Recombinant Bacillus pumilus UPF0754 membrane protein BPUM_0927 (BPUM_0927)

How is recombinant BPUM_0927 protein typically produced and purified?

The recombinant BPUM_0927 protein is commonly expressed in Escherichia coli expression systems rather than native Bacillus hosts. This heterologous expression approach takes advantage of E. coli's rapid growth and high protein yield capabilities. The protein is engineered with an N-terminal His-tag, which enables single-step affinity purification using nickel or cobalt-based chromatography.

Standard purification protocol:

• Culture transformed E. coli cells in appropriate media until optimal induction point

• Induce protein expression (typically with IPTG for T7-based systems)

• Harvest cells and disrupt using sonication or mechanical methods

• Solubilize membrane proteins with appropriate detergents

• Perform immobilized metal affinity chromatography (IMAC)

• Verify purity (>90%) using SDS-PAGE

• Lyophilize the purified protein for long-term storage

Due to its membrane protein nature, special considerations must be given to detergent selection during extraction and purification to maintain native protein folding and function.

What is known about the biological function of BPUM_0927 in Bacillus pumilus?

• It may function in membrane integrity or transport processes, given its predicted transmembrane domains

• It could potentially be involved in stress response mechanisms, as B. pumilus is known for exceptional resistance to oxidative stress

• It may participate in species-specific adaptations that differentiate B. pumilus from related Bacillus species

Research on other B. pumilus membrane proteins suggests potential roles in environmental adaptation, such as the high oxidative stress resistance characteristic of this species. B. pumilus demonstrates remarkable resistance to hydrogen peroxide, which involves various membrane-associated mechanisms and stress response pathways . Further research using gene knockout or overexpression studies would be needed to elucidate BPUM_0927's specific function.

What are the optimal storage and handling conditions for recombinant BPUM_0927 protein?

Optimal storage and handling of recombinant BPUM_0927 protein requires careful consideration of its membrane protein nature. Based on empirical data, the following protocol is recommended:

The reconstituted protein should be kept at 4°C for short-term use (up to one week). Repeated freeze-thaw cycles should be strictly avoided as they can compromise protein integrity and functionality. For experiments requiring native membrane protein conformation, consider incorporating appropriate detergents or lipid environments during reconstitution .

How can researchers verify the functional activity of recombinant BPUM_0927?

Verifying the functional activity of BPUM_0927 presents challenges due to its uncharacterized function. A systematic approach using multiple complementary methods is recommended:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure elements

  • Size exclusion chromatography to verify proper oligomeric state

  • Limited proteolysis to evaluate proper folding

• Membrane incorporation studies:

  • Liposome reconstitution assays

  • Detergent micelle incorporation efficiency

  • Membrane protein orientation analysis

• Functional hypotheses testing:

  • Transport assays using artificial membrane systems

  • Binding studies with potential ligands

  • Ion flux measurements if channel/transporter activity is suspected

• Comparative analyses:

  • Complement B. pumilus BPUM_0927 knockout strains with recombinant protein

  • Assess stress response parameters (particularly oxidative stress resistance)

  • Measure cell viability under various environmental conditions

While the specific function remains unknown, researchers can validate that the recombinant protein maintains native-like properties through these approaches. Combining structural verification with hypothesis-driven functional assays provides the most comprehensive validation strategy.

How can CRISPR-Cas9 be utilized to investigate BPUM_0927 function in Bacillus pumilus?

CRISPR-Cas9 technology offers powerful approaches for investigating BPUM_0927 function through precise genome editing. Based on successful application in B. pumilus for other genes, the following methodological framework is recommended:

• Target sequence identification and sgRNA design:

  • Identify unique 20-nucleotide sequences within BPUM_0927 followed by PAM sites (NGG)

  • Design 2-3 different sgRNAs targeting different regions to increase success probability

  • Verify target specificity through genome-wide off-target analysis

• CRISPR-Cas9 delivery system optimization:

  • Construct expression vectors containing Cas9 and sgRNA under appropriate promoters

  • Optimize transformation protocols specifically for B. pumilus (electroporation parameters)

  • Include appropriate selection markers (e.g., antibiotic resistance)

• Gene knockout strategy:

  • Design donor DNA with homology arms flanking BPUM_0927 to facilitate homology-directed repair

  • Include reporter genes or selection markers between homology arms

  • Verify knockout through PCR, sequencing, and protein expression analysis

• Phenotypic characterization:

  • Compare growth dynamics between wild-type and knockout strains

  • Assess membrane integrity and composition changes

  • Evaluate stress response, particularly to oxidative stress conditions

  • Analyze proteome and metabolome changes using MS-based approaches

Similar CRISPR-Cas9 approaches have been successfully employed for investigating other B. pumilus genes related to antimicrobial peptide production, such as bacilysin (bac) and bacteriocin (bact) genes . The knockout of these genes resulted in measurable phenotypic changes, including altered growth dynamics and decreased proteolytic activity. A comparable approach for BPUM_0927 would likely yield valuable insights into its functional role.

