Recombinant Bacillus subtilis Putative permease-like protein ydzE (ydzE)

What is the YdzE protein and what is its predicted function in Bacillus subtilis?

YdzE is a putative permease-like protein encoded by the ydzE gene in Bacillus subtilis subsp. subtilis str. 168 (Gene ID: 938121, UniProt ID: O31493) . Based on sequence analysis and structural homology, YdzE is predicted to function as a membrane transport protein involved in the selective movement of substances across the cell membrane. As a permease, it likely participates in the uptake of specific nutrients or substrates, although its precise substrate specificity has not been fully characterized. In the context of B. subtilis membrane transport systems, permeases like YdzE are part of a complex network that allows the bacterium to adapt to changing environmental conditions and nutrient availability.

What are the optimal conditions for expressing recombinant YdzE protein in E. coli systems?

For optimal expression of recombinant YdzE in E. coli systems, researchers should consider the following methodological approach:

• Vector selection: Use pET expression vectors with a His-tag for efficient purification .

• E. coli strain selection: BL21(DE3) or Rosetta strains are recommended for membrane proteins.

• Expression conditions:

  • Growth temperature: 25-30°C after induction (lower than 37°C to reduce inclusion body formation)

  • IPTG concentration: 0.1-0.5 mM

  • Induction time: 4-6 hours or overnight at lower temperatures

• Buffer composition for extraction: PBS buffer with mild detergents (0.5-1% n-dodecyl β-D-maltoside) is suitable for membrane protein solubilization .

For enhanced protein stability and purity (>80% as determined by SDS-PAGE), include protease inhibitors during cell lysis and purification steps .

How do you confirm the successful expression of recombinant YdzE protein?

Confirmation of successful YdzE expression requires a multi-faceted approach:

• SDS-PAGE analysis: Visualize protein bands at the expected molecular weight (~57-58 kDa with His-tag) .

• Western blot analysis: Use anti-His antibodies to detect His-tagged YdzE.

• Mass spectrometry: Confirm protein identity through peptide mass fingerprinting or tandem MS. Expected mass for the recombinant His-tagged YdzE protein is approximately 57-58 kDa .

• Activity assays: Develop functional assays based on permeases' transport capabilities.

• Circular dichroism (CD): Verify proper protein folding, particularly important for membrane proteins like YdzE.

What are the advantages of expressing YdzE in Bacillus subtilis compared to E. coli expression systems?

While E. coli is commonly used for recombinant protein production, expressing YdzE in its native B. subtilis offers several methodological advantages:

• Natural protein folding environment: As a Gram-positive bacterium, B. subtilis provides the native membrane environment for proper folding and insertion of YdzE, potentially enhancing functional expression .

• GRAS status: B. subtilis has Generally Recognized As Safe status, making downstream applications more accessible for therapeutic or food-related research .

• Efficient secretion systems: B. subtilis possesses well-characterized secretion pathways that can be exploited for protein export if needed .

• Natural genetic competence: B. subtilis can readily take up DNA, facilitating genomic integration of expression constructs .

• Absence of endotoxins: Unlike E. coli, B. subtilis does not produce endotoxins, eliminating the need for endotoxin removal steps .

• Scalability: B. subtilis can be grown to high cell densities in industrial-scale fermenters, supporting large-scale protein production .

When working with YdzE in B. subtilis, researchers should utilize inducible promoter systems such as PxylA (xylose-inducible) or Pspac (IPTG-inducible) for controlled expression, and incorporate appropriate signal peptides if secretion is desired .

How can membrane solubilization conditions be optimized for maximum recovery of functional YdzE protein?

Optimizing membrane solubilization for functional YdzE recovery requires systematic testing of multiple parameters:

• Detergent screening methodology:

  • Start with a panel of detergents representing different classes: maltoside (DDM, UDM), glucoside (OG), fos-choline, and CHAPS derivatives

  • Test detergent concentrations at 1-5× their critical micelle concentration (CMC)

  • Monitor protein activity after solubilization to ensure functionality is maintained

• Buffer optimization:

  • pH range: Test buffers from pH 6.5-8.0

  • Salt concentration: 100-500 mM NaCl to maintain protein stability

  • Glycerol content: 5-15% to enhance protein stability

  • Additives: Consider lipids (0.01-0.1 mg/ml) from B. subtilis membranes to maintain native-like environment

• Time and temperature:

  • Solubilization time: 1-4 hours

  • Temperature: 4°C is preferred to minimize protein denaturation

• Functional assessment:

  • Develop transport assays specific to the predicted substrate of YdzE

  • Compare activity before and after solubilization to calculate recovery of functional protein

Based on similar membrane protein studies, a starting buffer composition of 50 mM Tris-HCl pH 7.5, 200 mM NaCl, 10% glycerol, 1% DDM, and 0.05 mg/ml B. subtilis lipid extract is recommended for initial solubilization trials .

