Recombinant Bacillus subtilis Uncharacterized protein ysdA (ysdA)

What is ysdA and why is it classified as "uncharacterized"?

The ysdA protein (UniProt ID: P94519) is a small protein (89 amino acids) encoded by the ysdA gene in Bacillus subtilis. It is classified as "uncharacterized" because its precise biological function has not been experimentally validated, despite the protein sequence being known. The amino acid sequence (MIIAAYLVLINLCGFWVMGIDKRKAQQHKWRISEDRLWLIAIVFGALGVWLGMQTFRHKTKHASFKYGVPLLLVIEAILIAIYYSPFDL) suggests it may be a membrane-associated protein based on its hydrophobicity profile and predicted transmembrane domains . This classification is common for many bacterial proteins whose genes have been identified through genome sequencing but whose functions remain to be elucidated through targeted research.

What are the basic physicochemical properties of recombinant ysdA?

The recombinant ysdA protein has the following key physicochemical properties:

The high hydrophobicity and predicted transmembrane domains suggest this protein likely associates with or is embedded in membranes, which has implications for both expression systems and purification strategies .

What expression systems are optimal for recombinant ysdA production?

For recombinant production of ysdA, E. coli has been successfully employed as an expression host . The optimal expression system typically includes:

• Vector selection: pET series vectors with an N-terminal His-tag for purification

• Host strain: E. coli BL21(DE3) or Rosetta(DE3) for proteins with rare codons

• Induction conditions: 0.5-1.0 mM IPTG at OD600 of 0.6-0.8

• Temperature: Reduced temperature (16-25°C) post-induction to minimize inclusion body formation

• Duration: 4-16 hours induction time

For membrane-associated proteins like ysdA, expression optimization may require:

• Addition of membrane-mimicking detergents in the growth medium

• Use of specialized E. coli strains like C41(DE3) or C43(DE3) designed for membrane protein expression

• Fusion to solubility-enhancing partners like MBP (maltose-binding protein) or SUMO

The expression level should be monitored using SDS-PAGE analysis followed by Western blotting using anti-His antibodies to detect the recombinant protein .

What purification strategies are effective for recombinant His-tagged ysdA?

Purification of His-tagged ysdA can be achieved through the following protocol:

• Cell lysis: Sonication or pressure-based disruption in a buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and protease inhibitors

• Solubilization: For membrane-associated proteins, addition of mild detergents (0.5-1% DDM or CHAPS)

• Primary purification: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Binding buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole

  • Wash buffer: Same with 50 mM imidazole

  • Elution buffer: Same with 250-500 mM imidazole

• Secondary purification: Size exclusion chromatography to remove aggregates

• Quality control: SDS-PAGE analysis with >90% purity standard

For long-term storage, the purified protein should be buffer-exchanged into a stabilizing buffer (Tris/PBS-based with 6% trehalose, pH 8.0) and stored as aliquots at -80°C to avoid repeated freeze-thaw cycles .

How can researchers predict the secondary structure of ysdA?

Several computational approaches can be used to predict ysdA's secondary structure:

• Ab initio prediction algorithms:

  • PSIPRED, JPred, and PREDATOR for alpha-helices and beta-sheets

  • TMHMM and HMMTOP for transmembrane helices

• Homology-based methods:

  • HHpred for distant homology detection

  • I-TASSER for threading-based structure prediction

• Physicochemical analysis:

  • Hydrophobicity plots (Kyte-Doolittle)

  • Amphipathic helix prediction

Based on sequence analysis, ysdA likely contains 2-3 transmembrane helices with short connecting loops. The amino acid sequence shows patterns consistent with membrane-spanning regions, particularly in the segments IIAAYLVLINLCGFWVMG and WLIAIVFGALGVWLGM .

What experimental methods are suitable for determining ysdA tertiary structure?

Given the challenges of membrane protein crystallization, several complementary approaches are recommended:

• X-ray crystallography:

  • Requires detergent screening for optimal solubilization

  • Lipidic cubic phase (LCP) crystallization may be necessary

• NMR spectroscopy:

  • Solution NMR for detergent-solubilized protein

  • Solid-state NMR for membrane-embedded protein

• Cryo-electron microscopy:

  • Particularly useful if ysdA forms higher-order complexes

• Small-angle X-ray scattering (SAXS):

  • For low-resolution envelope determination in solution

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • For mapping solvent-accessible regions and structural dynamics

Each method has advantages and limitations for membrane proteins like ysdA. A multi-technique approach is recommended, starting with computational predictions to guide experimental design.

What approaches can identify potential binding partners of ysdA?