What protein-protein interaction studies would be most informative for elucidating BPUM_0927 function?

Given the limited knowledge about BPUM_0927 function, comprehensive protein-protein interaction (PPI) studies would provide valuable insights. The following methodological approach is recommended:

• In vivo crosslinking and co-immunoprecipitation (Co-IP):

  • Introduce epitope-tagged BPUM_0927 into B. pumilus

  • Perform chemical crosslinking to capture transient interactions

  • Immunoprecipitate BPUM_0927 complexes and identify interacting partners through mass spectrometry

  • Validate interactions through reciprocal Co-IP experiments

• Bacterial two-hybrid (B2H) screening:

  • Use BPUM_0927 as bait against a B. pumilus genomic library

  • Screen for positive interactions based on reporter gene activation

  • Confirm interactions through secondary validation methods

  • Map interaction domains through truncation mutants

• Membrane-specific interaction analysis:

  • Apply techniques specialized for membrane protein interactions:

    • Bimolecular fluorescence complementation (BiFC)

    • Förster resonance energy transfer (FRET)

    • Split-ubiquitin membrane yeast two-hybrid system

• Computational predictions and validation:

  • Utilize STRING database predictions for potential interactors

  • Perform co-expression analysis across different conditions

  • Validate top candidates through targeted experimental approaches

Based on comparative genomic analyses of the B. pumilus group, potential interaction partners might include components of membrane transport systems, particularly K+ transporters (TrK) and ABC transporters, which show differences between marine-derived and terrestrial strains . Transcriptional regulators unique to B. pumilus could also be prioritized as potential interaction candidates.

How does BPUM_0927 compare structurally and functionally to homologous proteins in other Bacillus species?

A comprehensive comparative analysis of BPUM_0927 with homologous proteins from other Bacillus species provides evolutionary and functional insights. The recommended methodological approach includes:

• Sequence-based comparative analysis:

  • Perform BLAST searches against Bacillus genomes to identify homologs

  • Conduct multiple sequence alignment to identify conserved residues

  • Calculate sequence conservation scores across different Bacillus clades

  • Generate phylogenetic trees to visualize evolutionary relationships

• Structural comparison:

  • Predict secondary and tertiary structures using computational methods

  • Compare predicted structures with known structures of homologous proteins

  • Identify conserved structural motifs that might indicate functional domains

  • Model protein-membrane interactions across different species

• Genomic context analysis:

  • Examine gene neighborhoods of BPUM_0927 homologs across species

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

  • Compare operon structures and potential co-regulation patterns

• Expression pattern comparison:

  • Analyze available transcriptomic data to compare expression patterns

  • Identify conditions under which BPUM_0927 and its homologs are differentially regulated

  • Correlate expression patterns with species-specific environmental adaptations

Based on genomic analyses, the B. pumilus group has shown significant differentiation from other Bacillus species, particularly in genes related to environmental adaptation. Marine-derived strains demonstrate enrichment in genes related to transcription, phage defense, and DNA recombination and repair compared to their terrestrial counterparts . BPUM_0927 might participate in these adaptation mechanisms, making comparative analysis particularly valuable for understanding its role in B. pumilus' unique environmental niche adaptations.

How might BPUM_0927 contribute to the exceptional oxidative stress resistance of Bacillus pumilus?

B. pumilus demonstrates remarkable resistance to oxidative stress, particularly to hydrogen peroxide. While BPUM_0927's specific role is not fully characterized, several methodological approaches can be used to investigate its potential contribution:

• Comparative expression analysis:

  • Measure BPUM_0927 expression levels under varying oxidative stress conditions

  • Compare expression patterns with known oxidative stress response genes

  • Correlate expression changes with physiological stress resistance parameters

• Functional analysis in oxidative stress response:

  • Generate BPUM_0927 knockout strains and assess H₂O₂ sensitivity

  • Complement knockout strains with wild-type or mutated BPUM_0927

  • Measure key oxidative stress markers (e.g., ROS levels, lipid peroxidation)

• Protein modification studies:

  • Assess post-translational modifications of BPUM_0927 during oxidative stress

  • Investigate potential redox-sensitive residues within the protein

  • Determine if BPUM_0927 undergoes structural changes under oxidative conditions

Unlike B. subtilis, B. pumilus lacks several key oxidative stress response proteins including catalase KatA, DNA-protection protein MrgA, and alkyl hydroperoxide reductase AhpCF. Instead, B. pumilus appears to rely on alternative mechanisms, including catalase KatX2 and high intracellular levels of the protective metabolite bacillithiol (Cys-GlcN-malate, BSH) . As a membrane protein, BPUM_0927 might participate in:

• Maintaining membrane integrity during oxidative stress

• Facilitating transport of protective compounds across the membrane

• Sensing environmental oxidative conditions and triggering appropriate responses

• Regulating ion homeostasis which is often disrupted under oxidative stress

Testing these hypotheses would require a combination of genetic, biochemical, and physiological approaches tailored to membrane protein analysis.

