Recombinant Bacillus subtilis Uncharacterized protein ywhH (ywhH)

What is known about the structural characteristics of ywhH protein and how does it compare to characterized B. subtilis proteins?

The ywhH protein represents one of several uncharacterized proteins in B. subtilis. While specific literature on ywhH is limited, researchers can apply similar characterization approaches used for other B. subtilis proteins. Structural prediction should include analysis for potential signal peptides, transmembrane domains, and sorting signals such as those found in YhcR (LPDTS) and YfkN (LPDTA) proteins .

For effective characterization, employ multiple prediction tools:

• Signal peptide prediction (SignalP, TatP)

• Transmembrane domain analysis (TMHMM, HMMTOP)

• Secondary structure prediction (PSIPRED, JPred)

• Domain architecture identification (InterPro, Pfam)

• Tertiary structure modeling (AlphaFold, I-TASSER)

How should researchers design experiments to determine the expression pattern of ywhH in B. subtilis?

To characterize the expression of ywhH, consider implementing these methodological approaches:

• Transcriptional analysis techniques:

  • Construct promoter-reporter fusions (similar to yhcS-lacZ fusions described in the literature)

  • Perform Northern blot analysis to detect and quantify transcripts during different growth phases

  • Implement qRT-PCR to measure expression under various conditions

• Analysis at different growth phases:

  • Examine expression throughout growth curve from early exponential to late stationary phase

  • Note that some B. subtilis genes show differential expression during stationary phase, as observed with the putative sortase gene yhcS

• Environmental condition screening:

  • Test expression under various stress conditions (temperature, pH, nutrients)

  • Analyze transcription in different media formulations

What techniques are most effective for detecting native ywhH protein expression in B. subtilis?

For detecting native ywhH expression, implement a multi-faceted approach:

• Antibody-based detection methods:

  • Develop specific antibodies against purified recombinant ywhH

  • Use Western blotting with appropriate controls

  • Consider epitope tagging approaches if antibody development is challenging

• Mass spectrometry-based approaches:

  • Perform targeted proteomics to detect ywhH in cellular fractions

  • Use MALDI-TOF mass spectrometry as demonstrated for identification of YhcR and YfkN proteins

  • Implement quantitative proteomics to measure relative abundance

• Subcellular localization analysis:

  • Fractionate B. subtilis cells into cytoplasmic, membrane, and cell wall components

  • Use the cell wall protein extraction procedure described for sortase-dependent proteins

  • Analyze fractions by appropriate detection methods

• Methodological considerations:

  • Include protease inhibitors during extraction

  • Consider using protease-deficient strains like WB800N for improved detection, as demonstrated for other B. subtilis proteins

  • Use appropriate controls (knockout strains, overexpression controls)

What expression systems are optimal for producing recombinant ywhH protein for structural and functional studies?

When selecting an expression system for ywhH, consider these evidence-based options:

• B. subtilis expression systems:

  • Reduced-genome strain MGB874 has demonstrated improved recombinant protein production capacity

  • IPTG-inducible promoters like Pgrac provide controlled expression, as successfully used for expression of sortases in B. subtilis

  • Consider stationary phase-specific promoters if ywhH is difficult to express

• Expression vector design:

  • Include strong ribosome binding sites for optimal translation

  • Incorporate appropriate secretion signals if secreted expression is desired

  • Design fusion constructs with detection/purification tags

• Expression conditions optimization:

  • Test expression at different growth phases based on information from native expression studies

  • Optimize induction timing and inducer concentration

  • Consider co-expression with chaperones if folding issues arise

How can researchers optimize the purification of recombinant ywhH to ensure protein quality for downstream applications?

For optimal purification of ywhH, implement this methodological workflow:

• Initial extraction considerations:

  • For potential membrane-associated proteins (if ywhH contains hydrophobic regions):

    • Use appropriate detergents for solubilization

    • Consider enzymatic cell wall digestion with lysozyme as used for sortase-dependent proteins

  • For cytoplasmic proteins:

    • Use sonication for cell disruption as described in protocols for B. subtilis protein extraction

    • Include protease inhibitors to prevent degradation

• Chromatography strategy:

  • Primary capture: Affinity chromatography using fusion tags

  • Intermediate purification: Ion exchange based on theoretical pI

  • Polishing: Size exclusion chromatography

• Quality control assessment:

  • SDS-PAGE for purity evaluation

  • Western blotting for identity confirmation

  • Mass spectrometry for accurate mass determination

  • Size exclusion chromatography for aggregation analysis

• Storage optimization:

  • Test buffer conditions for optimal stability

  • Determine appropriate storage temperature

  • Evaluate the need for stabilizing additives (glycerol, reducing agents)

What are the most effective approaches for troubleshooting expression problems with recombinant ywhH protein?

