Recombinant Bacillus subtilis Uncharacterized membrane protein ywzB (ywzB)

What is Bacillus subtilis and why is it important for biological research?

Bacillus subtilis is a Gram-positive, rod-shaped bacterium commonly found in soil and the gastrointestinal tract of ruminants, humans, and marine sponges. It is considered the best-studied Gram-positive bacterium and serves as a model organism for studying bacterial chromosome replication and cell differentiation . B. subtilis is often regarded as the Gram-positive equivalent of Escherichia coli in terms of laboratory popularity and has become an essential tool in both fundamental and applied research. Its genetic tractability and ease of culture have made it particularly valuable for investigating endospore formation, asymmetric cell division, biofilm formation, and multicellular behavior. Beyond basic research, B. subtilis has significant applications in probiotics for plants and animals, industrial protein production, and as a foundational organism for biotechnology applications .

What are uncharacterized membrane proteins in B. subtilis and why should researchers study them?

Uncharacterized membrane proteins like ywzB represent proteins identified in the B. subtilis genome through sequencing and annotation but whose specific functions remain unknown. These proteins constitute a significant portion of the B. subtilis proteome and represent an untapped resource for discovering novel biological mechanisms. Studying these proteins is crucial for completing our understanding of bacterial physiology, identifying new drug targets, and discovering novel biotechnological applications. Membrane proteins are particularly significant as they mediate interactions between the cell and its environment, often playing critical roles in signaling, transport, and cellular homeostasis. Their location at the interface between the cell and its surroundings makes them excellent candidates for sensing environmental changes, transporting molecules, and participating in cell-cell interactions. For researchers, uncharacterized membrane proteins offer opportunities to discover entirely new biological pathways and mechanisms unique to Gram-positive bacteria.

How does genetic code expansion enhance the study of membrane proteins in B. subtilis?

Genetic code expansion (GCE) represents a powerful approach for studying membrane proteins in B. subtilis. This technique allows for the incorporation of non-standard amino acids (nsAAs) with specialized chemical properties into proteins. According to recent research, B. subtilis can efficiently incorporate at least 20 distinct nsAAs using three different families of aminoacyl-tRNA synthetase systems . This capability enables several advanced experimental approaches for membrane protein research:

The incorporation of nsAAs allows for click-labeling, photo-crosslinking, and translational titration, providing precise tools to study protein structure, interactions, and function . Photo-crosslinking nsAAs are particularly valuable for capturing transient protein-protein interactions involving membrane proteins, which are traditionally difficult to study. Additionally, nsAAs can serve as spectroscopic probes, fluorescent tags, or provide chemical handles for specific modifications.

Importantly, B. subtilis shows efficient incorporation of nsAAs at amber stop codons in native genes, which differs from the behavior observed in non-recoded E. coli strains . This characteristic makes B. subtilis potentially advantageous for certain genetic code expansion applications, especially when studying membrane proteins in their native context.

What expression systems are most effective for recombinant membrane protein production in B. subtilis?

For optimal expression of recombinant membrane proteins like ywzB in B. subtilis, researchers should consider several specialized expression systems that have been optimized for membrane proteins. A critical consideration is the selection of appropriate vectors and promoters that allow controlled expression to prevent toxicity or aggregation issues common with membrane proteins. The following table summarizes effective expression systems for B. subtilis membrane proteins:

B. subtilis offers several advantages as an expression host for its own membrane proteins, including compatibility with the protein's native membrane environment, well-developed genetic tools, and high secretion capacity . For membrane proteins specifically, it's crucial to optimize induction timing, temperature, and media composition to enhance proper folding and membrane insertion. Incorporation of affinity tags (such as His-tag or Strep-tag) facilitates purification while appropriate signal sequences may be necessary if the protein contains extracytoplasmic domains. When expressing ywzB, researchers should be particularly attentive to its hydrophobicity profile to ensure proper membrane targeting and insertion.

What are the most effective purification strategies for B. subtilis membrane proteins?

Purification of membrane proteins from B. subtilis requires specialized strategies to maintain protein structure and function throughout the isolation process. A systematic approach involves several critical steps:

First, cell lysis and membrane isolation must be optimized specifically for B. subtilis. While mechanical disruption methods like French press or sonication are effective, enzymatic lysis using lysozyme is particularly efficient for B. subtilis due to its cell wall composition. Following lysis, differential centrifugation is used to isolate membrane fractions containing the target protein.