What role might BPUM_0927 play in B. pumilus adaptation to marine environments?

The Bacillus pumilus group includes strains found in diverse marine environments, from coastal waters to deep-sea sediments. Investigating BPUM_0927's potential role in marine adaptation requires specialized approaches:

• Comparative genomic analysis across ecological niches:

  • Compare BPUM_0927 sequences from marine versus terrestrial B. pumilus strains

  • Identify marine-specific sequence variations or selection signatures

  • Analyze gene neighborhood conservation patterns across ecological boundaries

• Expression profiling under marine-mimicking conditions:

  • Examine BPUM_0927 expression under varying salinity, pressure, and temperature

  • Compare expression patterns between marine and terrestrial isolates

  • Correlate expression changes with adaptive physiological responses

• Functional characterization in simulated marine conditions:

  • Assess growth phenotypes of BPUM_0927 knockout strains under marine-like conditions

  • Measure membrane properties and ion transport capabilities

  • Evaluate protein-protein interactions unique to marine conditions

Phylogenomic analysis reveals that marine B. pumilus group strains generally cluster into three species: B. pumilus, B. altitudinis, and B. safensis, all sharing a common ancestor . Marine-derived strains show enrichment in genes related to transcription, phage defense, and DNA recombination/repair, which may reflect adaptations to marine environmental challenges .

As a membrane protein, BPUM_0927 might be particularly important for maintaining cellular homeostasis in marine environments, potentially through osmoregulation, specialized transport, or membrane integrity maintenance under high-pressure or high-salinity conditions.

What are the recommended approaches for studying BPUM_0927 membrane integration and topology?

As a membrane protein, determining BPUM_0927's integration pattern and topology is essential for functional understanding. The following methodological approaches are recommended:

• Computational topology prediction:

  • Apply multiple prediction algorithms (TMHMM, HMMTOP, Phobius)

  • Generate consensus topology models highlighting transmembrane segments

  • Predict orientation relative to membrane (cytoplasmic vs. extracellular domains)

  • Identify potential functional motifs within predicted domains

• Experimental topology mapping:

  • Cysteine scanning mutagenesis with membrane-impermeable thiol reagents

  • Reporter fusion approach (PhoA/LacZ dual reporters at various positions)

  • Protease protection assays with reconstituted protein

  • Site-directed fluorescence labeling combined with quenching studies

• Advanced structural approaches:

  • Cryo-electron microscopy of membrane-reconstituted protein

  • Site-specific crosslinking to constrain conformational possibilities

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

  • Solid-state NMR studies of reconstituted protein

• In vivo validation approaches:

  • Epitope tagging at predicted loop regions

  • Fluorescent protein insertions at terminal domains

  • FRET-based distance measurements between domains

These approaches should be applied in combination, as each method has limitations when used in isolation. Integrating computational predictions with multiple experimental validation techniques provides the most reliable topology model. For membrane proteins like BPUM_0927, determining topology is a crucial first step toward understanding function, as it reveals which domains interact with different cellular compartments and identifies potential functional sites.

How can researchers integrate multi-omics approaches to elucidate BPUM_0927 function in the context of B. pumilus biology?

A comprehensive multi-omics strategy offers powerful insights into BPUM_0927 function within the broader biological context of B. pumilus. The recommended integrated approach includes:

• Comparative genomics foundation:

  • Analyze BPUM_0927 conservation, variation, and genomic context across B. pumilus strains

  • Identify co-evolving genes that might function in related pathways

  • Map genetic variations to potential functional domains

• Transcriptomics layer:

  • Perform RNA-seq under diverse conditions (environmental stresses, growth phases)

  • Identify co-expressed gene clusters containing BPUM_0927

  • Generate condition-specific expression profiles for regulatory insights

• Proteomics dimension:

  • Apply quantitative proteomics to BPUM_0927 wild-type and knockout strains

  • Perform membrane proteome analysis to identify interaction partners

  • Use phosphoproteomics to detect potential regulatory modifications

• Metabolomics component:

  • Compare metabolite profiles between wild-type and BPUM_0927 mutants

  • Focus on membrane-associated metabolites and lipid composition

  • Track metabolic shifts under conditions where BPUM_0927 is highly expressed

• Data integration framework:

  • Apply network analysis to identify functional modules involving BPUM_0927

  • Use machine learning approaches to predict functional relationships

  • Develop testable hypotheses based on integrated data patterns

This integrated approach has proven valuable in characterizing other B. pumilus proteins. For example, multi-omics studies of oxidative stress response revealed that B. pumilus induces bacillithiol-related genes and maintains high intracellular levels of this protective metabolite during peroxide stress . Similar approaches applied to BPUM_0927 would place it within its broader biological context and generate specific functional hypotheses for targeted validation.