Recombinant Bacillus subtilis Uncharacterized protein yddI (yddI), partial

How should recombinant YddI protein be stored for optimal stability?

Recombinant YddI protein stability is affected by multiple factors including buffer composition, storage temperature, and freeze-thaw cycles. For lyophilized preparations, the protein remains stable for approximately 12 months when stored at -20°C/-80°C . For liquid formulations, the shelf life decreases to approximately 6 months at the same temperatures . It is strongly recommended to avoid repeated freeze-thaw cycles, as this significantly reduces protein stability. For short-term work, aliquots can be stored at 4°C for up to one week . When reconstituting the protein, it should be centrifuged briefly before opening, then reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL with the addition of 5-50% glycerol (final concentration) before aliquoting for long-term storage .

What expression systems are suitable for producing recombinant YddI protein?

Escherichia coli has been successfully used as an expression system for recombinant YddI protein production . When designing expression constructs, researchers should consider incorporating affinity tags to facilitate purification, though the specific tag type may vary depending on the experimental requirements and will typically be determined during the manufacturing process . For laboratory-scale production, standard IPTG-inducible promoter systems have proven effective for regulating expression of B. subtilis proteins, as demonstrated in studies of other B. subtilis regulators . Expression optimization should include testing various induction temperatures, IPTG concentrations, and harvest times to maximize yield while maintaining proper folding of the protein.

What approaches can be used to elucidate the function of the uncharacterized YddI protein?

Determining the function of YddI requires a multi-faceted approach combining genomic, proteomic, and structural biology techniques:

• Comparative genomics: Analyze conservation patterns of YddI across different Bacillus species and strains. According to available data, there are at least 4 identical sequences across Bacillus subtilis strains including NCIB 3610, suggesting functional importance .

• Transcriptomic analysis: Employ DNA microarray or RNA-seq approaches similar to those used for studying DegU, ComA, and PhoP regulons in B. subtilis . These methods can reveal co-expression patterns with genes of known function.

• Protein-protein interaction studies: Use pull-down assays, yeast two-hybrid systems, or proximity labeling methods to identify interaction partners, which may provide functional context.

• Gene knockout studies: Generate yddI deletion mutants and analyze phenotypic changes across various growth conditions, particularly examining effects on sporulation, germination, or stress response pathways known to be variable among B. subtilis strains .

• Structural homology modeling: While current models exist with QMEAN scores of 0.51-0.62 , obtaining higher-resolution structures through X-ray crystallography could reveal structural motifs associated with specific functions.

• Biochemical activity assays: Test for enzymatic activities including protease, amylase, or other hydrolytic functions common in B. subtilis proteins .

Integration of these complementary approaches provides the most robust strategy for functional characterization of this currently uncharacterized protein.

How might YddI contribute to the genetic diversity observed among B. subtilis strains?

The YddI protein may play a role in the substantial genetic heterogeneity observed among B. subtilis strains. Microarray-based comparative genomic hybridization (M-CGH) studies have revealed that nearly one-third of B. subtilis strain 168-specific genes exhibit variability across different strains . While yddI itself was not specifically highlighted among variable genes in the available data, proteins involved in sporulation, cell wall synthesis, and adaptation to diverse environments show considerable variation between strains .

To investigate YddI's potential contribution to strain diversity, researchers should:

• Compare yddI sequence conservation and expression levels across diverse B. subtilis isolates from different ecological niches

• Determine if yddI exhibits evidence of horizontal gene transfer using computational methods that analyze GC content and codon usage bias

• Examine whether yddI expression is regulated by environmental conditions characteristic of specific niches

• Assess whether YddI function relates to sporulation, germination, or stress response pathways—processes that often vary between strains to enable adaptation to different environments

If YddI proves to be part of the variable gene pool, it may contribute to the adaptive capabilities that allow B. subtilis to thrive in diverse natural settings ranging from soil to plant surfaces .

What experimental approaches could determine if YddI is regulated by two-component systems in B. subtilis?

Two-component regulatory systems play crucial roles in B. subtilis gene regulation. To determine if YddI is regulated by these systems, researchers should implement the following experimental approach:

• Transcriptomic analysis: Perform RNA-seq or quantitative PCR to measure yddI expression levels in wild-type B. subtilis compared to strains with mutations in major two-component systems (DegS/DegU, ComP/ComA, PhoR/PhoP) . This approach successfully identified target genes in previous studies examining these regulons.

