Recombinant Bacillus subtilis Uncharacterized protein yxaI (yxaI)

What is the YxaI protein in Bacillus subtilis?

YxaI is an uncharacterized protein from Bacillus subtilis subsp. subtilis str. 168 with a sequence length of 151 amino acids. The protein has been computationally modeled using AlphaFold, with a very high global confidence score (pLDDT) of 92.3, indicating that the predicted structure is likely to be accurate despite the lack of experimental validation . The gene encoding this protein is designated as yxaI in the B. subtilis genome. Unlike some other related proteins in B. subtilis (such as YxaG, which has been characterized as a Fe-containing quercetin 2,3-dioxygenase), the specific function of YxaI remains to be experimentally determined .

How does YxaI compare to other characterized proteins in Bacillus subtilis?

While YxaI remains uncharacterized, other proteins in B. subtilis such as YxaG have been functionally characterized. YxaG has been identified as a novel Fe-containing quercetin 2,3-dioxygenase, making it the first prokaryotic carbon monoxide-forming enzyme that utilizes a flavonol to be characterized . This contrasts with the eukaryotic version of this enzyme, which contains a Cu ion instead of Fe. YxaG was subsequently renamed as qdoI based on its characterized function . Unlike YxaG, which belongs to the bicupin family, the specific protein family and potential enzymatic activity of YxaI have not yet been experimentally determined. Sequence alignment and structural prediction tools may provide preliminary insights into potential functional similarities with characterized proteins.

What expression systems are most suitable for recombinant production of YxaI?

For recombinant production of YxaI from B. subtilis, several expression systems can be considered, with B. subtilis itself being a particularly advantageous host. Based on successful approaches with other B. subtilis proteins, the following methodology is recommended:

• B. subtilis WB800N expression system: This strain is deficient in multiple extracellular proteases, making it particularly suitable for heterologous protein expression. The strain has been successfully used for displaying recombinant proteins on spore surfaces .

• Expression vector selection: For intracellular expression, vectors containing strong promoters like P43 or PXYL are recommended. For surface display, fusion with coat proteins such as CotG has proven effective in B. subtilis WB800N .

• Cloning strategy: The yxaI gene should be amplified using specific primers with appropriate restriction sites (such as SpeI and HindIII), digested, and ligated into the selected expression vector .

• Transformation and verification: Heat shock transformation followed by confirmation through colony PCR and Sanger sequencing ensures successful integration of the construct .

• Induction conditions: For optimal expression, culture in DSM media with appropriate antibiotics for 36-48 hours at 37°C has shown good results for similar proteins .

The expression can be verified using SDS-PAGE and Western blot analysis, with the expected molecular weight of the recombinant protein being calculated based on the YxaI sequence (151 amino acids) plus any fusion tags or proteins .

What purification strategies are recommended for isolating recombinant YxaI?

Based on successful purification approaches for similar proteins from B. subtilis, the following purification strategy is recommended:

Table 1: Recommended Purification Strategy for Recombinant YxaI

For spore surface-displayed YxaI, the extraction procedure would involve:

• Centrifugation of the culture at 8000 rpm for 15 minutes

• Treatment with Buffer GTE containing lysozyme (20 mg/mL) at 37°C for one hour

• Washing twice with PBS buffer

• Confirmation of successful display through immunofluorescence techniques

The purification strategy should be optimized based on the specific fusion tags used and the intended downstream applications. The presence of metal ions such as Ni²⁺, Zn²⁺, and K⁺ might enhance protein stability and activity, as observed with other recombinant proteins displayed on B. subtilis spores .

What structural characterization methods should be employed to validate the AlphaFold model of YxaI?

To validate and refine the AlphaFold-predicted structure of YxaI (which has a high confidence score of 92.3 ), the following structural characterization methods are recommended:

• X-ray Crystallography:

  • Crystallization screening using commercially available kits

  • Optimization of crystallization conditions (pH, temperature, precipitants)

  • Data collection at synchrotron radiation facilities

  • Structure determination using molecular replacement with the AlphaFold model as a template

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Expression of ¹⁵N and/or ¹³C-labeled YxaI

  • Collection of 2D and 3D heteronuclear spectra

  • Chemical shift assignments and structure calculation

  • Comparison with the AlphaFold model

• Cryo-Electron Microscopy (Cryo-EM):

  • Sample preparation on grids

  • Data collection and processing

  • Model building using the AlphaFold prediction as initial model

• Small-Angle X-ray Scattering (SAXS):

  • Solution-based measurements of protein shape

  • Comparison of experimental scattering with theoretical scattering from the AlphaFold model

  • Assessment of oligomerization state

• Circular Dichroism (CD) Spectroscopy:

  • Analysis of secondary structure content

  • Comparison with predictions from the AlphaFold model

  • Thermal stability assessment

The integration of data from multiple methods would provide a comprehensive validation of the computational model and potentially reveal dynamic properties not captured by the static AlphaFold prediction .