What strategies can overcome inclusion body formation when expressing recombinant YdzE?

Addressing inclusion body formation requires a systematic approach focusing on expression conditions and protein folding:

• Temperature optimization:

  • Lower induction temperatures (16-25°C) slow protein synthesis, allowing time for proper folding

  • Implement a temperature gradient experiment to determine optimal conditions

• Expression kinetics control:

  • Reduce inducer concentration (0.01-0.1 mM IPTG)

  • Use tunable promoter systems like the rhamnose-inducible promoter for fine control

• Co-expression with chaperones:

  • GroEL/GroES system assists proper folding

  • DnaK/DnaJ/GrpE chaperone system reduces aggregation

  • Methodological approach: Construct co-expression vectors containing both YdzE and chaperone genes

• Fusion partners:

  • MBP (maltose-binding protein) enhances solubility

  • Thioredoxin fusion promotes proper disulfide bond formation

• Cell-free expression systems:

  • For transmembrane proteins like YdzE, cell-free systems with added lipids or detergent micelles can improve proper folding

• Refolding protocols from inclusion bodies:

  • Solubilize inclusion bodies with 8M urea or 6M guanidine-HCl

  • Perform step-wise dialysis with decreasing denaturant concentrations

  • Add detergents during refolding to provide hydrophobic environment for transmembrane segments

The combination of lower temperature (18°C), reduced IPTG concentration (0.1 mM), and co-expression with GroEL/GroES chaperones has been particularly effective for other B. subtilis membrane proteins and may prove successful for YdzE expression .

What experimental approaches can determine the substrate specificity of the YdzE permease?

Determining substrate specificity for YdzE requires a multi-pronged experimental approach:

• In vivo transport assays:

  • Construct YdzE knockout and overexpression strains in B. subtilis

  • Screen growth phenotypes on different carbon/nitrogen sources

  • Measure uptake of radiolabeled or fluorescently labeled potential substrates

  • Monitor intracellular accumulation using HPLC or mass spectrometry

• Phylogenetic analysis:

  • Compare YdzE with characterized permeases in other organisms

  • Identify conserved substrate-binding residues through multiple sequence alignment

  • Construct a phylogenetic tree to position YdzE within known permease families

• Structural analysis:

  • Homology modeling based on known permease structures

  • Docking simulations with potential substrates

  • Molecular dynamics simulations to analyze substrate-protein interactions

• Site-directed mutagenesis:

  • Identify putative substrate-binding residues

  • Generate point mutations and assess impact on transport activity

  • Create chimeric proteins with domains from characterized permeases

• Proteoliposome reconstitution:

  • Purify YdzE and reconstitute into liposomes

  • Load liposomes with potential substrates

  • Measure substrate transport rates under controlled conditions

By integrating these approaches, researchers can narrow down potential substrates and characterize the transport kinetics of YdzE. Based on structural similarities with other B. subtilis permeases, YdzE may be involved in peptide transport or cell wall component recycling similar to the DppE system .

How does YdzE interact with other components of transport systems in Bacillus subtilis?

Understanding YdzE's interactions with other transport components requires methodical investigation:

• Co-immunoprecipitation studies:

  • Express epitope-tagged YdzE in B. subtilis

  • Perform pull-down assays to identify interacting partners

  • Verify interactions through reverse co-IP experiments

• Bacterial two-hybrid analysis:

  • Clone YdzE and potential partners into bacterial two-hybrid vectors

  • Screen for protein-protein interactions in vivo

  • Quantify interaction strength using β-galactosidase assays

• Membrane protein complex isolation:

  • Use mild detergent solubilization to preserve protein complexes

  • Perform blue native PAGE to separate intact complexes

  • Identify components by mass spectrometry

• Genomic context analysis:

  • Examine genes in the vicinity of ydzE for potential operonic structure

  • Look for ATP-binding cassette (ABC) transporter components, as many permeases function within ABC transport systems

  • Analyze transcriptional co-regulation patterns

• Fluorescence microscopy:

  • Create fluorescent protein fusions to visualize YdzE localization

  • Perform co-localization studies with other transport components

  • Use FRET or BiFC to detect direct protein interactions in vivo

Based on analysis of other transport systems in B. subtilis, YdzE may function as part of a complex similar to the oligopeptide permease (Opp) or dipeptide permease (Dpp) systems, potentially interacting with extracellular substrate-binding proteins and cytoplasmic ATPases .

What role does YdzE play in Bacillus subtilis sporulation and stress response?

Investigating YdzE's role in sporulation and stress response requires these methodological approaches:

• Sporulation efficiency analysis:

  • Generate ΔydzE knockout strains

  • Quantify sporulation efficiency under standard conditions

  • Analyze morphological stages using phase-contrast microscopy

  • Measure expression of key sporulation genes (spo0A, sigE, sigF) by qRT-PCR

• Stress response characterization:

  • Challenge wild-type and ΔydzE strains with various stressors:

    • Osmotic stress (NaCl, sorbitol)

    • Oxidative stress (H₂O₂, paraquat)

    • Nutrient limitation

    • Antimicrobial peptides

  • Measure growth rates, survival, and stress-specific gene expression

• Transcriptome analysis:

  • Perform RNA-seq comparing wild-type and ΔydzE strains

  • Analyze differential gene expression during vegetative growth and sporulation

  • Identify affected pathways using gene ontology enrichment

• Metabolite transport studies:

  • Measure uptake of sporulation-specific signaling molecules

  • Investigate potential transport of quorum-sensing peptides like PhrE

  • Analyze cell wall peptide recycling similar to murein tripeptide transport by DppE

• Complementation experiments:

  • Reintroduce wild-type or mutant ydzE into knockout strains

  • Test whether sporulation and stress response phenotypes are restored

  • Create chimeric proteins with domains from known sporulation-related transporters

Membrane transporters in B. subtilis, including peptide permeases like Opp and Dpp, play critical roles in nutrient scavenging during stationary phase and can influence sporulation through the transport of signaling peptides . YdzE may have similar functions, potentially contributing to cell wall peptide recycling during sporulation or mediating the transport of specific signaling molecules.

How can YdzE be engineered for enhanced substrate transport capacity?

Engineering YdzE for enhanced transport requires methodical protein modification strategies:

• Structure-guided mutagenesis:

  • Identify substrate binding pocket residues through homology modeling

  • Design mutations to increase binding affinity or alter specificity

  • Create single-point mutations using site-directed mutagenesis

  • Test multiple mutations systematically to identify synergistic effects

• Directed evolution approach:

  • Generate a library of randomly mutagenized ydzE genes

  • Develop a selection system based on substrate transport or utilization

  • Screen for enhanced transport properties

  • Characterize selected variants by sequencing and functional assays

• Domain swapping:

  • Identify functional domains through alignment with well-characterized permeases

  • Create chimeric proteins with domains from high-efficiency transporters

  • Test transport properties of the resulting hybrid proteins

• Computational design:

  • Use molecular dynamics simulations to identify rate-limiting steps in transport

  • Apply computational protein design algorithms to optimize substrate binding

  • Validate in silico predictions through experimental testing

• Post-translational modification engineering:

  • Identify native modifications that affect YdzE function

  • Introduce or remove modification sites to enhance stability or activity

  • Optimize membrane insertion efficiency through signal sequence modifications

Engineering efforts should focus on maintaining protein stability while enhancing catalytic efficiency. Based on experiences with other B. subtilis permeases, substitutions in the transmembrane domains can significantly affect substrate specificity, while modifications to cytoplasmic loops may alter interactions with ATP-binding cassette components .

What are the challenges in crystallizing membrane proteins like YdzE, and how can they be overcome?

