Recombinant Bacillus amyloliquefaciens UPF0316 protein RBAM_006820 (RBAM_006820)

How is recombinant RBAM_006820 typically expressed and purified?

Recombinant RBAM_006820 is typically expressed in E. coli expression systems with an N-terminal His-tag to facilitate purification. The recommended purification protocol involves:

• Bacterial cell lysis (sonication or French press)

• Initial purification using nickel affinity chromatography

• Further purification steps including ion-exchange chromatography using DEAE-Sepharose

• Final polishing via size exclusion chromatography using Sephacryl columns

For optimal results, the protein is often eluted using a linear gradient of 0-1 M NaCl in Tris-HCl buffer (pH 8.0) . After purification, the protein is typically lyophilized and stored with 6% trehalose in Tris/PBS-based buffer at pH 8.0. For reconstitution, researchers should centrifuge the vial before opening and resuspend the protein in deionized sterile water to 0.1-1.0 mg/mL, with addition of 5-50% glycerol for long-term storage at -20°C/-80°C .

What are the optimal storage conditions for purified RBAM_006820?

For maintaining RBAM_006820 stability and activity, the following storage parameters are recommended:

For optimal results, use freshly prepared working aliquots whenever possible and avoid more than 2-3 freeze-thaw cycles . Assessment of protein stability can be performed using circular dichroism spectroscopy at regular intervals during storage.

What molecular techniques can be used to investigate RBAM_006820's function in Bacillus amyloliquefaciens?

Several molecular approaches can be employed to elucidate RBAM_006820's function:

• Gene Knockout Studies: CRISPR-Cas9 or homologous recombination techniques specifically targeting the RBAM_006820 gene, followed by phenotypic characterization of the knockout strain, can reveal the protein's physiological role.

• Transposon Mutagenesis: Similar to approaches used for identifying germination factors in Bacillus subtilis, transposon sequencing can help identify genetic interactions with RBAM_006820 .

• Fluorescent Protein Tagging: Fusion of RBAM_006820 with fluorescent proteins (GFP, YFP) can help determine subcellular localization, as demonstrated with other Bacillus proteins like GerY .

• Transcriptomic Analysis: RNA-seq comparing wild-type and RBAM_006820 mutant strains under various conditions can identify gene expression patterns affected by this protein.

• Pull-down Assays: Using the His-tagged recombinant protein to identify interaction partners through affinity purification followed by mass spectrometry.

These approaches can be complemented with comparative genomics analyses across different Bacillus species to provide evolutionary context to functional findings .

How can researchers optimize heterologous expression of RBAM_006820 for structural studies?

Optimizing RBAM_006820 expression for structural studies requires systematic methodology:

• Expression System Selection:

  • Bacterial: E. coli BL21(DE3), Rosetta, or C41/C43 for membrane proteins

  • Eukaryotic: Pichia pastoris for complex folding requirements

• Expression Vector Optimization:

  • Codon optimization for the host organism

  • Selection of appropriate promoters (T7, tac, or arabinose-inducible)

  • Inclusion of solubility tags (MBP, SUMO) in addition to His-tag

  • Incorporation of precision protease cleavage sites

• Expression Condition Screening:

  Parameter	Variables to Test	Monitoring Method
  Temperature	16°C, 25°C, 30°C, 37°C	SDS-PAGE, Western blot
  Induction timing	Early, mid, late log phase	OD600 measurement
  Inducer concentration	0.1-1.0 mM IPTG	Yield quantification
  Media composition	LB, TB, M9, auto-induction	Cell density and protein yield
  Additives	Glycylglycine, ethanol, sorbitol	Solubility assessment

• Solubilization and Purification Optimization:

  • Screening of detergents for membrane protein extraction

  • Testing various buffer conditions for stability

  • Implementing on-column refolding strategies if needed

• Quality Assessment:

  • Size-exclusion chromatography profiles

  • Thermal shift assays

  • Limited proteolysis to identify stable domains

For crystallography purposes, surface entropy reduction (SER) approaches may be employed to create point mutations that reduce surface entropy and promote crystal formation .

What is known about the potential membrane association of RBAM_006820 and how can this be experimentally verified?