What approaches can be used to determine the potential enzymatic activity of YxaI?

Since YxaI is an uncharacterized protein, a systematic approach to identify its potential enzymatic activity should include:

• Bioinformatic Analysis:

  • Sequence similarity searches against characterized proteins

  • Structural alignment with known enzymes

  • Identification of conserved catalytic motifs or domains

  • Analysis of genomic context (neighboring genes)

• Substrate Screening:

  • Based on structural similarity to YxaG (a quercetin 2,3-dioxygenase ), testing of flavonols and related compounds

  • High-throughput screening with diverse substrate libraries

  • Activity-based protein profiling

• Enzymatic Assays:

  • Spectrophotometric assays for common enzymatic activities

  • HPLC or LC-MS analysis for product identification

  • Isothermal titration calorimetry (ITC) for binding studies

  • Oxygen consumption measurement (if dioxygenase activity is suspected)

• Metal Ion Dependency:

  • Testing activity in the presence of various metal ions (Fe²⁺, Cu²⁺, Zn²⁺, Mn²⁺, etc.)

  • ICP-MS analysis to identify co-purifying metals

  • Metal chelation and reconstitution experiments

• Mutagenesis Studies:

  • Site-directed mutagenesis of predicted catalytic residues

  • Activity comparison between wild-type and mutant proteins

Given that YxaG, another B. subtilis protein, was characterized as an Fe-containing quercetin 2,3-dioxygenase , it would be prudent to specifically test YxaI for similar activities, while considering its unique structural features that may indicate alternative functions.

How can protein-protein interaction studies help elucidate the function of YxaI?

Identifying the interaction partners of YxaI can provide crucial insights into its cellular function and biological role. The following approaches are recommended:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Expression of tagged YxaI in B. subtilis

  • Affinity purification of YxaI complexes

  • Mass spectrometric identification of co-purifying proteins

  • Validation of interactions using reciprocal pulldowns

• Yeast Two-Hybrid (Y2H) Screening:

  • Construction of YxaI bait plasmid

  • Screening against B. subtilis genomic library

  • Validation of positive interactions using co-immunoprecipitation

• Bacterial Two-Hybrid (B2H) System:

  • Adaptation of Y2H methodology for prokaryotic proteins

  • Particularly suitable for membrane-associated interactions

• Proximity-Dependent Biotin Identification (BioID):

  • Fusion of YxaI with a biotin ligase

  • Expression in B. subtilis and biotinylation of proximal proteins

  • Streptavidin pulldown and mass spectrometry identification

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Quantitative measurement of binding kinetics

  • Validation of specific interactions identified by high-throughput methods

• Co-crystallization:

  • Structural determination of YxaI in complex with interaction partners

  • Identification of binding interfaces

The integration of multiple interaction detection methods would provide a comprehensive interaction network of YxaI, potentially connecting it to known cellular pathways and functions in B. subtilis.

What transcriptomic and proteomic approaches can help understand the physiological role of YxaI?

A comprehensive understanding of YxaI's physiological role requires investigation of its expression patterns and responses to various conditions. The following omics approaches are recommended:

• RNA-Seq Analysis:

  • Comparison of wild-type and yxaI knockout strains under various conditions

  • Identification of differentially expressed genes

  • Construction of co-expression networks

  • Time-course analysis during growth phases

• Quantitative Proteomics:

  • SILAC or TMT-based quantitative proteomics

  • Comparison of protein abundance in wild-type vs. knockout strains

  • Post-translational modification profiling

• ChIP-Seq Analysis (if relevant):

  • If YxaI is suspected to have DNA-binding properties

  • Identification of genomic binding sites

  • Integration with transcriptomic data

• Metabolomics:

  • Comparison of metabolite profiles between wild-type and knockout strains

  • Identification of altered metabolic pathways

  • Integration with enzymatic activity data

• Phenotypic Microarray:

  • Testing growth of wild-type vs. knockout strains under hundreds of conditions

  • Identification of conditional phenotypes

  • Linking to specific metabolic or stress response pathways

Table 2: Recommended Experimental Conditions for Omics Studies

Integration of data from these approaches would provide a systems-level understanding of YxaI's role in B. subtilis physiology and potentially reveal conditions where its function is particularly important.

