR-based imaging to track BSC2 localization in live cells

• Single-cell technologies:

  • Apply single-cell RNA-seq to capture heterogeneity in BSC2 response

  • Use single-cell proteomics to identify cell-specific BSC2 effects

  • Implement microfluidic approaches for real-time phenotypic tracking

  • Develop reporter systems visible at the single-cell level

• Advanced imaging:

  • Apply super-resolution microscopy to study BSC2 localization

  • Use correlative light and electron microscopy for ultrastructural context

  • Implement live-cell imaging with fluorescent translational reporters

  • Apply expansion microscopy to resolve subcellular BSC2 distributions

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Implement spatial transcriptomics for biofilm structure-function relationships

  • Develop computational models integrating multiple data types

  • Apply machine learning for pattern recognition across complex datasets

These approaches can help overcome current limitations in understanding BSC2 function, particularly regarding its subcellular localization, temporal dynamics, and heterogeneous effects within cellular populations .

What are the potential applications of BSC2 research in biotechnology and medicine?

BSC2 research holds significant potential for applications in various fields:

• Antifungal drug development:

  • Target BSC2 to overcome resistance in pathogenic fungi

  • Develop combination therapies targeting BSC2-dependent pathways

  • Create screening platforms to identify compounds affecting BSC2 function

  • Design BSC2 inhibitors as adjuvants for existing antifungals

• Biofilm control strategies:

  • Apply BSC2-related insights to disrupt harmful fungal biofilms

  • Develop biofilm prevention approaches for medical devices

  • Create biotechnological tools for controlled biofilm formation

  • Design targeted approaches for biofilm dispersal

• Protein production optimization:

  • Exploit BSC2's role in translation to enhance recombinant protein yield

  • Optimize stop codon readthrough for specific applications

  • Develop strains with controlled translational fidelity

  • Enhance production of difficult-to-express proteins

• Cell wall engineering:

  • Modify cell surface properties for industrial applications

  • Enhance cell resistance to industrial processing conditions

  • Develop strains with tailored adhesion properties

  • Create specialized cell surfaces for biotechnological applications

While BSC2 has been primarily studied in S. cerevisiae, the mechanisms it influences (drug resistance, biofilm formation, translation) are broadly relevant across fungal species, including pathogenic ones. Translating research findings to species like Candida or Aspergillus could lead to significant medical advances in antifungal therapy .

How might synthetic biology approaches be used to further characterize and exploit BSC2 functions?

Synthetic biology offers powerful approaches to expand BSC2 research:

• Modular domain engineering:

  • Create chimeric proteins combining BSC2 domains with other functional modules

  • Develop synthetic BSC2 variants with enhanced or novel functions

  • Design orthogonal BSC2-based regulatory systems

  • Implement domain swapping to test functional hypotheses

• Synthetic genetic circuits:

  • Design feedback loops involving BSC2 for precise regulation

  • Create oscillatory systems to study temporal aspects of BSC2 function

  • Implement synthetic promoters responsive to BSC2-regulated pathways

  • Develop bistable switches for controlled biofilm formation

• Minimal system reconstitution:

  • Identify minimal components required for BSC2-mediated phenotypes

  • Reconstitute these systems in heterologous hosts

  • Implement bottom-up approaches to understand complex phenotypes

  • Create simplified model systems for mechanistic studies

• Directed evolution:

  • Develop selection schemes for enhanced BSC2 functions

  • Evolve BSC2 variants with specialized properties

  • Apply continuous evolution systems for rapid optimization

  • Implement deep mutational scanning to comprehensively map function

These approaches could help isolate and enhance specific BSC2 functions for both fundamental research and applied biotechnology. For example, creating synthetic variants of BSC2 with enhanced ability to regulate cell wall properties could lead to strains with improved industrial characteristics or novel biotechnological applications .