Membrane protein solubilization represents the most critical step, requiring careful selection of detergents. Common choices include n-dodecyl-β-D-maltoside (DDM), lauryl maltose neopentyl glycol (LMNG), or CHAPS. The optimal detergent concentration, pH, and ionic strength must be determined empirically for each membrane protein, including ywzB. Addition of stabilizing agents such as glycerol or specific lipids can enhance protein stability during extraction.

For affinity purification, C-terminal tags are generally preferred for membrane proteins to avoid interfering with membrane insertion signals. His-tag purification using Ni-NTA or TALON resins is commonly employed, though Strep-tag or FLAG-tag systems may provide higher purity in some cases. Following initial purification, size exclusion chromatography helps remove aggregates and assess protein homogeneity, while ion exchange chromatography can provide additional purification if needed.

How can non-standard amino acids be effectively incorporated into ywzB for functional studies?

The incorporation of non-standard amino acids (nsAAs) into membrane proteins like ywzB provides powerful tools for functional characterization. Recent research has demonstrated successful incorporation of up to 20 different nsAAs in B. subtilis using three major aminoacyl-tRNA synthetase/tRNA pair systems :

• MjTyrRS variants (derived from Methanococcus jannaschii tyrosyl-tRNA synthetase)

• ScWRS (Saccharomyces cerevisiae tryptophan synthetase)

• MaPylRS (Methanomethylophilus alvus pyrrolysine synthetase)

To incorporate nsAAs into ywzB, researchers should first identify the specific positions where nsAA incorporation would provide functional insights. For membrane proteins, positions at predicted protein-protein interaction interfaces, within transmembrane domains, or at sites of potential post-translational modifications are particularly informative. The amber stop codon (UAG) is typically used as the reassignment codon for nsAA incorporation in B. subtilis.

The experimental procedure involves genomic integration of the selected aminoacyl-tRNA synthetase/tRNA pair, modification of the ywzB gene to include amber codons at desired positions, and supplementation of the growth medium with the appropriate nsAA. The efficiency of nsAA incorporation in B. subtilis appears to be higher than in non-recoded E. coli strains, with efficient incorporation observed at amber stop codons in native B. subtilis genes .

For membrane proteins specifically, nsAAs enabling photo-crosslinking (such as p-benzoyl-L-phenylalanine) are particularly valuable for capturing transient interaction partners. Additionally, nsAAs containing bioorthogonal handles allow for specific labeling with fluorophores or other probes for localization and trafficking studies.

What approaches are most effective for determining the function of uncharacterized membrane proteins like ywzB?

Determining the function of uncharacterized membrane proteins requires a multi-faceted approach combining computational prediction, genetic manipulation, and biochemical characterization. For proteins like ywzB, researchers should implement a systematic workflow:

Begin with comprehensive bioinformatic analysis, including sequence homology searches, structural prediction using tools like AlphaFold2, and analysis of genomic context. For membrane proteins, transmembrane topology prediction is essential to understand membrane orientation. Gene neighborhood analysis may reveal functional relationships with neighboring genes that could suggest potential functions.

Genetic approaches provide crucial functional insights. Creating clean deletion mutants allows observation of phenotypic changes that may indicate the protein's role. For potentially essential genes, conditional expression systems or CRISPRi-based depletion should be employed. Complementation studies with mutated versions can pinpoint critical residues for function.

Localization studies using fluorescent protein fusions or immunofluorescence microscopy reveal the protein's subcellular distribution, which often correlates with function. For membrane proteins, determining whether they localize to specific membrane microdomains can be particularly informative. Fractionation studies confirm membrane association and topology.

Protein-protein interaction studies using techniques like bacterial two-hybrid systems, co-immunoprecipitation, or photo-crosslinking with non-standard amino acids can identify interaction partners that may suggest function . For membrane proteins specifically, photo-crosslinking using nsAAs has proven particularly effective in B. subtilis .

How can researchers effectively study protein-protein interactions involving membrane proteins in B. subtilis?