How can CRISPR-Cas9 genome editing be optimized for studying YxaI function in Bacillus subtilis?

CRISPR-Cas9 genome editing offers precise genetic manipulation capabilities for studying YxaI function in B. subtilis. The following optimized methodology is recommended:

• sgRNA Design and Optimization:

  • Design multiple sgRNAs targeting the yxaI gene using tools optimized for B. subtilis

  • Verify specificity against the B. subtilis genome

  • Test efficiency using a reporter system

• CRISPR-Cas9 Delivery System:

  • Construction of a plasmid containing Cas9 under an inducible promoter

  • Integration of sgRNA expression cassette

  • Inclusion of homology-directed repair (HDR) template for precise modifications

• Genetic Modifications to Study YxaI:

  • Complete gene knockout to assess loss-of-function phenotypes

  • Point mutations of predicted catalytic residues

  • C-terminal or N-terminal tagging for localization and interaction studies

  • Promoter replacement for controlled expression

• Transformation and Selection:

  • Optimization of transformation protocol for B. subtilis

  • Two-step selection process using appropriate markers

  • Colony PCR and sequencing verification of edits

• Phenotypic Characterization:

  • Growth curve analysis under various conditions

  • Competitive fitness assays with wild-type

  • Stress response profiling

  • Microscopic analysis of cell morphology and protein localization

The CRISPR-Cas9 system should be optimized for efficiency in B. subtilis, potentially by using a codon-optimized Cas9 and testing various promoters for sgRNA expression. The efficiency of genome editing can be significantly improved by temporary inactivation of the DNA repair systems in B. subtilis.

What are the challenges in resolving discrepancies between computational predictions and experimental data for YxaI?

Resolving discrepancies between the computational model of YxaI (from AlphaFold DB, with a high confidence score of 92.3 ) and experimental data requires a systematic approach:

• Sources of Discrepancies:

  • Post-translational modifications not captured in the AlphaFold model

  • Conformational dynamics and protein flexibility

  • Interactions with cofactors, ligands, or other proteins

  • Effects of experimental conditions (pH, ionic strength, temperature)

  • Technical limitations of experimental methods

• Methodological Approaches to Resolution:

  • Integration of multiple experimental techniques (X-ray, NMR, Cryo-EM, SAXS)

  • Molecular dynamics simulations to explore conformational space

  • Hybrid modeling approaches combining computational and experimental data

  • Analysis of protein in different conditions to capture state-dependent conformations

• Specific Strategies for YxaI:

  • Focus on regions with lower pLDDT scores in the AlphaFold model

  • Test the effects of potential cofactors or metal ions on structure

  • Perform HDX-MS (Hydrogen-Deuterium Exchange Mass Spectrometry) to identify flexible regions

  • Use cross-linking mass spectrometry to validate tertiary structure predictions

• Data Integration Framework:

  • Develop a systematic approach to weight and integrate different data sources

  • Use Bayesian statistical methods to update the computational model with experimental data

  • Apply ensemble modeling to represent multiple conformational states

• Validation Metrics:

  • Establish quantitative metrics to assess model-data agreement

  • Perform blind tests with new experimental data

  • Cross-validate with orthogonal techniques

By systematically addressing these challenges, researchers can develop a more accurate and complete understanding of YxaI structure and function, reconciling computational predictions with experimental reality.

How can evolutionary analysis provide insights into the functional conservation of YxaI across bacterial species?