• Promoter analysis: Clone the yddI promoter region upstream of a reporter gene (such as lacZ) and transform this construct into wild-type and sensor kinase-deficient mutants (degS, comP, phoR) . Complementation with plasmid-expressed response regulators can then demonstrate direct regulation.

• Chromatin immunoprecipitation (ChIP): Perform ChIP-seq with antibodies against phosphorylated response regulators to determine if they directly bind to the yddI promoter region.

• Electrophoretic mobility shift assays (EMSA): Use purified response regulators to test for direct binding to the yddI promoter sequence in vitro.

• Bioinformatic analysis: Examine the yddI promoter region for consensus binding sequences known to be recognized by B. subtilis response regulators.

This combined approach has proven effective for identifying regulon members of two-component systems in B. subtilis as demonstrated in previous whole-genome microarray studies .

What purification strategies are most effective for recombinant YddI protein?

Purification of recombinant YddI protein requires a strategic approach based on its biochemical properties. While the specific purification protocol will depend on the expression system and tags used, the following general strategy is recommended:

• Affinity chromatography: The primary purification step should utilize affinity tags incorporated during expression. The tag type will be determined during the manufacturing process . Common options include:

  • His-tag purification using immobilized metal affinity chromatography (IMAC)

  • GST-tag purification using glutathione sepharose

  • MBP-tag purification using amylose resin

• Secondary purification: Following affinity purification, additional chromatography steps may be required to achieve higher purity:

  • Ion exchange chromatography based on YddI's predicted isoelectric point

  • Size exclusion chromatography to separate monomeric from potential dimeric forms, as suggested by structural models

• Quality control: Purity assessment using SDS-PAGE should confirm >85% purity, consistent with commercially available preparations . Western blotting can be used for identity confirmation.

• Tag removal: If the affinity tag might interfere with functional studies, consider incorporating a protease cleavage site between the tag and YddI, followed by a second affinity step to remove the cleaved tag.

• Buffer optimization: Final buffer conditions should be optimized for stability, with consideration of including glycerol (5-50%) for storage preparations .

This purification strategy should yield high-quality protein suitable for both structural and functional characterization studies.

How can researchers design experiments to investigate potential antimicrobial properties of YddI?

While the function of YddI remains uncharacterized, investigating its potential antimicrobial properties is warranted given that B. subtilis produces various antibacterial and antifungal compounds . A systematic experimental approach should include:

• Growth inhibition assays: Test purified recombinant YddI against a panel of microorganisms including:

  • Gram-positive bacteria (e.g., Staphylococcus aureus)

  • Gram-negative bacteria (e.g., Escherichia coli)

  • Fungi (e.g., Aspergillus flavus, which B. subtilis is known to inhibit)

  • Plant pathogens (e.g., Rhizoctonia, against which B. subtilis shows biocontrol activity)

  Measure zones of inhibition or minimum inhibitory concentrations (MICs) using standard protocols.

• Mode of action studies:

  • Membrane permeabilization assays using fluorescent dyes

  • Macromolecular synthesis inhibition assays (DNA, RNA, protein, cell wall)

  • Time-kill kinetics to determine bactericidal vs. bacteriostatic effects

• Structure-function analysis:

  • Generate truncated variants to identify active domains

  • Perform site-directed mutagenesis of conserved residues

  • Assess antimicrobial activity of synthetic peptides derived from YddI sequence

• Resistance development: Expose sensitive organisms to sub-inhibitory concentrations of YddI to assess the risk of resistance development.

• Synergy testing: Evaluate combinations of YddI with known antibiotics or other B. subtilis antimicrobial compounds like difficidin and oxydifficidin .

Proper controls should include heat-denatured YddI, buffer-only treatments, and known antimicrobial compounds produced by B. subtilis as positive controls.

What analytical techniques are best suited for studying potential interactions between YddI and other B. subtilis proteins?

Investigating protein-protein interactions involving YddI requires a comprehensive approach utilizing complementary techniques:

• Co-immunoprecipitation (Co-IP):

  • Express tagged YddI in B. subtilis to capture physiologically relevant interaction partners

  • Use anti-tag antibodies to precipitate YddI complexes

  • Identify co-precipitated proteins by mass spectrometry

  • Validate interactions with reverse Co-IP using antibodies against identified partners

• Bacterial two-hybrid (B2H) system:

  • Create fusion constructs of YddI with one domain of a split reporter protein

  • Screen against a library of B. subtilis proteins fused to the complementary domain

  • Interaction reconstitutes reporter activity (typically transcription activation)

  • This approach is advantageous for detecting direct binary interactions

• Surface Plasmon Resonance (SPR):