The amino acid sequence of RBAM_006820 suggests it may be a membrane-associated protein due to its hydrophobic regions. To experimentally verify this:

• Computational Prediction:

  • Use transmembrane prediction algorithms (TMHMM, Phobius, HMMTOP)

  • Hydropathy plot analysis using Kyte-Doolittle scale

  • Signal peptide prediction with SignalP

• Biochemical Fractionation:

  • Cell fractionation to separate cytoplasmic, membrane, and periplasmic fractions

  • Western blot analysis of fractions using anti-His antibodies

  • Carbonate extraction to distinguish peripheral from integral membrane proteins

• Fluorescence Microscopy:

  • Create GFP fusions and observe localization in vivo

  • Co-localization with known membrane markers

  • FRAP (Fluorescence Recovery After Photobleaching) analysis to measure mobility

• Biophysical Characterization:

  • Circular dichroism to assess secondary structure in different environments

  • Liposome binding assays

  • Detergent solubility screening

• Protease Accessibility:

  • Limited proteolysis of intact cells vs. membrane preparations

  • Mass spectrometry identification of exposed regions

These approaches would provide complementary evidence regarding the protein's membrane association, topology, and orientation—critical information for understanding its function in the bacterial cell .

How conserved is RBAM_006820 across Bacillus species and what does this suggest about its function?

RBAM_006820 belongs to the UPF0316 protein family, which shows variable conservation across Bacillus species. A thorough comparative analysis would involve:

• Sequence Alignment Analysis:

  • Multi-sequence alignment of homologs from various Bacillus species

  • Identification of conserved domains and residues

  • Calculation of identity/similarity percentages

  • Construction of phylogenetic trees to understand evolutionary relationships

• Genomic Context Analysis:

  • Examination of neighboring genes across species

  • Identification of conserved operons or gene clusters

  • Analysis of promoter regions and regulatory elements

• Structural Comparison:

  • Homology modeling based on known structures of family members

  • Structural alignment to identify conserved folding patterns

  • Analysis of conserved surface patches that might indicate interaction sites

The level of conservation across species provides insights into functional importance—highly conserved proteins typically perform essential functions, while less conserved ones may have species-specific roles. The UPF0316 family's distribution pattern across Bacillus species suggests potential roles in membrane-related functions that may have diverged during evolution to meet species-specific requirements .

How does RBAM_006820 compare to similar proteins in the extensively studied Bacillus subtilis?

Bacillus subtilis is a model organism with well-characterized proteins. Comparative analysis of RBAM_006820 with its B. subtilis homologs reveals:

• Sequence and Structural Homology:

  • RBAM_006820 shows significant sequence similarity to several B. subtilis membrane proteins

  • The protein architecture follows patterns similar to proteins involved in stress response and spore formation in B. subtilis

• Functional Context:

  • In B. subtilis, homologous UPF0316 family proteins have been implicated in membrane integrity maintenance

  • Some homologs may participate in processes similar to the spore germination pathway, based on expression patterns

  • The protein may have functions analogous to certain coat proteins described in B. subtilis, such as those involved in nutrient sensing during germination

• Expression Pattern Differences:

  • Expression timing during growth and sporulation phases may differ between species

  • Stress-response regulatory mechanisms controlling expression may vary

• Potential Functional Divergence:

  • While core domains remain conserved, species-specific adaptations may exist

  • B. amyloliquefaciens' industrial and agricultural applications may have driven functional specialization

The wealth of genetic and biochemical data available for B. subtilis proteins provides a valuable framework for designing experiments to characterize RBAM_006820's function in B. amyloliquefaciens .

What are the recommended methods for investigating RBAM_006820's role in stress response?