Studying protein-protein interactions involving membrane proteins presents unique challenges due to their hydrophobic nature and membrane environment. For B. subtilis membrane proteins like ywzB, several specialized approaches have proven effective:

Photo-crosslinking using non-standard amino acids represents one of the most powerful approaches for capturing membrane protein interactions in their native environment. B. subtilis has been shown to efficiently incorporate photo-reactive nsAAs like p-benzoyl-L-phenylalanine (pBpa) or p-azido-L-phenylalanine (pAzF), which form covalent bonds with nearby molecules upon UV irradiation . This technique allows for the capture of even transient or weak interactions that might be lost during traditional co-immunoprecipitation experiments.

For a comprehensive interaction analysis, implementing proximity-based labeling approaches like BioID or APEX2 provides an alternative strategy. These methods involve fusing the enzyme to the membrane protein of interest, which then biotinylates proximal proteins that can be identified by streptavidin pulldown and mass spectrometry. While primarily developed in eukaryotic systems, these approaches have been adapted for bacterial systems including B. subtilis.

Split-protein complementation assays modified for membrane proteins, such as the bacterial adenylate cyclase two-hybrid (BACTH) system, offer another approach. These systems have been optimized to accommodate membrane topology constraints and can detect interactions in vivo.

Computational approaches can complement experimental methods, including co-evolution analysis to identify potentially interacting partners based on correlated mutations across species, and structural modeling to predict interaction interfaces using tools like AlphaFold-Multimer.

What methodologies can be used to determine the structure of membrane proteins like ywzB?

Determining the structure of membrane proteins remains challenging but several complementary approaches can be employed for proteins like ywzB:

X-ray crystallography continues to be a powerful technique for membrane protein structure determination, though it requires optimization of detergent conditions, crystallization parameters, and often the use of specialized approaches like lipidic cubic phase crystallization. For B. subtilis membrane proteins specifically, identifying stabilizing lipids from the native membrane may enhance crystallization success.

Cryo-electron microscopy (cryo-EM) has revolutionized membrane protein structural biology by eliminating the need for crystallization. Single-particle cryo-EM is particularly effective for larger membrane proteins or complexes (typically >100 kDa), while advances in microscopy and detection now enable structure determination of increasingly smaller proteins. For smaller membrane proteins like ywzB, incorporating it into a larger scaffold such as an antibody complex may facilitate analysis.

Nuclear magnetic resonance (NMR) spectroscopy provides an alternative for smaller membrane proteins or specific domains. Solution NMR typically works for proteins <25 kDa, while solid-state NMR can accommodate larger structures. NMR also offers the advantage of studying protein dynamics and conformational changes.

Computational structure prediction has advanced significantly with tools like AlphaFold2 and RoseTTAFold now capable of producing remarkably accurate predictions even for membrane proteins. These predictions can guide experimental design and provide structural insights when experimental structures are not available.

Hybrid approaches combining low-resolution experimental data (such as crosslinking constraints, EPR measurements, or hydrogen-deuterium exchange) with computational modeling often provide the most comprehensive structural understanding.

How can genetic code expansion be leveraged to study membrane protein dynamics in B. subtilis?

Genetic code expansion offers unique capabilities for studying membrane protein dynamics by enabling site-specific incorporation of non-standard amino acids (nsAAs) with specialized properties. For B. subtilis membrane proteins like ywzB, several approaches have proven particularly valuable:

Fluorescent nsAAs provide a minimally perturbative way to monitor protein dynamics compared to bulky fluorescent protein fusions. Amino acids like L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap) can be directly incorporated into specific positions within ywzB, allowing for precise tracking of conformational changes through changes in fluorescence properties. This approach is especially valuable for membrane proteins where traditional fluorescent protein tags may disrupt membrane insertion or function.

Environmentally sensitive probes incorporated via nsAAs can report on local microenvironment changes during protein conformational shifts. These probes exhibit altered fluorescence properties based on solvent polarity, making them ideal for monitoring transitions between hydrophobic (membrane-embedded) and hydrophilic environments during protein function.

Photoswitchable or photocaged nsAAs enable temporal control over protein function, allowing researchers to activate or inactivate specific protein domains with light pulses. This capability permits precise investigation of rapid conformational changes or signaling events involving membrane proteins like ywzB.

The ability to incorporate bioorthogonal reactive groups (azides, alkynes) through nsAAs facilitates selective labeling of the target protein with minimal background. This approach permits pulse-chase experiments to track protein turnover, trafficking, and localization dynamics with high temporal resolution.