When encountering expression challenges with ywhH, systematically address potential issues:

• Low expression yield troubleshooting:

  • Analyze mRNA levels to determine if the issue is transcriptional

  • Check for rare codons that might limit translation efficiency

  • Test different promoter strengths and ribosome binding sites

  • Consider using protease-deficient strains (like WB800N) that have demonstrated improved protein detection

• Insolubility troubleshooting:

  • Reduce expression temperature (16-25°C)

  • Lower inducer concentration for slower expression

  • Test different fusion partners (MBP, SUMO, thioredoxin)

  • Optimize lysis buffer components (salt, pH, additives)

• Instability troubleshooting:

  • Add protease inhibitors during all purification steps

  • Identify and mutate protease-sensitive sites

  • Test expression as fusion with stabilizing domains

• Systematic optimization approach:

How can researchers determine the potential cellular localization and function of ywhH through experimental approaches?

To determine ywhH localization and function, implement these complementary approaches:

• Subcellular localization studies:

  • Generate fluorescent protein fusions for microscopy

  • Perform subcellular fractionation and detection in different cellular compartments

  • If ywhH contains potential sorting signals, test whether it is anchored to the cell wall using the methodology described for YhcR and YfkN

• Cell wall anchoring assessment:

  • If ywhH contains sequences similar to the LPDTS or LPDTA sorting signals found in YhcR and YfkN, investigate whether it is processed by the YhcS sortase

  • Generate fusion constructs with reporter enzymes (similar to YhcR123-AmyQ described in the literature)

  • Assay for surface display using whole-cell activity assays

• Interactome analysis:

  • Perform pull-down assays with tagged ywhH

  • Implement bacterial two-hybrid screens

  • Use crosslinking followed by mass spectrometry

  • Analyze genetic interactions through synthetic genetic arrays

• Function prediction validation:

  • Design activity assays based on bioinformatic predictions

  • Test phenotypes of knockout strains under diverse conditions

  • Perform complementation studies with mutated variants

What genetic approaches should be used to investigate the physiological role of ywhH in B. subtilis?

For comprehensive genetic characterization of ywhH:

• Gene knockout and complementation:

  • Generate clean deletion mutants (ΔywhH)

  • Perform complementation with wild-type and mutant versions

  • Create conditional depletion strains if knockout is lethal

• Phenotypic characterization:

  • Assess growth under various environmental conditions

  • Test responses to different stressors

  • Examine morphological changes by microscopy

  • Implement experimental evolution approaches as described for B. subtilis adaptation studies

• Multi-omics profiling:

  • Compare transcriptomic profiles of wild-type and ΔywhH strains

  • Perform comparative proteomics to identify altered protein levels

  • Analyze metabolomic changes to identify affected pathways

• Genetic interaction mapping:

  • Construct double mutants with genes in predicted pathways

  • Perform suppressor screens to identify compensatory mutations

  • Implement Tn-seq for genome-wide interaction mapping

How can researchers exploit sortase-based systems for studying potential cell wall association of ywhH?

If ywhH is predicted to interact with the cell wall, researchers can utilize B. subtilis sortase systems:

• Sortase-mediated anchoring analysis:

  • Examine ywhH sequence for sorting signals similar to those in YhcR (LPDTS) and YfkN (LPDTA)

  • Test dependence on YhcS sortase by expressing in wild-type and ΔyhcS strains

  • Use the methodologies described for isolation of sortase-dependent cell wall proteins

• Surface display applications:

  • Create fusion proteins with the YhcR123 sorting sequence, which has been demonstrated to efficiently display recombinant proteins on B. subtilis cell surface

  • Implement the α-amylase reporter system described for verification of surface display

  • Quantify surface display efficiency using activity-based assays

• Interaction studies on cell surface:

  • Use sortase-mediated labeling techniques

  • Apply cell surface crosslinking approaches

  • Implement surface-sensitive biophysical methods

• Methodological considerations:

  • Ensure expression of yhcS, as it shows growth phase-dependent expression with highest levels in late stationary phase

  • Consider using IPTG-inducible expression systems for both ywhH and sortase

  • Include appropriate controls (non-sorting signal variants)

What approaches can determine if ywhH plays a role in B. subtilis adaptation to environmental conditions?