  • Immobilize purified YddI on a sensor chip

  • Flow potential interaction partners over the surface

  • Measure real-time binding kinetics (kon and koff rates)

  • Determine binding affinities (KD values)

• Isothermal Titration Calorimetry (ITC):

  • Directly measure thermodynamic parameters of interactions

  • Determine binding stoichiometry, enthalpy, and entropy changes

  • Provides label-free quantification of interactions in solution

• Proximity-dependent biotin identification (BioID):

  • Fuse YddI to a promiscuous biotin ligase

  • Express the fusion in B. subtilis

  • Identify biotinylated proteins (proximity partners) by streptavidin pull-down and mass spectrometry

  • Particularly useful for detecting transient or weak interactions

• Crosslinking Mass Spectrometry (XL-MS):

  • Use chemical crosslinkers to stabilize protein complexes in vivo

  • Identify crosslinked peptides by mass spectrometry

  • Map interaction interfaces at amino acid resolution

The selection of appropriate techniques should consider the potential oligomeric state of YddI, as structural models suggest both monomeric and dimeric forms may exist .

How should researchers design control experiments when studying the functional role of YddI?

Rigorous experimental design with appropriate controls is essential when investigating the uncharacterized YddI protein:

• Genetic manipulation controls:

  • Empty vector controls for overexpression studies

  • Complementation of yddI knockouts to confirm phenotype specificity

  • Non-targeting guide RNA controls for CRISPR-based approaches

  • Marker gene replacements to control for positional effects in the genome

• Protein-based controls:

  • Heat-denatured YddI protein to control for non-specific effects

  • Structurally similar but functionally distinct proteins from B. subtilis

  • Point mutants affecting predicted functional residues or domains

  • Tagged vs. untagged protein comparisons to assess tag interference

• Growth condition controls:

  • Testing multiple media compositions and environmental conditions

  • Time-course experiments to capture temporal dynamics

  • Comparative analysis across multiple B. subtilis strains that contain identical yddI sequences

  • Inclusion of known regulon members as positive controls when studying regulatory pathways

• Technical controls:

  • Biological replicates (minimum n=3) with appropriate statistical analysis

  • Technical replicates to assess methodological variation

  • Randomization and blinding procedures where applicable

  • Calibration controls for quantitative measurements

• Validation approaches:

  • Orthogonal methods to confirm key findings

  • In vitro confirmation of in vivo observations

  • Dose-response relationships to establish causality

Implementation of these controls will help distinguish genuine functional roles of YddI from experimental artifacts and establish the specificity of any observed phenotypes.

What troubleshooting strategies can be employed when expression of recombinant YddI yields insoluble protein?

Insoluble expression of recombinant YddI presents a common challenge that can be addressed through systematic troubleshooting:

• Optimize expression conditions:

  • Reduce expression temperature (16-20°C) to slow folding and prevent aggregation

  • Decrease inducer concentration to reduce expression rate

  • Test different E. coli host strains, particularly those designed for difficult proteins (e.g., SHuffle®, Origami™, or ArcticExpress™)

  • Use auto-induction media for gradual protein expression

• Modify the construct design:

  • Add solubility-enhancing fusion partners (MBP, SUMO, or TrxA) rather than simple affinity tags

  • Remove potential hydrophobic regions based on structural predictions

  • Try expressing discrete domains rather than the full-length protein

  • Codon-optimize the sequence for E. coli expression

• Adjust buffer conditions during lysis and purification:

  • Include mild detergents (0.1% Triton X-100 or 0.5% CHAPS)

  • Add stabilizing agents (glycerol, arginine, or low concentrations of urea)

  • Test various pH conditions around the theoretical pI of YddI

  • Include molecular chaperones (BSA or recombinant chaperones) in buffers

• Recovery from inclusion bodies:

  • Develop a denaturation/refolding protocol using stepwise dialysis

  • Use high-pressure refolding techniques

  • Attempt on-column refolding during affinity purification

• Alternative expression systems:

  • Consider homologous expression in B. subtilis itself

  • Test eukaryotic expression systems for complex proteins

  • Explore cell-free protein synthesis systems

Each approach should be evaluated with small-scale test expressions before scaling up, with solubility assessed via SDS-PAGE analysis of supernatant versus pellet fractions after cell lysis. Commercial preparations have achieved >85% purity using SDS-PAGE validation , suggesting that soluble expression is achievable with appropriate optimization.

How can researchers resolve data interpretation challenges when studying an uncharacterized protein like YddI?