Evolutionary analysis can provide valuable insights into the functional conservation and potential role of YxaI. The following comprehensive approach is recommended:

• Phylogenetic Analysis:

  • Identification of YxaI homologs across bacterial species

  • Multiple sequence alignment using MUSCLE or MAFFT

  • Construction of phylogenetic trees using maximum likelihood or Bayesian methods

  • Mapping of known functional data onto the phylogenetic tree

• Evolutionary Rate Analysis:

  • Calculation of site-specific evolutionary rates

  • Identification of conserved residues under purifying selection

  • Detection of sites under positive selection

  • Correlation of evolutionary conservation with structural features

• Synteny Analysis:

  • Examination of genomic context conservation across species

  • Identification of consistently co-occurring genes

  • Correlation with known operons or functional pathways

• Domain Architecture Analysis:

  • Identification of conserved domains or motifs

  • Analysis of domain shuffling events across evolution

  • Correlation with functional diversification

• Ancestral Sequence Reconstruction:

  • Inference of ancestral YxaI sequences

  • Experimental characterization of reconstructed proteins

  • Analysis of functional shifts during evolution

Table 3: Evolutionary Conservation Analysis Framework for YxaI

This comprehensive evolutionary analysis would provide a framework for understanding the functional conservation of YxaI and potentially transfer functional annotations from better-characterized homologs in other bacterial species.

What are the optimal parameters for heterologous expression of YxaI on Bacillus subtilis spore surfaces?

Heterologous expression of YxaI on B. subtilis spore surfaces requires optimization of multiple parameters. Based on successful approaches with other proteins, the following optimized protocol is recommended:

• Selection of Coat Protein Anchor:

  • CotG as the primary choice for surface display based on successful previous applications

  • CotB, CotC, or CotZ as alternatives if CotG fusion is not functional

  • Comparative testing of N-terminal versus C-terminal fusions

• Expression Vector Design:

  • Construction of pHS-CotG-YxaI plasmid similar to previously successful designs

  • Incorporation of appropriate restriction sites (e.g., SpeI and HindIII) for cloning

  • Inclusion of linker sequences between CotG and YxaI to ensure proper folding

  • Addition of epitope tags for detection (e.g., His-tag or FLAG-tag)

• Transformation and Expression:

  • Transformation into B. subtilis WB800N strain (deficient in extracellular proteases)

  • Culture in DSM media with appropriate antibiotics for 36-48 hours

  • Induction of sporulation by nutrient depletion

• Optimization Parameters:

  • Media composition (concentrations of metals, carbon sources, nitrogen sources)

  • Temperature and pH conditions

  • Duration of sporulation phase

  • Harvest timing

• Verification Methods:

  • SDS-PAGE and Western blot analysis of spore coat proteins

  • Immunofluorescence microscopy for visualization of surface display

  • Functional assays if the enzymatic activity of YxaI is known

  • Flow cytometry quantification of display efficiency

Table 4: Optimization Matrix for YxaI Spore Surface Display

The successful display of YxaI on spore surfaces should be evaluated by its stability, recyclability (maintaining >70% activity after multiple cycles as observed with other immobilized enzymes ), and functional properties if known.

How can single-molecule techniques be applied to study the structural dynamics of YxaI?

Single-molecule techniques offer unique insights into protein dynamics that are not accessible through bulk measurements. For studying YxaI, the following approaches are recommended:

• Single-Molecule FRET (smFRET):

  • Strategic placement of fluorophore pairs at key positions in YxaI

  • Measurement of distance dynamics between labeled residues

  • Detection of conformational substates and transitions

  • Correlation with functional states if known

• Atomic Force Microscopy (AFM):

  • Imaging of individual YxaI molecules on surfaces

  • Force spectroscopy to probe mechanical properties

  • Measurement of protein-protein or protein-ligand interactions

  • High-speed AFM to capture conformational dynamics

• Single-Molecule Pull-Down (SiMPull):

  • Detection of native interaction partners at single-molecule level

  • Quantification of binding stoichiometry

  • Analysis of complex assembly kinetics

• Nanopore Analysis:

  • Translocation of YxaI through nanopores

  • Electrical detection of structural features

  • Potential for sensing ligand binding or conformational changes

• Total Internal Reflection Fluorescence (TIRF) Microscopy:

  • Visualization of individual fluorescently labeled YxaI molecules

  • Tracking of diffusion dynamics

  • Analysis of oligomerization states

• Experimental Design Considerations:

  • Selection of labeling sites based on the AlphaFold model

  • Verification that labels do not disrupt protein function

  • Control experiments with known structural perturbations

  • Integration with computational models for interpretation

The application of these techniques to YxaI would reveal dynamic properties not captured in static structural models, potentially providing insights into function-related conformational changes and interactions with binding partners or substrates.