Multiple complementary strategies can be employed to identify interaction partners:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express His-tagged ysdA in B. subtilis

  • Perform crosslinking to capture transient interactions

  • Purify using Ni-NTA and identify co-purifying proteins by MS

• Bacterial two-hybrid (B2H) system:

  • Create fusion constructs with split reporter domains

  • Screen against a B. subtilis genomic library

• Proximity-dependent biotin identification (BioID):

  • Fuse ysdA to a biotin ligase

  • Identify proximal proteins through biotinylation

• Co-immunoprecipitation with specific antibodies:

  • Generate anti-ysdA antibodies

  • Pull down native protein complexes

• Protein microarrays:

  • Probe B. subtilis proteome arrays with purified ysdA

When performing these experiments, appropriate controls are crucial, including "bait-only" controls for background binding and validation of potential interactions using orthogonal methods.

How can genome-wide approaches help elucidate ysdA function?

Several genomic and transcriptomic approaches can provide functional insights:

• Transcriptional profiling:

  • RNA-seq comparing wild-type and ysdA deletion strains

  • Identify genes differentially expressed upon ysdA knockout

• Genome-wide fitness profiling:

  • Transposon sequencing (Tn-seq) to identify synthetic lethal or beneficial interactions

  • Screen in various growth conditions to identify condition-specific functions

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • If ysdA has DNA-binding properties

  • Map genome-wide binding sites

• Metabolomic profiling:

  • Compare metabolite profiles between wild-type and ysdA mutants

  • Identify metabolic pathways affected by ysdA

• Phenotypic microarrays:

  • Test growth of ysdA mutants across hundreds of conditions

  • Identify condition-specific growth defects

This multi-omics approach has been successfully applied to characterize previously uncharacterized proteins in B. subtilis, leveraging its genetic tractability as a model organism .

How conserved is ysdA across different bacterial species?

Comparative genomic analysis of ysdA reveals:

The high conservation within Bacillus suggests ysdA may serve a specific function relevant to the biology of this genus. Proteins with similar sequence characteristics in other Firmicutes often function in membrane organization, signaling, or stress response, which aligns with B. subtilis' role as a model organism for Gram-positive bacteria . The variable conservation pattern outside Bacillus could indicate functional specialization or adaptation to specific ecological niches.

What can genomic context reveal about potential ysdA function?

Analysis of the genomic neighborhood of ysdA across Bacillus species provides functional clues:

• Co-localization patterns:

  • Genes frequently co-located with ysdA include those involved in:

    • Membrane lipid biosynthesis

    • Cell envelope maintenance

    • Stress response regulation

• Operon structure:

  • In many Bacillus species, ysdA is part of an operon with genes involved in:

    • Membrane protein assembly

    • Cell wall synthesis

    • Biofilm formation components

• Regulatory elements:

  • Promoter analysis suggests regulation by:

    • Sigma factors associated with cell envelope stress

    • Two-component systems responding to environmental signals

This genomic context analysis suggests ysdA may function in membrane organization, potentially similar to flotillins (FloA and FloT) in B. subtilis, which regulate membrane fluidity and domain formation . This connection would be consistent with the predicted membrane-associated nature of ysdA based on its sequence properties .

How can CRISPR-Cas9 be optimized for genetic manipulation of ysdA in B. subtilis?

CRISPR-Cas9 editing of ysdA can be accomplished using the following optimized protocol:

• gRNA design:

  • Target unique 20-bp sequences in ysdA with NGG PAM sites

  • Avoid regions with off-target matches in the B. subtilis genome

  • Recommended tools: CHOPCHOP or CRISPRdirect with B. subtilis genome

• Delivery system:

  • Plasmid-based: pJOE8999 (contains Cas9 and sgRNA scaffold)

  • Integration-based: Use thrC locus for stable expression

• Editing strategy:

  • Gene knockout: Provide repair template with selection marker

  • Point mutations: ~1kb homology arms with desired mutation

  • Tagged versions: C-terminal tags preferred to avoid disrupting signal sequences

• Transformation protocol:

  • Use standard B. subtilis competence induction

  • Plate on selective media with appropriate antibiotics

  • Colony PCR and sequencing for verification

• Efficiency optimization:

  • Adding recombineering proteins (RecA, Beta) can increase HDR efficiency

  • Multiple gRNAs targeting same gene can improve knockout rates

This approach leverages B. subtilis' natural genetic competence and homologous recombination capabilities, making it an excellent model system for genetic manipulation . For membrane proteins like ysdA, special consideration should be given to potential lethal phenotypes by using inducible systems.

What phenotypic assays can detect functional changes in ysdA mutants?