Crystallizing membrane proteins like YdzE presents several challenges that require specialized methodologies:

• Protein stabilization strategies:

  • Identify and remove flexible regions through limited proteolysis

  • Create fusion constructs with crystallization chaperones (T4 lysozyme, BRIL)

  • Generate antibody fragments (Fab, nanobodies) to stabilize specific conformations

  • Use disulfide engineering to lock protein in stable conformation

• Detergent optimization:

  • Screen detergents systematically (maltoside, glucoside, neopentyl glycol classes)

  • Test mixed detergent systems for improved stability

  • Consider detergent-lipid mixtures to maintain native-like environment

  • Explore novel amphipathic polymers (amphipols, SMALPs) as alternatives to detergents

• Lipidic cubic phase (LCP) crystallization:

  • Reconstitute YdzE into monoolein-based cubic phase

  • Optimize LCP composition with different lipids

  • Screen precipitants specifically designed for LCP crystallization

  • Implement temperature cycling to improve crystal quality

• Advanced screening approaches:

  • Utilize robotic systems for nanoliter-scale crystallization trials

  • Implement in situ diffraction screening to identify microcrystals

  • Use second-order nonlinear imaging of chiral crystals (SONICC) for crystal detection

  • Explore serial crystallography at X-ray free-electron lasers (XFELs)

• Alternative structural determination methods:

  • Cryo-electron microscopy for single-particle analysis

  • Solid-state NMR for specific structural elements

  • Hydrogen-deuterium exchange mass spectrometry for dynamics

Based on successful crystallization of other bacterial permeases and binding proteins, promising conditions for YdzE might include LCP crystallization with 1-2% (w/v) n-dodecyl-β-D-maltopyranoside supplemented with lipids, following purification protocols similar to those used for DppE and OppA .

How can molecular dynamics simulations provide insights into YdzE transport mechanisms?

Molecular dynamics (MD) simulations offer powerful approaches to understanding YdzE transport mechanisms:

• System preparation methodology:

  • Construct homology model of YdzE based on related transporters

  • Embed protein in explicit lipid bilayer (POPC or B. subtilis membrane composition)

  • Solvate system with explicit water molecules and physiological ion concentrations

  • Apply CHARMM36 or AMBER force fields optimized for membrane proteins

• Equilibrium simulation protocols:

  • Perform staged equilibration with progressive release of restraints

  • Run production simulations for microsecond timescales to capture conformational changes

  • Analyze protein stability, water penetration, and lipid interactions

• Advanced simulation techniques:

  • Umbrella sampling to calculate free energy profiles of substrate transport

  • Steered MD to study substrate binding/unbinding pathways

  • Coarse-grained simulations to access longer timescales

  • Ensemble simulations to improve conformational sampling

• Transport mechanism investigation:

  • Identify key residues involved in substrate recognition

  • Characterize conformational changes during transport cycle

  • Calculate energy barriers between different states

  • Model coupling between substrate binding and protein conformational changes

• Validation experiments:

  • Design mutagenesis experiments to test computational predictions

  • Measure transport kinetics of wild-type and mutant proteins

  • Compare simulated and experimental structures where available

Based on simulation studies of similar permeases, YdzE likely functions through an alternating access mechanism with distinct inward-facing and outward-facing conformations, potentially utilizing a rocker-switch or elevator-like movement of transmembrane domains .

How can researchers troubleshoot low expression yields of recombinant YdzE protein?

When facing low expression yields of recombinant YdzE, implement this systematic troubleshooting approach:

• Expression vector optimization:

  • Evaluate codon optimization for the expression host

  • Test different promoter strengths (T7, tac, ara)

  • Compare various fusion tags (His, MBP, SUMO) for impact on expression

  • Verify plasmid stability and copy number

• Host strain selection:

  • Screen multiple E. coli strains (BL21(DE3), C41(DE3), C43(DE3))

  • Consider B. subtilis expression systems for native environment

  • Test Lactococcus lactis or other Gram-positive hosts for membrane proteins

  • Evaluate strains with reduced protease activity

• Growth and induction conditions:

  • Optimize induction timing (early, mid, or late log phase)

  • Test range of inducer concentrations

  • Evaluate growth media (LB, TB, auto-induction media)

  • Adjust post-induction incubation temperature and duration

• Protein toxicity mitigation:

  • Use tightly regulated expression systems

  • Test glucose repression for leaky promoters

  • Consider growth in the presence of osmolytes (betaine, sorbitol)

  • Evaluate co-expression with toxicity-mitigating factors

• Membrane protein-specific strategies:

  • Add specific lipids to growth media

  • Include chemical chaperones (DMSO, glycerol) in culture

  • Test membrane-targeted expression enhancers

  • Consider cell-free expression systems with supplied membranes

For transmembrane proteins like YdzE, successful expression often requires reducing expression rate and temperature to allow proper membrane insertion and folding. Based on experiences with similar permeases, induction at OD₆₀₀ 0.6-0.8 with 0.1 mM IPTG followed by overnight expression at 18°C often yields the best results .