To investigate potential roles in environmental adaptation:

• Experimental evolution strategies:

  • Subject wild-type and ΔywhH strains to laboratory evolution under selective conditions, as described for B. subtilis adaptation studies

  • Test adaptation to challenges such as low atmospheric pressure, high UV radiation, and unfavorable growth temperatures

  • Sequence evolved strains to identify compensatory mutations

• Stress response characterization:

  • Examine expression of ywhH under various stress conditions

  • Test survival of ΔywhH mutants during exposure to:

    • Temperature extremes

    • Osmotic stress

    • Oxidative stress

    • Nutrient limitation

• Competitive fitness assays:

  • Perform competition experiments between wild-type and ΔywhH strains

  • Measure selection coefficients under different conditions

  • Implement long-term evolution experiments to detect subtle fitness effects

• Comparative genomics approach:

  • Analyze conservation of ywhH across Bacillus species from different environmental niches

  • Identify co-evolving genes that might function with ywhH

  • Examine genomic context conservation across related species

How should researchers analyze conflicting results from different experimental approaches when characterizing ywhH?

When faced with contradictory results during ywhH characterization:

• Systematic validation process:

  • Verify protein identity and integrity in all experiments

  • Test multiple independent protein preparations

  • Implement additional controls to rule out artifacts

  • Consider tag interference if fusion proteins were used

• Methodological considerations:

  • Evaluate the sensitivity and specificity of each assay

  • Assess whether different methods measure different aspects of function

  • Consider whether buffer components or experimental conditions affect results

  • Examine whether post-translational modifications play a role

• Condition-dependent function analysis:

  • Test whether activity varies across different:

    • pH ranges

    • Temperature conditions

    • Salt concentrations

    • Redox environments

  • Consider potential cofactor requirements

• Integrated data interpretation approach:

What statistical approaches are appropriate for analyzing experimental data related to ywhH characterization?

For robust statistical analysis of ywhH research data:

• Experimental design considerations:

  • Determine appropriate sample sizes through power analysis

  • Include biological replicates (different cultures/preparations)

  • Implement technical replicates to assess method variability

  • Design experiments to control for batch effects

• Statistical methods by data type:

  • Expression data: ANOVA with post-hoc tests, regression analysis

  • Growth/phenotype data: Growth curve modeling, area under curve analysis

  • Interaction data: Significance testing against appropriate controls

  • Evolutionary data: Population genetic statistics, selection coefficient calculations

• Multiple testing correction:

  • Apply FDR correction for high-throughput screens

  • Use Bonferroni correction for multiple planned comparisons

  • Implement permutation tests for complex datasets

• Data visualization approaches:

  • Create appropriate visualizations for different data types

  • Include error bars representing biological variability

  • Use consistent scales when comparing conditions

  • Implement heat maps for multidimensional data

How can researchers determine the physiological significance of biochemical findings about ywhH?

To establish physiological relevance:

• Correlation of biochemical and in vivo data:

  • Compare in vitro activity with phenotypes of knockout strains

  • Analyze structure-function relationships through complementation with mutant versions

  • Test whether conditions that affect in vitro activity match conditions where knockout shows phenotypes

• Physiological context analysis:

  • Determine when and where ywhH is expressed during growth and development

  • Examine expression changes in response to physiologically relevant stimuli

  • Analyze evolutionary conservation across Bacillus species from different niches

  • Test relevance in conditions resembling natural habitats

• Systems-level analysis:

  • Integrate transcriptomic, proteomic, and metabolomic data

  • Examine effects on known pathways

  • Model potential roles in metabolic networks

  • Consider adaptation to specific environmental conditions as studied in B. subtilis evolution experiments

• Validation in complex conditions:

  • Test phenotypes in mixed cultures or biofilms

  • Examine competitiveness in soil microcosms

  • Analyze function during interactions with other microorganisms

  • Study role during sporulation or germination

How can researchers leverage laboratory evolution approaches to understand ywhH function?