Based on the predicted membrane association of ysdA, the following phenotypic assays are recommended:

• Membrane integrity assays:

  • Propidium iodide permeability

  • SYTOX Green uptake kinetics

  • Membrane potential measurements using DiBAC4(3)

• Microscopy-based approaches:

  • Lipid domain visualization using fluorescent lipid analogs

  • Localization of membrane proteins using fluorescent fusions

  • High-resolution imaging of cell envelope architecture

• Stress response tests:

  • Osmotic shock survival rates

  • Detergent sensitivity assays

  • Temperature sensitivity growth curves

  • Antibiotic susceptibility profiling

• Biofilm formation analysis:

  • Crystal violet staining quantification

  • Confocal microscopy of biofilm architecture

  • Expression analysis of matrix production genes

• Membrane fluidity measurements:

  • Fluorescence anisotropy using DPH or TMA-DPH

  • Laurdan generalized polarization

These assays are particularly relevant given B. subtilis' established roles in membrane organization research and biofilm formation studies . Comparing wild-type and ysdA mutant strains across these phenotypic dimensions can provide insights into the protein's function.

What methods can determine if ysdA interacts with specific membrane lipids?

Given ysdA's predicted membrane association, several techniques can identify lipid interactions:

• Liposome binding assays:

  • Prepare liposomes with different lipid compositions

  • Incubate with purified ysdA

  • Separate bound and unbound protein by centrifugation

  • Quantify protein distribution by Western blotting

• Lipid overlay assays (PIP strips):

  • Spot various lipids on nitrocellulose membranes

  • Probe with purified His-tagged ysdA

  • Detect binding using anti-His antibodies

• Surface plasmon resonance (SPR):

  • Immobilize lipids on sensor chips

  • Measure real-time binding kinetics

  • Determine affinity constants for different lipids

• Monolayer insertion experiments:

  • Spread lipid monolayers at air-water interface

  • Inject ysdA into subphase

  • Monitor surface pressure changes

• Native mass spectrometry:

  • Analyze protein-lipid complexes directly

  • Identify specifically bound lipid species

These methods should be performed with lipids known to be present in B. subtilis membranes, potentially focusing on those involved in functional membrane microdomains, which have been extensively studied in this organism .

How can researchers investigate ysdA's potential role in membrane organization?

B. subtilis has emerged as a model system for bacterial membrane organization research . To investigate ysdA's potential role in this process:

• Fluorescence microscopy with lipid dyes:

  • FM4-64 for general membrane visualization

  • NAO for cardiolipin-rich domains

  • Comparison between wild-type and ΔysdA strains

• Super-resolution microscopy:

  • PALM/STORM imaging of fluorescently tagged ysdA

  • Co-localization with known domain markers (FloT, FloA)

  • Single-particle tracking to assess dynamics

• Membrane fractionation:

  • Detergent-resistant membrane isolation

  • Sucrose gradient ultracentrifugation

  • Western blot analysis of fraction composition

• Atomic force microscopy (AFM):

  • Direct visualization of membrane topography

  • Force spectroscopy to measure mechanical properties

  • Comparison of wild-type and mutant membranes

• Genetic interaction studies:

  • Create double mutants with flotillin genes (floA, floT)

  • Synthetic phenotypes would suggest functional relationships

This approach is particularly relevant given that B. subtilis has been established as a leading model for studying membrane organization proteins like flotillins that regulate membrane fluidity and domain formation .

How is ysdA expression regulated in response to environmental conditions?

Understanding ysdA regulation requires comprehensive expression analysis:

• Quantitative RT-PCR:

  • Design primers specific to ysdA

  • Measure expression changes under various conditions:

    • Different growth phases

    • Nutrient limitation

    • Temperature stress

    • Osmotic stress

    • Cell wall targeting antibiotics

• Promoter fusion studies:

  • Clone ysdA promoter region upstream of reporter genes

  • Use luciferase or fluorescent proteins as reporters

  • Monitor expression dynamics in real-time

• Transcription factor binding:

  • Identify potential regulatory proteins using:

    • Electrophoretic mobility shift assays (EMSA)

    • DNase footprinting

    • ChIP-seq with antibodies against regulatory proteins

• Regulation in different genetic backgrounds:

  • Measure expression in strains lacking key sigma factors

  • Test dependency on two-component systems

  • Examine expression in biofilm vs. planktonic states

What proteomics approaches can verify ysdA production and modification?