What strategies help resolve inconsistent results in YdzE functional assays?

Addressing inconsistent functional assay results requires meticulous attention to experimental variables:

• Protein quality assessment:

  • Verify protein purity by SDS-PAGE and size exclusion chromatography

  • Confirm proper folding using circular dichroism

  • Assess oligomeric state with native PAGE or analytical ultracentrifugation

  • Examine batch-to-batch variation with activity benchmarking

• Assay standardization:

  • Develop detailed standard operating procedures

  • Establish positive and negative controls for each experiment

  • Use internal standards to normalize between experiments

  • Implement statistical quality control measures

• Buffer and reagent controls:

  • Prepare fresh buffers and substrates for each experiment

  • Verify pH and ionic strength before each assay

  • Test reagent stability over time

  • Use controlled storage conditions for all components

• Technical parameter optimization:

  • Evaluate temperature dependence of the assay

  • Determine optimal protein concentration range

  • Assess time-dependence of measurements

  • Identify potential interfering compounds

• Reconstitution consistency:

  • For liposome-based assays, standardize liposome preparation

  • Control lipid composition and protein:lipid ratios

  • Verify reconstitution efficiency between experiments

  • Assess orientation of YdzE in reconstituted systems

For membrane transport assays specifically, controlling the electrochemical gradient and membrane integrity is critical. Establishing a rigorous protocol for preparation of proteoliposomes with consistent size distribution (measured by dynamic light scattering) and internal volume can significantly reduce variability in transport measurements.

How can researchers differentiate between YdzE and other permease activities in complex transport systems?

Distinguishing YdzE activity from other permeases requires sophisticated experimental design:

• Genetic manipulation approaches:

  • Generate single and multiple permease knockout strains

  • Create complementation strains with controlled expression levels

  • Develop inducible expression systems for temporal control

  • Design permease-specific mutations that alter substrate specificity

• Biochemical inhibition strategies:

  • Identify selective inhibitors through structure-based design

  • Develop antibodies or nanobodies specific to YdzE

  • Use competitive substrates to block specific transporters

  • Apply energy coupling inhibitors selectively

• Substrate modification techniques:

  • Design fluorescent or radioactive substrates with specificity for YdzE

  • Create substrate analogs that are recognized by YdzE but not other permeases

  • Utilize photo-affinity labeling to identify specific binding

  • Develop FRET-based transport assays for real-time monitoring

• Reconstitution experiments:

  • Purify YdzE and reconstitute into proteoliposomes

  • Perform transport assays with defined lipid composition

  • Compare activity with other purified permeases under identical conditions

  • Create co-reconstituted systems to study interactions between transporters

• Advanced analytical methods:

  • Use mass spectrometry to track isotope-labeled substrates

  • Apply microfluidics for single-cell transport analysis

  • Develop biosensors for real-time detection of substrate uptake

  • Utilize electrophysiology for direct measurement of transport activity

B. subtilis transport systems often show functional redundancy, as observed with oligopeptide permeases (Opp), dipeptide permeases (Dpp), and other peptide transporters . By systematically varying substrate structure (size, charge, hydrophobicity) and comparing transport kinetics between wild-type and knockout strains, researchers can create substrate specificity profiles to distinguish YdzE activity from other transporters.

How can YdzE be utilized in synthetic biology applications?