Based on recent research, B. subtilis shows efficient incorporation of nsAAs at amber stop codons in native genes , suggesting that these approaches can be readily implemented for studying membrane protein dynamics in this organism.

How should researchers analyze proteomics data for membrane proteins like ywzB?

Analysis of proteomics data for membrane proteins requires specialized approaches to address the unique challenges these hydrophobic proteins present. For B. subtilis membrane proteins like ywzB, researchers should implement the following strategies:

During database searching, special consideration must be given to membrane protein-specific issues. These include adjusting digestion enzyme specificity parameters, as membrane-embedded regions often display incomplete digestion with trypsin. Chymotrypsin or other alternative proteases may provide better coverage of hydrophobic domains. Search parameters should account for potential post-translational modifications common in membrane proteins, and higher mass tolerance settings may be necessary for modified peptides.

Validation of membrane protein identifications requires stringent criteria. For low-abundance proteins like many uncharacterized membrane proteins, manual verification of MS/MS spectra for key peptides is essential. Requiring multiple peptides for protein identification increases confidence, and statistical validation using target-decoy approaches helps establish false discovery rates. Comparison across technical and biological replicates can further validate findings.

Quantitative analysis of membrane proteins presents unique challenges. Normalization strategies must account for extraction biases that often affect hydrophobic proteins differently. While label-free quantification is commonly used, isotopic labeling approaches like SILAC or TMT can provide more accurate quantification, especially for comparative studies involving ywzB under different conditions.

Topology analysis based on proteomic data can provide insights into membrane protein orientation. This includes accessibility-based methods where surface-exposed regions show different labeling patterns compared to membrane-embedded domains. Integration of proteomics data with predicted transmembrane topology models enhances structural understanding.

What bioinformatic tools and databases are most valuable for predicting the function of uncharacterized membrane proteins?

Bioinformatic analysis provides crucial insights for uncharacterized membrane proteins like ywzB through a combination of specialized tools and databases:

For sequence-based analysis, sensitive homology detection tools like HHpred, which uses hidden Markov models, often identify distant relationships missed by standard BLAST searches. Position-specific scoring matrices through PSI-BLAST can similarly detect remote homologs that might suggest functional similarities. Domain identification using databases like Pfam, SMART, or the Conserved Domain Database helps identify functional modules within the protein sequence. For membrane proteins specifically, tools like TMHMM, HMMTOP, or Phobius predict transmembrane topology, which is essential for understanding protein orientation and identifying potential functional regions.

Structural prediction has advanced significantly with AlphaFold2 and RoseTTAFold now capable of generating highly accurate structural models even for membrane proteins. These predictions can reveal potential binding pockets, interaction surfaces, and structural motifs associated with specific functions. For membrane proteins like ywzB, structural models should be analyzed in the context of the membrane environment, considering how specific regions interact with lipids versus aqueous environments.

Genomic context analysis provides functional clues through examination of neighboring genes, which often have related functions. Tools like STRING, GeConT, or DOOR² help visualize and analyze gene neighborhood conservation across species. This approach is particularly powerful when combined with expression correlation data, which identifies genes with similar expression patterns that may function in the same pathway.

Specialized databases including SubtiWiki (specific to B. subtilis), TransportDB (for membrane transporters), and TCDB (Transporter Classification Database) provide curated information highly relevant to membrane protein function prediction. These resources often include experimental data and annotations not available in more general databases.

How can researchers validate computational predictions for membrane protein function?

Validating computational predictions for uncharacterized membrane proteins requires a systematic experimental approach that builds from simple to more complex methodologies:

The foundation of validation begins with targeted gene knockout or depletion studies. For non-essential genes like many uncharacterized membrane proteins, clean deletion mutants can reveal phenotypic changes indicating the protein's role. For potentially essential genes, conditional expression systems or CRISPRi-based depletion provide alternatives. These genetic approaches should examine multiple phenotypes including growth under various conditions, morphology, and specific functional assays suggested by computational predictions.

Localization and expression studies provide crucial contextual information. Fluorescent protein fusions or immunolocalization reveal subcellular distribution patterns that often correlate with function. Quantitative analysis of expression under different conditions using RT-qPCR or proteomics can identify regulatory patterns that suggest function. For membrane proteins specifically, determining whether they localize to particular membrane domains can be particularly informative.