Interpreting experimental data for an uncharacterized protein presents unique challenges that require rigorous analytical approaches:

• Distinguish correlation from causation:

  • Implement genetic complementation studies to verify that observed phenotypes are specifically due to YddI

  • Use dose-response relationships to establish causality

  • Apply multiple independent methods to confirm key findings

  • Design experiments that test specific mechanistic hypotheses rather than general associations

• Address potential artifacts:

  • Control for tag interference by comparing tagged and untagged versions of YddI

  • Validate protein-protein interactions with reciprocal pull-downs and competition assays

  • Use appropriate statistical methods to distinguish signal from noise

  • Consider how experimental conditions might create non-physiological behaviors

• Contextual interpretation:

  • Compare findings against known protein families with similar structural characteristics

  • Use the extensive knowledge base of B. subtilis biology to provide context

  • Consider evolutionary conservation patterns across bacterial species

  • Examine YddI in relation to the genetic heterogeneity observed among B. subtilis strains

• Resolve contradictory results:

  • Systematically identify variables that might explain discrepancies

  • Consider strain-specific differences, as B. subtilis shows considerable genetic heterogeneity

  • Examine temporal aspects, as protein function may be growth-phase dependent

  • Investigate condition-specific effects, as B. subtilis adapts to diverse environments

• Integrate diverse data types:

  • Develop a computational framework to integrate structural, genomic, and functional data

  • Use network analysis to position YddI within known cellular pathways

  • Apply machine learning approaches to predict function from multiple data sources

  • Create testable models that explain the cumulative evidence

By applying these strategies, researchers can progressively build a coherent functional model of YddI despite the initial absence of characterization.

How might YddI be involved in B. subtilis sporulation or germination processes?

The potential role of YddI in sporulation or germination warrants investigation, as these processes are critical to B. subtilis survival and show considerable variation between strains . A methodical approach to exploring this connection includes:

• Expression pattern analysis:

  • Determine if yddI expression changes during sporulation or germination using qRT-PCR and reporter fusions

  • Compare expression profiles with known sporulation genes across the developmental timeline

  • Analyze whether yddI is regulated by key sporulation transcription factors (σE, σF, σG, σK)

  • Examine if yddI is co-regulated with other spore coat proteins like CotP and CotX, which show strain variability

• Phenotypic analysis of mutants:

  • Construct precise yddI deletion mutants and assess:

    • Sporulation efficiency and timing

    • Spore resistance properties (heat, chemicals, radiation)

    • Germination rate and responsiveness to germinants

    • Spore coat ultrastructure using electron microscopy

• Localization studies:

  • Create fluorescent protein fusions to determine subcellular localization during sporulation

  • Perform immunogold electron microscopy to precisely localize YddI in mature spores

  • Conduct fractionation experiments to determine if YddI associates with specific spore structures

• Protein interaction studies:

  • Identify potential interactions with known sporulation proteins, particularly BofA (which inhibits pro-σK processing)

  • Investigate interactions with spore coat proteins showing strain variability

  • Examine potential associations with germination receptors or cortex-lytic enzymes

• Comparative analysis across strains:

  • Assess whether YddI function in sporulation differs among various B. subtilis strains

  • Determine if strains with different ecological niches show altered YddI expression or function during sporulation

Through this systematic investigation, researchers can determine whether YddI contributes to the diversity in sporulation and germination mechanisms that enable B. subtilis to thrive in diverse environmental conditions .

What approaches can determine if YddI plays a role in B. subtilis antibiotic production or resistance?

B. subtilis produces various antimicrobial compounds including difficidin, oxydifficidin, bacitracin, bacillin, and bacillomycin B . Investigating YddI's potential role in antibiotic production or resistance requires a structured experimental approach:

• Antibiotic production assessment:

  • Compare antibiotic production profiles of wild-type and yddI mutant strains using bioassays against indicator organisms

  • Employ liquid chromatography-mass spectrometry (LC-MS) to quantify specific antibiotics

  • Analyze culture supernatants for antimicrobial activity during different growth phases

  • Test production under various environmental conditions that typically induce antibiotic synthesis

• Gene expression analysis:

  • Determine if yddI expression correlates with expression of known antibiotic biosynthetic gene clusters

  • Assess whether transcription factors regulating antibiotic production also affect yddI expression

  • Use reporter fusions to monitor real-time expression patterns

  • Perform RNA-seq analysis comparing wild-type and regulatory mutants

• Antibiotic resistance testing:

  • Determine sensitivity of yddI mutants to various antibiotics using minimum inhibitory concentration (MIC) assays