What integrative approaches can link YxaI structure to its cellular function in Bacillus subtilis?

Integrating multiple experimental approaches is essential for establishing a comprehensive understanding of the relationship between YxaI structure and its cellular function. The following integrative framework is recommended:

• Structure-Function Correlation Pipeline:

  • Systematic mutagenesis based on structural features

  • Functional assays to determine effects of mutations

  • Computational prediction of functional sites

  • Experimental validation of predictions

• In Vivo Structural Biology:

  • In-cell NMR to study YxaI structure in native environment

  • Fluorescence microscopy with genetically encoded sensors

  • Cross-linking mass spectrometry in native B. subtilis cells

  • Correlation with physiological states

• Systems Biology Integration:

  • Network analysis combining transcriptomics, proteomics, and metabolomics data

  • Identification of condition-specific YxaI functions

  • Construction of predictive models for YxaI-dependent processes

  • Experimental validation of model predictions

• Multi-scale Computational Modeling:

  • Molecular dynamics simulations of YxaI in various environments

  • Coarse-grained models of YxaI interactions with cellular components

  • Integration of experimental constraints into models

  • Prediction of cellular-level effects of YxaI perturbations

• Temporal and Spatial Regulation Analysis:

  • Time-resolved studies of YxaI expression and localization

  • Correlation with cell cycle stages or developmental processes

  • Identification of regulatory networks controlling YxaI

  • Subcellular localization studies using fluorescent fusion proteins

This integrative approach would bridge the gap between molecular structure and cellular function, providing a comprehensive understanding of YxaI's role in B. subtilis biology and potentially revealing novel functional paradigms for this uncharacterized protein.

What are the most promising research directions for further characterization of YxaI?

Based on the current understanding of YxaI as an uncharacterized protein with a high-confidence computational structure model , several promising research directions emerge:

• Experimental Structure Determination: While the AlphaFold model provides a strong starting point with a global pLDDT score of 92.3 , experimental validation through X-ray crystallography or cryo-EM would provide definitive structural information and potentially reveal features not captured in the computational model.

• Functional Assignment: Systematic substrate screening, combined with structural analysis and comparison to characterized proteins like YxaG , offers the most direct path to functional characterization. The potential role as an enzyme involved in flavonol metabolism or related pathways should be particularly investigated.

• Physiological Role Determination: Creation of knockout strains and comprehensive phenotypic characterization under various conditions would reveal the biological importance of YxaI. Integration with omics approaches would place YxaI in the context of cellular networks.

• Development as a Biotechnological Tool: If enzymatic activity is identified, the promising results with B. subtilis spore surface display systems suggest YxaI could be developed for biotechnological applications, particularly if it demonstrates recyclability similar to other immobilized enzymes.

• Evolutionary Analysis: Comparative genomics across diverse bacterial species could reveal patterns of conservation and co-evolution that provide insights into YxaI function and importance.

These research directions, pursued in parallel with an integrated approach, would transform YxaI from an uncharacterized protein into a well-understood component of B. subtilis biology, potentially revealing novel biological functions and applications.

How should researchers prioritize experimental approaches when characterizing an uncharacterized protein like YxaI?

When characterizing an uncharacterized protein like YxaI, researchers should prioritize experimental approaches using the following strategic framework:

• Initial Characterization Phase:

  • Express and purify the recombinant protein (highest priority)

  • Verify the predicted structure through circular dichroism or limited proteolysis

  • Determine oligomerization state and basic biochemical properties

  • Identify potential cofactors or metal dependencies

• Functional Discovery Phase:

  • Conduct bioinformatic analysis to generate functional hypotheses

  • Perform targeted substrate screening based on structural features

  • Create and phenotype gene knockout strains

  • Identify interaction partners through pulldown experiments

• Detailed Characterization Phase:

  • Determine experimental structure if function warrants the investment

  • Perform detailed enzyme kinetics or binding studies

  • Conduct site-directed mutagenesis of key residues

  • Analyze expression patterns and regulation mechanisms

• Integration and Application Phase:

  • Place protein in cellular pathway context using omics approaches

  • Explore potential biotechnological applications

  • Investigate evolutionary conservation across species

  • Develop tools or methods based on the characterized protein