Biochemical characterization should follow to directly test predicted functions. If a transport function is predicted, researchers should conduct transport assays using purified protein reconstituted in liposomes or membrane vesicles. For predicted enzymatic activities, in vitro assays with purified protein can test specific substrate interactions. Binding assays can validate predicted interactions with other proteins, small molecules, or nucleic acids.

Site-directed mutagenesis targeting predicted functional residues provides powerful validation. If computational models suggest specific amino acids involved in substrate binding or catalysis, mutating these residues should alter the protein's function in predictable ways. This approach is particularly valuable when combined with structural information, whether predicted through AlphaFold2 or determined experimentally.

What strategies can researchers use to overcome expression and purification difficulties with membrane proteins?

Membrane protein expression and purification present numerous challenges that researchers must systematically address. For recombinant B. subtilis membrane proteins like ywzB, several strategies have proven effective:

When facing low expression levels, a methodical optimization approach is essential. Testing multiple promoter systems with different induction strengths and kinetics can identify optimal expression conditions. The search results indicate that B. subtilis has "a broad range of available genetic tools including inducible promoters and protein tags" that can be leveraged for membrane protein expression. Modulating growth temperature significantly impacts membrane protein folding, with lower temperatures (20-25°C) often improving proper folding by slowing the production rate. Optimizing media composition, particularly osmolytes and ion concentrations, can enhance membrane protein stability during expression.

For proteins that exhibit toxicity when overexpressed, implementing tight expression control through precisely regulated promoter systems is crucial. Leaky expression can be minimized using repressor titration or dual-control systems. Fusion partners that enhance folding or reduce toxicity, such as MBP or Mistic, have proven beneficial for challenging membrane proteins. The co-expression of chaperones specific to membrane proteins can also improve folding and reduce toxicity.

Purification difficulties often stem from inefficient solubilization or instability in detergent solutions. Systematic screening of detergents is essential, progressing from mild (DDM, LMNG) to more aggressive options (SDS, sarkosyl) if necessary. Detergent mixtures sometimes perform better than individual detergents. Adding specific lipids from the native B. subtilis membrane during purification can significantly enhance stability. For proteins that remain challenging, alternative solubilization approaches such as styrene-maleic acid lipid particles (SMALPs) or native nanodiscs can maintain a more native-like lipid environment.

How can researchers address inconsistent or contradictory results when characterizing uncharacterized membrane proteins?

When facing inconsistent or contradictory results during the characterization of membrane proteins like ywzB, researchers should implement a systematic troubleshooting approach:

First, conduct a comprehensive analysis of experimental variables across different studies or experiments. Differences in expression systems, tags, purification methods, or buffer conditions can dramatically affect membrane protein behavior. The lipid environment is particularly critical for membrane proteins, so variations in membrane composition between experiments may explain contradictory results. Temperature, pH, and ionic strength can also significantly impact membrane protein structure and function.

Consider protein heterogeneity as a source of contradictions. Membrane proteins often exist in multiple conformational states or oligomeric forms that may have different functional properties. Post-translational modifications can also vary between expression systems or growth conditions, affecting function. Advanced techniques like native mass spectrometry or analytical ultracentrifugation can assess protein homogeneity and identify different populations that might explain contradictory results.

Biological context matters significantly for membrane proteins. The function of ywzB might differ depending on growth phase, stress conditions, or interaction partners present in different experimental setups. Testing the protein's behavior across a range of physiologically relevant conditions may resolve apparent contradictions by revealing condition-specific functions.

What quality control measures should be implemented when working with recombinant membrane proteins?

During expression and purification, multiple analytical techniques should be employed to assess protein quality. Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) provides information about both size and homogeneity, critical for detecting aggregation or mixed oligomeric states common in membrane proteins. Circular dichroism spectroscopy verifies proper secondary structure formation, particularly important for helical membrane proteins. Thermal stability assays using differential scanning fluorimetry or nanoDSF help optimize buffer conditions and identify stabilizing ligands.

Functional verification is essential even before detailed characterization begins. For proteins with predicted activities, simple activity assays confirm that the purified protein is functionally active. Ligand binding assays using techniques like isothermal titration calorimetry or microscale thermophoresis verify that the protein retains its binding capabilities. For membrane proteins specifically, reconstitution into liposomes or nanodiscs followed by functional testing provides a more native-like environment for quality assessment.