  • Test susceptibility to B. subtilis-produced antibiotics and those from other microorganisms

  • Investigate whether YddI overexpression affects antibiotic resistance profiles

  • Examine potential protein-protein interactions with known resistance determinants

• Functional characterization:

  • Test if purified YddI directly modifies antibiotics through enzymatic action

  • Investigate potential regulatory roles in signaling pathways controlling antibiotic production

  • Examine YddI localization during antibiotic production phases

  • Assess if YddI affects quorum sensing systems that regulate antibiotic biosynthesis

• Comparative genomics:

  • Compare yddI conservation in B. subtilis strains with different antibiotic production profiles

  • Analyze genomic context of yddI for proximity to antibiotic biosynthesis or resistance genes

  • Examine co-evolution patterns with known antimicrobial systems

This methodology will help elucidate whether YddI contributes to the antimicrobial arsenal that makes B. subtilis effective as a biocontrol agent against fungal plant pathogens and other microorganisms .

How can structural models of YddI inform rational design of experiments to elucidate its function?

The available structural models of YddI can guide targeted experimental approaches:

• Structure-guided mutagenesis:

  • Analyze the three available SWISS-MODEL structures of YddI (based on templates 5uwb.1.A, 6h2d.1.B, and 7aed.1.A) to identify:

    • Conserved surface residues that may form functional sites

    • Potential binding pockets for ligands or substrates

    • Interface residues that may mediate protein-protein interactions in the homo-2-mer model

  • Create point mutations at these sites and assess functional consequences

• Domain analysis and truncation studies:

  • Identify discrete structural domains within YddI models

  • Express and characterize individual domains to determine their specific functions

  • Create chimeric proteins with domains from related proteins to test functional hypotheses

• Ligand binding prediction:

  • Use computational docking to predict potential substrates or cofactors that might bind to YddI

  • Validate predictions through binding assays (isothermal titration calorimetry, microscale thermophoresis)

  • Perform structure-activity relationship studies with related compounds

• Conformational dynamics:

  • Assess whether YddI might undergo conformational changes between monomeric and dimeric states

  • Use molecular dynamics simulations to predict flexible regions

  • Test predictions with hydrogen-deuterium exchange mass spectrometry or other experimental approaches

• Structural homology functional inference:

  • Compare YddI models with structurally similar proteins of known function

  • Identify key residues conserved between YddI and functional homologs

  • Design experiments to test whether YddI shares functional properties with structural homologs

The QMEAN scores of the current models (0.51-0.62) indicate moderate reliability, suggesting that experimental validation of structural predictions is essential. Researchers should consider obtaining high-resolution experimental structures through X-ray crystallography or cryo-electron microscopy to increase confidence in structure-guided experimental design.

What advanced genomic approaches could reveal the evolutionary significance of YddI across Bacillus species?

Understanding the evolutionary context of YddI requires sophisticated genomic analyses:

• Comprehensive phylogenetic analysis:

  • Construct phylogenetic trees based on YddI sequences across Bacillus species and related genera

  • Compare YddI phylogeny with species phylogeny to identify potential horizontal gene transfer events

  • Calculate selection pressures (dN/dS ratios) to determine if YddI is under purifying, neutral, or positive selection

  • Map conservation patterns to structural models to identify functionally constrained regions

• Synteny analysis:

  • Examine conservation of genomic context around yddI across bacterial genomes

  • Identify co-evolved gene clusters that might indicate functional relationships

  • Determine if yddI appears in known mobile genetic elements or genomic islands

• Population genomics:

  • Analyze yddI sequence variation across strains of B. subtilis from diverse ecological niches

  • Determine if particular variants correlate with specific environmental adaptations

  • Connect to the known genetic heterogeneity among B. subtilis strains

  • Assess if yddI contributes to strain-specific phenotypic differences

• Ancient sequence reconstruction:

  • Use ancestral sequence reconstruction methods to infer the evolutionary trajectory of YddI

  • Express and characterize reconstructed ancestral YddI proteins to understand functional evolution

  • Identify key mutational events that may have altered YddI function over evolutionary time

• Metagenomics approaches:

  • Survey environmental metagenomes to identify novel YddI variants in uncultured Bacillus relatives

  • Compare sequences from different environments to identify niche-specific adaptations

  • Use single-cell genomics to link YddI variants to specific bacterial lineages in complex communities

These approaches can reveal whether YddI represents a core component of Bacillus biology or contributes to the adaptive diversification that allows B. subtilis to thrive in diverse environments .