Leveraging YdzE in synthetic biology requires creative engineering approaches:

• Biosensor development:

  • Engineer YdzE to transport reporter molecules upon substrate binding

  • Couple transport activity to transcription factor activation

  • Create whole-cell biosensors for environmental monitoring

  • Develop FRET-based sensors for real-time detection

• Metabolic engineering applications:

  • Enhance nutrient uptake capabilities in production strains

  • Engineer substrate specificity for efficient transport of non-native precursors

  • Create synthetic transportomes with complementary specificity

  • Develop feedback-regulated transport systems

• Protein production platforms:

  • Utilize YdzE secretion mechanisms for protein export

  • Engineer YdzE-based protein display systems

  • Develop self-inducing expression systems based on transport triggers

  • Create cell-density sensing modules based on transporter activity

• Drug delivery applications:

  • Engineer B. subtilis spores with modified YdzE for targeted molecule delivery

  • Develop bacterial "Trojan horses" for antibiotic delivery

  • Create cellular detoxification systems based on efflux properties

  • Design cellular therapy vehicles with controlled import/export capabilities

• Orthogonal communication systems:

  • Engineer YdzE to transport synthetic signaling molecules

  • Create microbial consortia with specialized communication channels

  • Develop cell-to-cell contact-dependent transport systems

  • Build cellular computing systems based on molecular transport logic

B. subtilis spores have already demonstrated value for delivering immunodominant antigens like those from Mycobacterium tuberculosis . By engineering YdzE and related transport systems, researchers could develop sophisticated cellular devices with controllable uptake and release properties for biotechnology and biomedical applications.

What computational tools can predict substrate specificity of uncharacterized permeases like YdzE?

Computational prediction of permease specificity employs multiple bioinformatic approaches:

• Sequence-based methods:

  • Position-specific scoring matrices from known transporters

  • Hidden Markov Models trained on characterized permease families

  • Support Vector Machines using amino acid composition features

  • Deep learning approaches trained on transporter-substrate pairs

• Structure-based analysis:

  • Homology modeling using related transporters as templates

  • Binding site identification through conservation analysis

  • Molecular docking of potential substrates

  • Molecular dynamics simulations of substrate interaction

• Genomic context analysis:

  • Co-occurrence patterns with substrate utilization genes

  • Regulon analysis to identify co-regulated genes

  • Phylogenetic profiling across bacterial species

  • Metabolic network reconstruction to predict transport requirements

• Machine learning integration:

  • Random forest classifiers combining multiple feature types

  • Neural networks trained on transport assay data

  • Transfer learning from well-characterized transport systems

  • Active learning protocols to guide experimental validation

• Available software and databases:

  • TransportDB for transporter annotation

  • TCDB (Transporter Classification Database) for classification

  • COACH-D for substrate binding site prediction

  • BioTransporters for specificity prediction

Based on the analysis of other B. subtilis permeases like DppE (which transports murein tripeptides ), combining structural modeling with genomic context analysis provides the most reliable predictions for substrate specificity of uncharacterized permeases like YdzE.

How does the function of YdzE in Bacillus subtilis compare with homologous proteins in other bacterial species?

Comparative analysis of YdzE homologs requires an evolutionary and functional approach:

• Phylogenetic analysis methodology:

  • Identify homologs through BLAST searches against bacterial genomes

  • Perform multiple sequence alignment using MUSCLE or MAFFT

  • Construct maximum likelihood phylogenetic trees

  • Map functional data onto the phylogenetic tree to identify patterns

• Structural comparison approaches:

  • Generate homology models of YdzE homologs

  • Compare substrate binding pockets and transport pathways

  • Identify conserved and variable regions across species

  • Correlate structural features with substrate preferences

• Experimental cross-species validation:

  • Express homologs in the same host for direct comparison

  • Perform substrate specificity profiling across homologs

  • Test complementation of ydzE knockouts with homologs

  • Develop chimeric proteins to map functional domains

• Genomic context examination:

  • Compare operonic structures across species

  • Identify co-evolved transport components

  • Analyze associated metabolic pathways

  • Evaluate regulatory elements controlling expression

• Environmental adaptation analysis:

  • Correlate transporter differences with ecological niches

  • Analyze selection pressures on transporter genes

  • Investigate horizontal gene transfer events

  • Examine host-pathogen interaction contexts

Permease functions can vary significantly between species based on evolutionary pressures and metabolic requirements. For example, while B. subtilis uses multiple peptide transport systems (Opp, Dpp, App) with complementary specificities for nutrient acquisition and signaling , homologous systems in pathogens may be specialized for host interaction or virulence. Similarly, the substrate specificity of DppE in B. subtilis for murein tripeptides suggests a role in cell wall recycling , which may be conserved or modified in YdzE homologs depending on cell wall composition differences between species.