Recombinant Bacillus phage SPbeta Protein bhlB (bhlB)

What is Bacillus phage SPbeta Protein bhlB and what is its genetic context?

Bacillus phage SPbeta Protein bhlB (also known as yomA) is a phage-encoded protein found in the SPβ prophage of Bacillus subtilis 168. The protein is classified as a holin-like bacteriophage protein, which suggests involvement in cell membrane disruption during the phage lytic cycle. The gene exists under two primary nomenclatures: bhlB and yomA, with bhlB being the preferred designation in most databases .

SPβ is a well-characterized prophage with a genome ranging between 128 and 140 kb, exhibiting terminal redundancy in its dsDNA. The phage belongs to the Siphoviridae morphotype and requires approximately 90 minutes after induction to produce and release about 30 virions .

What expression systems are most effective for producing functional recombinant bhlB protein?

Several expression systems have been successfully employed for recombinant bhlB production, each with distinct advantages:

All systems typically achieve ≥85% purity as determined by SDS-PAGE when properly optimized .

How should researchers design experiments to study bhlB function in the context of SPβ phage biology?

When designing experiments to study bhlB function, consider the following methodological framework:

• Genetic Manipulation Approach: Create knockout mutants of bhlB in SPβ lysogenic strains to assess its role in the phage life cycle. This can be accomplished through homologous recombination or CRISPR-Cas9 gene editing.

• Induction Protocol: Standardize induction conditions using either mitomycin C or UV radiation exposure, which are known to activate the SPβ prophage. This allows for controlled study of bhlB expression during the lytic cycle .

• Comparative Analysis: Compare wild-type and bhlB-mutant phage for differences in:

  • Lysis timing and efficiency

  • Phage particle assembly

  • Burst size (number of virions released)

  • Host range specificity

• Localization Studies: Use fluorescently-tagged bhlB to track its subcellular localization during the phage life cycle, which may provide insights into its functional role .

• Time-Course Analysis: Implement a time-resolved experimental design to capture the dynamic expression and activity of bhlB during the 90-minute lytic cycle of SPβ .

What purification strategies yield the highest purity and functional activity for recombinant bhlB?

A systematic purification approach for recombinant bhlB typically involves:

• Initial Clarification: Cell lysis followed by centrifugation and filtration (0.2 μm) to remove cellular debris.

• Capture Phase: Affinity chromatography using an appropriate tag system. If the recombinant protein contains a histidine tag, immobilized metal affinity chromatography (IMAC) is recommended.

• Intermediate Purification: Ion exchange chromatography based on the predicted isoelectric point of bhlB.

• Polishing Step: Size exclusion chromatography to achieve final purity ≥85% as determined by SDS-PAGE .

• Quality Control: SDS-PAGE analysis combined with Western blotting to confirm identity and purity. Mass spectrometry verification is recommended for critical applications.

Researchers should note that different host expression systems may require adjusted purification strategies due to varying cellular components and potential post-translational modifications .

How does bhlB contribute to the SPβ phage life cycle and what evidence supports this role?

Current evidence suggests bhlB functions as a holin-like protein, which typically forms pores in the bacterial cytoplasmic membrane during the late stages of infection. This function is critical for allowing endolysins to access the peptidoglycan layer, facilitating cell lysis and virion release.

The life cycle of SPβ involves:

• Integration as a prophage into B. subtilis genome

• Maintenance in the lysogenic state

• Induction upon DNA damage (e.g., mitomycin C exposure)

• Excision from the host genome

• DNA replication and virion assembly

• Cell lysis and phage release

bhlB is thought to be involved in the final lysis step, though direct experimental validation is still emerging. The protein's classification as "holin-like" is based on sequence homology rather than confirmed functional studies .

What is known about the regulation of bhlB expression during the SPβ life cycle?

The regulation of bhlB expression involves complex mechanisms:

• SOS Response Connection: SPβ contains several SOS boxes that provide binding sites for LexA (also known as DinR, damage-inducible regulator). While bhlB itself is not directly associated with an SOS box, its expression may be indirectly regulated through the SOS response pathway .