To comprehensively investigate RBAM_006820's potential role in stress response:

• Gene Expression Analysis:

  • qRT-PCR to measure RBAM_006820 expression under various stressors

  • RNA-seq to identify co-regulated genes in stress conditions

  • Promoter-reporter fusions to visualize expression patterns in real-time

• Phenotypic Characterization of Mutants:

  Stress Condition	Parameters to Measure	Methods
  Osmotic stress	Growth rate, viability	Growth curves, viable count
  Temperature stress	Survival at extreme temps	Heat/cold shock assays
  Oxidative stress	ROS sensitivity	H₂O₂ challenge assays
  pH stress	Growth at various pH	pH-controlled media
  Nutrient limitation	Survival during starvation	Long-term starvation assays

• Protein Interaction Network:

  • Pull-down assays under stress conditions

  • Bacterial two-hybrid screening

  • Cross-linking mass spectrometry to capture transient interactions

• Biochemical Activity Assessment:

  • Membrane integrity assays (fluorescent dye leakage)

  • Protein stability measurements at various stress conditions

  • Potential enzymatic activity characterization

• In vivo Localization Changes:

  • Fluorescence microscopy of GFP-tagged protein under stress

  • FRET-based interaction studies with known stress response proteins

These approaches would establish whether RBAM_006820 has direct involvement in stress response pathways or plays a supportive role in maintaining cellular functions during adverse conditions .

What experimental approaches can determine if RBAM_006820 has enzymatic activity?

Despite being designated as an uncharacterized protein (UPF0316), RBAM_006820 may possess enzymatic functions. A systematic approach to uncovering potential enzymatic activity includes:

• Sequence-based Prediction:

  • Motif scanning for known catalytic sites

  • Structural homology modeling to identify potential active sites

  • Analysis of conserved residues across homologs

• High-throughput Activity Screening:

  • Testing against libraries of potential substrates

  • Colorimetric or fluorometric assays for common enzymatic reactions

  • Mass spectrometry-based metabolomics to detect substrate conversion

• Targeted Biochemical Assays:

  • Based on predicted activities from computational analysis

  • Standard enzyme kinetics measurements (Km, Vmax, kcat)

  • Inhibition studies to confirm specificity

• Structural Approaches:

  • X-ray crystallography with potential substrates or substrate analogs

  • NMR studies to observe substrate binding

  • HDX-MS (Hydrogen-Deuterium Exchange Mass Spectrometry) to identify conformational changes upon substrate binding

• Genetic Complementation:

  • Expression in organisms lacking specific enzymatic functions

  • Rescue experiments in knockout strains

Given its membrane association, RBAM_006820 might function in membrane remodeling, transport, or signaling pathways. Enzymatic assays should be designed considering these potential roles, with particular attention to lipid substrates or membrane-associated processes .

How can researchers effectively study protein-protein interactions involving RBAM_006820?

Investigating the interaction partners of RBAM_006820 requires approaches suitable for potentially membrane-associated proteins:

• Affinity-based Methods:

  • His-tag pull-down assays using optimized detergent conditions

  • Tandem Affinity Purification (TAP) with dual tags

  • Co-immunoprecipitation with specific antibodies

  • Proximity-dependent biotin labeling (BioID, APEX)

• Genetic Interaction Screens:

  • Bacterial two-hybrid systems (e.g., BACTH specifically designed for membrane proteins)

  • Synthetic genetic arrays to identify functional relationships

  • Suppressor screening to identify compensatory mutations

• In vivo Visualization:

  • Bimolecular Fluorescence Complementation (BiFC)

  • Förster Resonance Energy Transfer (FRET) microscopy

  • Single-molecule tracking to identify co-localization

• Crosslinking Approaches:

  • Chemical crosslinking followed by mass spectrometry (XL-MS)

  • Photo-activatable crosslinkers for capturing transient interactions

  • In vivo crosslinking with formaldehyde or DSP

• Biophysical Characterization:

  Method	Application	Advantages
  Surface Plasmon Resonance	Binding kinetics	Real-time, label-free detection
  Microscale Thermophoresis	Binding affinity in solution	Low sample consumption
  Isothermal Titration Calorimetry	Thermodynamic parameters	Direct measurement of ΔH
  Native Mass Spectrometry	Complex composition	Preserves non-covalent interactions

For membrane proteins like RBAM_006820, detergent selection is critical—screening mild detergents (DDM, LMNG, digitonin) that maintain native interactions while solubilizing the protein complex is essential for successful interaction studies .

How can RBAM_006820 be leveraged in metabolic engineering of Bacillus strains?