Structural integrity verification using limited proteolysis helps confirm proper folding, as well-folded membrane proteins typically show distinct proteolytic patterns with protease-resistant core domains. Native gel electrophoresis assesses oligomeric state homogeneity, while negative stain electron microscopy provides visual confirmation of sample quality and homogeneity. For more detailed structural assessment, hydrogen-deuterium exchange mass spectrometry can probe protein dynamics and identify potentially misfolded regions.

Batch-to-batch consistency testing is particularly important for membrane proteins, which can be sensitive to minor variations in purification procedures. Establishing quantitative benchmarks for purity, activity, and stability facilitates comparison between preparations and ensures experimental reproducibility.

How might research on uncharacterized B. subtilis membrane proteins contribute to biotechnology applications?

Research on uncharacterized membrane proteins like ywzB has significant potential to advance biotechnology applications through several promising avenues:

In biocontrol applications, B. subtilis is already recognized as an effective biological control agent against plant pathogens such as Fusarium wilt of cucumber . Uncharacterized membrane proteins may play roles in the mechanisms underlying this antagonistic activity, including production of antimicrobial compounds, competition for nutrients, or direct interaction with pathogens. Characterizing these proteins could lead to engineered B. subtilis strains with enhanced biocontrol capabilities through optimized expression of key membrane proteins involved in these processes.

For protein production systems, B. subtilis is widely used in industrial settings due to its high secretion capacity and GRAS (Generally Recognized As Safe) status . Membrane proteins involved in protein translocation, quality control, or stress response could be manipulated to enhance the yield and quality of recombinant proteins. The genetic code expansion capabilities demonstrated in B. subtilis provide powerful tools for engineering these systems, as they allow incorporation of non-standard amino acids with specialized chemical properties .

Biosensor development represents another promising application area. Membrane proteins often function as sensors for environmental conditions, nutrients, or signaling molecules. Uncharacterized membrane proteins like ywzB may have binding capabilities for specific analytes that could be harnessed in whole-cell biosensors or incorporated into synthetic detector systems. The photo-crosslinking capabilities enabled by genetic code expansion in B. subtilis could facilitate identification of molecules that interact with these membrane proteins.

Synthetic biology applications benefit greatly from well-characterized membrane protein components. As researchers uncover the functions of proteins like ywzB, these proteins can be incorporated into synthetic circuits as input sensors, signal processors, or output actuators. The genetic tractability of B. subtilis and its expanding molecular toolkit make it an excellent chassis for these applications.

What emerging technologies are likely to accelerate characterization of uncharacterized membrane proteins?

Several cutting-edge technologies are poised to revolutionize the characterization of uncharacterized membrane proteins like ywzB in the coming years:

Cryo-electron microscopy (cryo-EM) continues to advance rapidly, with improvements in detectors, sample preparation, and image processing now enabling structure determination of increasingly smaller proteins at near-atomic resolution. These developments will make structural studies of membrane proteins more accessible without the need for crystallization. For B. subtilis membrane proteins specifically, cryo-EM tomography of intact cells can reveal the native context and organization of membrane protein complexes.

AI-powered structural prediction has already transformed protein research with tools like AlphaFold2 and RoseTTAFold generating remarkably accurate models even for membrane proteins. Future developments will likely improve prediction of protein complexes, protein-ligand interactions, and conformational dynamics, providing powerful starting points for functional hypotheses about uncharacterized proteins like ywzB.

Single-molecule techniques offer unique insights into membrane protein dynamics and heterogeneity. Advanced methods like single-molecule FRET, high-speed AFM, and single-particle tracking can reveal conformational changes, oligomerization states, and localization patterns that bulk measurements miss. The genetic code expansion capabilities demonstrated in B. subtilis facilitate site-specific labeling for these techniques.

Spatial transcriptomics and proteomics approaches are beginning to provide unprecedented insights into the subcellular localization and context-dependent expression of proteins. For membrane proteins like ywzB, these techniques can reveal co-localization with functional partners and condition-specific expression patterns that suggest function. As these technologies become more sensitive, they will increasingly capture low-abundance membrane proteins that are currently challenging to detect.

High-throughput functional screening methods using CRISPR-based approaches enable systematic testing of phenotypes associated with membrane protein mutations or expression changes. The successful implementation of CRISPRi in B. subtilis provides a foundation for these approaches, which can rapidly narrow down potential functions for uncharacterized proteins.