• Phage-Specific Regulation: The phage-derived component YonR may be involved in regulating bhlB expression, though this requires further investigation .

• Host Factor Involvement: The extracytoplasmic function (ECF) sigma factor SigY plays a role in SPβ maintenance. Deletion mutants of SigY spontaneously lose the SPβ prophage, suggesting it may influence the expression of phage genes including bhlB .

• Temporal Expression Pattern: As a predicted holin protein involved in cell lysis, bhlB is likely expressed late in the infection cycle, approximately 60-90 minutes after induction, immediately preceding virion release .

How can researchers investigate potential interactions between bhlB and host cellular components?

To investigate bhlB-host interactions, consider these methodological approaches:

• Protein-Protein Interaction Studies:

  • Bacterial two-hybrid systems

  • Co-immunoprecipitation followed by mass spectrometry

  • Surface plasmon resonance to measure binding kinetics

  • Fluorescence resonance energy transfer (FRET) for in vivo interaction detection

• Membrane Localization Analysis:

  • Fractionation studies to determine membrane association

  • Super-resolution microscopy with fluorescently tagged bhlB

  • Lipid interaction assays to determine membrane specificity

• Structural Biology Approaches:

  • X-ray crystallography of bhlB alone and in complex with potential partners

  • NMR studies for dynamic interaction analysis

  • Cryo-EM to visualize membrane-associated complexes

• Functional Genomics:

  • Transposon mutagenesis screening to identify host factors affecting bhlB function

  • RNA-seq to identify transcriptional changes in response to bhlB expression

  • CRISPR interference screens to identify essential host factors

What experimental controls are critical when evaluating bhlB function in phage infection models?

When designing experiments to evaluate bhlB function, the following controls are essential:

• Genetic Controls:

  • Wild-type SPβ phage (positive control)

  • bhlB deletion mutant

  • Complementation with wild-type bhlB (rescue control)

  • Complementation with mutated bhlB variants

  • Empty vector control

• Host Strain Controls:

  • B. subtilis 168 (natural host)

  • SPβ-free strain like CU1050 or its descendant YB886

  • Hosts with varying susceptibility (e.g., B. pumilus, B. amyloliquefaciens)

• Induction Controls:

  • Uninduced samples

  • Various induction methods (mitomycin C, UV radiation)

  • Time-course sampling

  • Quantification of phage particles by plaque assays

• Technical Controls:

  • Protein expression verification by Western blot

  • Membrane fractionation quality controls

  • Cell viability measures

What are common challenges when working with recombinant bhlB and how can they be addressed?

Researchers often encounter these challenges when working with recombinant bhlB:

• Protein Solubility Issues:

  • Challenge: As a membrane-associated protein, bhlB may form inclusion bodies.

  • Solution: Express at lower temperatures (16-20°C), use solubility tags (SUMO, MBP), or optimize buffer conditions with mild detergents like n-dodecyl β-D-maltoside (DDM).

• Toxicity to Expression Host:

  • Challenge: Holin proteins can be toxic to the expression host.

  • Solution: Use tightly controlled inducible systems, cell-free expression systems, or toxicity-resistant strains .

• Functional Verification:

  • Challenge: Confirming that purified bhlB retains its native function.

  • Solution: Develop membrane permeabilization assays using liposomes or bacterial spheroplasts.

• Stability During Storage:

  • Challenge: Membrane proteins often aggregate during storage.

  • Solution: Store at -20°C/-80°C with 50% glycerol, avoid repeated freeze-thaw cycles, and aliquot for single use .

How can researchers validate that their recombinant bhlB preparations maintain native structural and functional properties?