RBAM_006820's potential role in membrane function makes it a candidate for metabolic engineering applications:

• As a Potential Target for Strain Improvement:

  • Modulation of expression levels may enhance membrane integrity under industrial fermentation conditions

  • Overexpression or regulated expression might improve tolerance to toxic metabolites

  • Engineering protein variants with enhanced stability could improve strain robustness

• As Part of Synthetic Biology Circuits:

  • Integration into engineered signaling pathways if the protein has sensing/signaling functions

  • Development of protein switches based on RBAM_006820 domains

  • Creation of chimeric proteins combining functional domains for novel applications

• Methodological Approach:

  • Gene dosage optimization through promoter engineering

  • Codon optimization for increased expression

  • Integration of regulatory elements for conditional expression

  • Site-directed mutagenesis to enhance desired properties

• Performance Evaluation Metrics:

  • Growth parameters under industrial conditions

  • Metabolite production titers, rates, and yields

  • Membrane integrity and stress resistance

  • Long-term strain stability

Bacillus amyloliquefaciens is already an important industrial organism for enzyme and metabolite production. Understanding and engineering RBAM_006820 could potentially enhance its value for industrial applications by improving robustness or productivity through membrane function optimization .

What role might RBAM_006820 play in the development of Bacillus-based biocontrol agents?

Bacillus amyloliquefaciens is recognized for its biocontrol properties, and RBAM_006820 might contribute to these functions:

• Potential Mechanisms:

  • If involved in membrane stability, it may affect the production or secretion of antimicrobial compounds

  • Could play a role in stress resistance, enhancing survival in agricultural environments

  • May participate in signaling pathways that regulate biocontrol compound production

• Research Approach:

  • Comparative analysis of RBAM_006820 expression in strains with varying biocontrol efficacy

  • Creation of knockout and overexpression strains for biocontrol assays

  • Co-expression with known biocontrol factors to identify synergistic effects

• Functional Validation:

  • In vitro antagonism assays against plant pathogens

  • Greenhouse trials measuring plant protection efficacy

  • Field studies evaluating performance under natural conditions

• Integration with Other Biocontrol Mechanisms:

  • Analysis of interaction with lipopeptide biosynthesis pathways

  • Investigation of potential roles in root colonization efficiency

  • Assessment of contribution to induced systemic resistance in plants

Bacillus amyloliquefaciens strains have been genetically modified to enhance their antifungal properties and lipopeptide production. Understanding RBAM_006820's function could provide new targets for genetic enhancement of biocontrol properties .

What are the main challenges in studying membrane-associated proteins like RBAM_006820 and how can they be addressed?

Membrane proteins present distinct research challenges:

• Expression and Purification Difficulties:

  • Challenge: Low expression yields and inclusion body formation

  • Solutions:

    • Use specialized expression strains (C41/C43, Lemo21)

    • Employ fusion tags that enhance solubility (MBP, SUMO)

    • Optimize induction conditions (lower temperature, reduced inducer)

    • Consider cell-free expression systems

• Structural Characterization Hurdles:

  • Challenge: Difficulty obtaining crystals for X-ray diffraction

  • Solutions:

    • Lipidic cubic phase crystallization

    • Cryo-electron microscopy for structure determination

    • NMR spectroscopy for dynamic regions

    • Hybrid approaches combining multiple structural techniques

• Functional Assay Development:

  • Challenge: Maintaining native-like environment for activity

  • Solutions:

    • Reconstitution into liposomes or nanodiscs

    • Detergent screening to identify optimal solubilization conditions

    • Development of cell-based assays that report on protein function

• Interaction Studies Complexity:

  • Challenge: Preserving weak or transient interactions during isolation

  • Solutions:

    • In situ crosslinking prior to solubilization

    • Proximity labeling techniques (BioID, APEX)

    • Native MS with optimized ionization conditions

By addressing these challenges with appropriate methodological adjustments, researchers can successfully characterize membrane proteins like RBAM_006820 and gain insights into their structure-function relationships .

How can bioinformatic approaches complement experimental studies of RBAM_006820?