Building on B. subtilis evolution studies , researchers can:

• Experimental evolution design:

  • Subject wild-type and ΔywhH strains to parallel evolution under selective conditions

  • Focus on stressors like those described for B. subtilis adaptation studies:

    • Low atmospheric pressure

    • High UV radiation

    • Unfavorable growth temperatures

  • Implement long-term serial transfer protocols

• Evolved strain analysis:

  • Perform whole genome sequencing to identify adaptive mutations

  • Compare mutation spectra between wild-type and ΔywhH evolved lines

  • Search for compensatory mutations in ΔywhH backgrounds

  • Validate adaptive mutations through reconstruction experiments

• Fitness landscape mapping:

  • Measure fitness effects of ywhH variants

  • Construct libraries of ywhH mutants for selection experiments

  • Identify critical residues by deep mutational scanning

  • Map epistatic interactions with other genes

• Field-inspired approaches:

  • Test function in conditions mimicking natural environments

  • Isolate natural B. subtilis strains with ywhH variants

  • Compare functionality across environmental isolates

What protein engineering approaches can be applied to ywhH to enhance its potential biotechnological applications?

For protein engineering of ywhH:

• Rational design strategies:

  • Structure-guided mutagenesis of key residues

  • Domain swapping with functionally related proteins

  • Fusion to reporter domains for activity sensing

  • Surface modification for improved stability or solubility

• Display technology applications:

  • Fusion to YhcR123 sorting sequence for surface display on B. subtilis

  • Development of screening systems based on sortase-mediated anchoring

  • Creation of whole-cell catalysts with surface-displayed ywhH

  • Engineering cell surface interaction capabilities

• Directed evolution approaches:

  • Develop high-throughput screening methods for improved variants

  • Implement error-prone PCR for random mutagenesis

  • Use DNA shuffling with homologous proteins

  • Apply compartmentalized self-replication techniques

• Application-specific modifications:

  • Stability enhancement for industrial applications

  • Substrate specificity engineering

  • pH and temperature range expansion

  • Cofactor dependency modification

How can structural biology approaches contribute to understanding ywhH function and evolution?

Structural biology provides critical insights through:

• Structure determination methods:

  • X-ray crystallography of purified ywhH

  • NMR spectroscopy for dynamic regions

  • Cryo-EM for large complexes

  • Integrative structural biology combining multiple techniques

• Structure-function analysis:

  • Identification of potential active sites or binding pockets

  • Mapping of evolutionarily conserved surface patches

  • Structural classification and comparison with characterized proteins

  • Molecular docking for predicting interaction partners

• Evolutionary structure analysis:

  • Comparison of structural features across Bacillus species

  • Identification of structural adaptations in different environments

  • Analysis of co-evolution between interacting surfaces

  • Reconstruction of ancestral protein states

• Applied structural insights:

  • Structure-guided protein engineering

  • Rational design of inhibitors or activators

  • Prediction of post-translational modification sites

  • Understanding of stability determinants

What methodological approaches are most effective for determining if ywhH interacts with the B. subtilis cell wall or membrane?

To investigate potential cell wall/membrane interactions:

• Subcellular fractionation approaches:

  • Separate cytoplasmic, membrane, and cell wall fractions

  • Implement the cell wall protein extraction procedure used for sortase-dependent proteins

  • Use protease shaving of intact cells to identify surface-exposed domains

  • Perform detergent phase separation for membrane protein analysis

• Microscopy techniques:

  • Fluorescent protein fusions for localization studies

  • Immunofluorescence microscopy with specific antibodies

  • Super-resolution microscopy for precise localization

  • Electron microscopy with immunogold labeling

• Sortase-based analysis:

  • Examine ywhH for sorting signals similar to YhcR (LPDTS) and YfkN (LPDTA)

  • Test dependence on YhcS sortase by comparing wild-type and ΔyhcS strains

  • Generate fusion constructs with confirmed sorting signals for validation

  • Implement enzymatic reporters for surface accessibility testing, as demonstrated with α-amylase fusions

• Biochemical interaction studies:

  • Binding assays with purified cell wall components

  • Liposome association experiments for membrane interaction

  • Crosslinking studies in intact cells

  • Surface plasmon resonance with immobilized peptidoglycan or membrane mimetics