To ensure recombinant bhlB maintains native properties, implement these validation strategies:

• Structural Validation:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure

  • Dynamic light scattering to assess aggregation state

  • Limited proteolysis to evaluate proper folding

  • Native PAGE to analyze oligomeric state

• Functional Validation:

  • Liposome permeabilization assays

  • Conductance measurements in artificial membranes

  • Complementation of bhlB-deficient phage

  • Membrane localization in bacterial cells

• Quality Control Metrics:

  • Purity ≥85% by SDS-PAGE

  • Endotoxin testing for preparations used in cellular assays

  • Mass spectrometry to confirm intact mass and post-translational modifications

• Batch Consistency Testing:

  • Activity assays across multiple production batches

  • Stability assessment at various time points

  • Lot-to-lot comparison of critical quality attributes

How can phage display technologies be adapted to study bhlB structure-function relationships?

Phage display offers powerful approaches for bhlB research:

• Domain Mapping:

  • Create a library of bhlB fragments displayed on filamentous phage

  • Screen against potential interaction partners or membranes

  • Identify minimal functional domains for membrane interaction

• Mutagenesis Scanning:

  • Generate libraries with randomized mutations in specific regions

  • Select for variants with altered host specificity or improved stability

  • Map critical residues for function

• Directed Evolution:

  • Apply selection pressure to identify bhlB variants with novel properties

  • Evolve variants with expanded host range or enhanced lytic activity

  • Characterize mutations that alter specificity

• Structural Constraint Analysis:

  • Use cysteine scanning mutagenesis combined with phage display

  • Identify regions tolerant to modification versus structurally crucial domains

  • Develop structure-function models based on selection results

What bioinformatic approaches can provide insights into bhlB evolution and function across different phage species?

Computational approaches offer valuable insights for bhlB research:

• Comparative Genomics:

  • Identify bhlB homologs across phage families

  • Analyze synteny and genomic context conservation

  • Map evolutionary relationships between holin proteins

• Structural Prediction:

  • Apply machine learning algorithms to predict membrane topology

  • Model bhlB tertiary structure using AlphaFold or similar tools

  • Simulate membrane interactions using molecular dynamics

• Functional Network Analysis:

  • Construct protein-protein interaction networks

  • Identify functional clusters associated with bhlB homologs

  • Predict functional partners based on co-evolution patterns

• Evolutionary Analysis:

  • Calculate selection pressures on different bhlB domains

  • Identify rapidly evolving regions that may indicate host adaptation

  • Compare bhlB evolution rates with other phage components

How might recombinant bhlB be utilized in phage therapy research against antibiotic-resistant bacteria?

Recombinant bhlB holds potential applications in phage therapy research:

• Engineered Lysis Systems:

  • Express recombinant bhlB alongside endolysins for synergistic bacterial killing

  • Develop controlled lysis systems for targeted bacterial elimination

  • Engineer chimeric holins with broader host specificity

• Host Range Expansion:

  • Study mutations in bhlB that alter host specificity

  • Apply directed evolution to develop variants with activity against resistant pathogens

  • Use bhlB structure-function knowledge to rationally design broader spectrum variants

• Resistance Mechanism Studies:

  • Investigate bacterial resistance mechanisms against bhlB

  • Identify compensatory mutations that overcome resistance

  • Develop combination strategies to prevent resistance emergence

• Delivery System Development:

  • Create liposomal delivery systems containing bhlB

  • Design controlled release formulations

  • Develop tissue-specific targeting strategies

What experimental designs would best address the role of bhlB in potential species-jumping capabilities of SPβ-related phages?

To investigate bhlB's role in host range determination, consider these experimental approaches:

• Comparative Host Range Analysis:

  • Test wild-type and bhlB-mutant phages against diverse Bacillus species

  • Measure adsorption rates and efficiency of plating

  • Determine if bhlB mutations correlate with expanded host range

• Experimental Evolution:

  • Passage SPβ on alternate Bacillus hosts

  • Sequence evolved phages to identify adaptive mutations in bhlB

  • Test if artificially introducing these mutations expands host range

• Domain Swapping Experiments:

  • Create chimeric bhlB proteins with domains from related phages

  • Test if specific domains confer host-specific functionality

  • Identify minimum genetic changes required for host switching

• Receptor Identification:

  • Use recombinant bhlB to pull down potential host receptors

  • Compare receptor binding across susceptible and resistant hosts

  • Map the interaction interfaces through mutational analysis