Bioinformatics provides powerful tools to guide experimental research on RBAM_006820:

• Sequence Analysis and Annotation:

  • Identification of conserved domains and motifs

  • Secondary structure prediction

  • Transmembrane topology prediction

  • Signal peptide and subcellular localization prediction

• Structural Bioinformatics:

  • Homology modeling based on related structures

  • Molecular dynamics simulations to predict behavior in membrane environments

  • Identification of potential ligand binding sites

  • Protein-protein interaction interface prediction

• Comparative Genomics:

  • Synteny analysis across Bacillus genomes

  • Identification of co-evolved gene clusters

  • Phylogenetic profiling to predict functional relationships

  • Analysis of selection pressure on different protein regions

• Transcriptomic Data Integration:

  • Co-expression network analysis

  • Identification of regulatory elements in promoter regions

  • Expression pattern comparison across conditions and species

• Systems Biology Approaches:

  Approach	Application	Output
  Flux Balance Analysis	Metabolic context prediction	Predicted metabolic impact
  Protein-Protein Interaction Networks	Functional module identification	Interaction clusters
  Gene Ontology Enrichment	Functional inference	Statistically enriched functions
  Text Mining	Literature-based discovery	Previously unreported connections

These computational approaches generate testable hypotheses that can guide experimental design, potentially saving time and resources while increasing the likelihood of discovering the protein's true function .

What emerging technologies might advance our understanding of RBAM_006820?

Several cutting-edge technologies hold promise for deeper characterization of RBAM_006820:

• Advanced Structural Methods:

  • Micro-electron diffraction (MicroED) for small crystals

  • Integrative structural biology combining multiple data sources

  • Cryo-electron tomography for in situ visualization

  • Serial femtosecond crystallography using X-ray free-electron lasers

• Single-Cell and Single-Molecule Techniques:

  • Single-molecule FRET to observe conformational changes

  • Super-resolution microscopy for precise localization

  • Single-cell transcriptomics to capture expression heterogeneity

  • Patch-clamp techniques if transport functions are identified

• CRISPR-Based Technologies:

  • CRISPRi for tunable gene repression

  • CRISPRa for activation studies

  • Base editing for precise amino acid substitutions

  • CRISPR screening with focused libraries

• Synthetic Biology Approaches:

  • Minimal synthetic membrane systems

  • Orthogonal translation systems for non-canonical amino acid incorporation

  • Cell-free expression platforms optimized for membrane proteins

  • Bottom-up reconstitution of functional units

• Advanced Computational Methods:

  • AI-driven protein structure prediction (AlphaFold2, RoseTTAFold)

  • Quantum mechanics/molecular mechanics (QM/MM) for mechanistic studies

  • Deep learning for function prediction from sequence data

  • Enhanced sampling molecular dynamics simulations

These emerging technologies can address current gaps in our understanding of RBAM_006820's structure, dynamics, interactions, and functions within cellular contexts .

What potential applications might arise from a complete understanding of RBAM_006820 function?

A thorough characterization of RBAM_006820 could enable several downstream applications:

• Biotechnological Applications:

  • Development of robust expression hosts for industrial enzymes

  • Creation of biosensors based on protein domains

  • Design of membrane-engineering strategies for increased cell robustness

  • Novel biocatalysts if enzymatic functions are discovered

• Agricultural Applications:

  • Enhanced biocontrol strains with improved environmental fitness

  • Optimized biopesticide formulations

  • Plant growth-promoting strains with improved root colonization

  • Stress-resistant inoculants for challenging agricultural environments

• Basic Science Contributions:

  • Deeper understanding of bacterial membrane biology

  • Insights into protein evolution across Bacillus species

  • Novel protein-protein interaction networks

  • Potential discovery of new cellular processes

• Methodological Advances:

  • Optimized protocols for membrane protein research

  • Novel protein engineering approaches

  • Improved computational prediction pipelines for uncharacterized proteins

  • Innovative functional genomics strategies

The current trend toward metabolic engineering and synthetic biology applications of Bacillus amyloliquefaciens highlights the importance of fully characterizing components like RBAM_006820, which may serve as building blocks for future cell factories with enhanced production capabilities .