Several proteomics techniques can characterize native ysdA expression and modifications:

• Targeted proteomics:

  • Selected reaction monitoring (SRM) or parallel reaction monitoring (PRM)

  • Develop specific peptide transitions for ysdA

  • Absolute quantification using isotope-labeled standards

• Post-translational modification analysis:

  • Phosphoproteomic analysis to identify phosphorylation sites

  • Glycoproteomic methods to detect glycosylation

  • MS/MS fragmentation patterns for detailed modification mapping

• Protein turnover assessment:

  • Pulse-chase experiments with stable isotope labeling

  • Determine half-life and degradation kinetics

  • Compare stability under different stress conditions

• Spatial proteomics:

  • Subcellular fractionation followed by MS analysis

  • Proximity labeling combined with proteomics

  • Membrane vs. cytosolic distribution quantification

These methods provide crucial information about the physiological context of ysdA production and potential regulatory mechanisms through post-translational modifications, which complements the genetic and transcriptomic analyses.

How might ysdA contribute to B. subtilis stress response mechanisms?

B. subtilis is known for its robust stress response systems . To investigate ysdA's potential role:

• Survival assays under stress conditions:

  • Compare wild-type and ΔysdA strains under:

    • Heat shock (42-54°C)

    • Osmotic stress (0.4-2.5M NaCl)

    • Oxidative stress (H₂O₂, paraquat)

    • Membrane-targeting compounds

  • Measure survival rates and recovery kinetics

• Transcriptional profiling during stress:

  • RNA-seq of wild-type vs. ΔysdA under stress

  • Focus on known stress regulons:

    • SigB (general stress)

    • SigW (envelope stress)

    • CssRS (secretion stress)

• Protein aggregation and quality control:

  • Monitor protein aggregation using reporters

  • Examine chaperone induction patterns

  • Assess membrane protein misfolding

• Physiological parameters under stress:

  • Membrane potential maintenance

  • Proton motive force measurements

  • ATP levels and energy homeostasis

This investigation is particularly relevant given B. subtilis' importance as a model for studying bacterial stress responses, especially those related to the cell envelope and membrane integrity .

Could ysdA play a role in B. subtilis biofilm formation?

B. subtilis forms complex biofilms with multicellular features . To explore ysdA's potential role:

• Biofilm architecture analysis:

  • Compare wild-type and ΔysdA biofilms using:

    • Confocal laser scanning microscopy

    • Scanning electron microscopy

    • Biomass quantification

• Matrix component production:

  • Quantify exopolysaccharide production

  • Measure protein components (TasA, BslA)

  • Evaluate hydrophobicity of biofilm surface

• Gene expression in biofilms:

  • Monitor key biofilm regulators:

    • SinR, SinI, AbrB

    • DegU, ComA

    • Spo0A phosphorylation state

  • Compare planktonic vs. biofilm expression patterns

• Cell differentiation within biofilms:

  • Examine matrix producers vs. motile cells

  • Assess sporulation frequency

  • Evaluate division of labor within the community

What collaborative research approaches would accelerate ysdA characterization?

Comprehensive characterization of ysdA would benefit from interdisciplinary collaboration:

• Structural biology and biophysics:

  • Determine high-resolution structure

  • Characterize dynamics and conformational states

  • Map interaction surfaces

• Systems biology:

  • Place ysdA in protein-protein interaction networks

  • Integrate multi-omics data

  • Develop predictive models of function

• Synthetic biology:

  • Engineer variants with modified properties

  • Create biosensors based on ysdA

  • Explore potential biotechnological applications

• Evolutionary biology:

  • Trace evolutionary history across bacterial species

  • Identify selective pressures on sequence conservation

  • Correlate with ecological adaptations

• Applied microbiology:

  • Investigate potential roles in industrial strains

  • Explore contributions to probiotic properties

  • Assess relevance to agricultural applications

This interdisciplinary approach leverages B. subtilis' status as a model organism with applications ranging from traditional food fermentation to industrial enzyme production and genetic manipulation techniques .

What emerging technologies could provide breakthrough insights into ysdA function?

Several cutting-edge technologies hold promise for ysdA characterization:

• Cryo-electron tomography:

  • Visualize ysdA in its native membrane context

  • Observe macromolecular assemblies in situ

  • Detect structural changes under different conditions

• Single-cell transcriptomics:

  • Reveal cell-to-cell variability in ysdA expression

  • Identify subpopulations with distinct expression patterns

  • Correlate with cellular phenotypes

• AlphaFold2 and related AI approaches:

  • Predict structural features with high confidence

  • Model protein-protein and protein-lipid interactions

  • Guide experimental design for validation

• Genome-wide CRISPRi screening:

  • Identify genetic interactions systematically

  • Discover condition-specific functional relationships

  • Map pathway connections

• Microfluidics and time-lapse microscopy:

  • Track ysdA dynamics in single cells over time

  • Correlate localization with cell cycle events

  • Monitor responses to controlled environmental perturbations