Recombinant Bacillus subtilis subsp. spizizenii Quinol oxidase subunit 2 (qoxA)

What is Bacillus subtilis subsp. spizizenii and how does it differ from other B. subtilis subspecies?

Bacillus subtilis subsp. spizizenii is one of four recognized subspecies within the B. subtilis group, alongside subsp. subtilis, subsp. inaquosorum, and subsp. stercoris. Recent genomic comparisons suggest these subspecies should be elevated to species status. The key differentiating factor between these subspecies is their production of distinct bioactive secondary metabolites. B. subtilis subsp. spizizenii specifically conserves genes for producing mycosubtilin, bacillaene, and 3,30-neotrehalosadiamine, which creates a unique metabolic profile compared to other subspecies . Phylogenetic analyses using 16S rRNA sequences have further supported the classification of B. spizizenii as a distinct group, with some evidence suggesting further subdivision into subspecies based on lanthipeptide structures and 16S rRNA gene organization .

What is quinol oxidase subunit 2 (qoxA) and what is its function in B. spizizenii?

Quinol oxidase subunit 2 (qoxA) is a membrane protein that functions as part of the respiratory chain in B. spizizenii. Its recommended name is Quinol oxidase subunit 2 with an EC number of 1.10.3.-. It is also known by alternative names including Oxidase aa(3)-600 subunit 2, Quinol oxidase aa3-600 subunit qoxA, and Quinol oxidase polypeptide II . The protein plays a crucial role in the terminal oxidase complex that transfers electrons from quinol to oxygen during aerobic respiration, contributing to the organism's energy metabolism. The protein is encoded by the qoxA gene (locus name BSUW23_18875) in the strain ATCC 23059/NRRL B-14472/W23 .

How is the B. spizizenii strain characterized taxonomically?

B. spizizenii strains can be taxonomically characterized through gene sequencing, particularly of the 16S rRNA encoding genes (rrn genes) and in the case of subtilin-producing strains, the subtilin encoding spaS gene . Several B. spizizenii strains have been identified and deposited in culture collections, including DSM 618, 1087, 6395, 6405, and 8439 from the German Collection of Microorganisms and Cell Cultures, as well as field-collected strains such as HS and N5 . The B. spizizenii type strain TU-B-10 T (DSM 15029 T) produces entianin rather than subtilin, suggesting a potential subspecies division within B. spizizenii . GC content calculation and GC profiling have also been used to differentiate B. spizizenii from related Bacillus species .

What are the storage conditions for recombinant qoxA protein?

The recombinant B. spizizenii qoxA protein requires specific storage conditions to maintain stability and activity. It is typically provided in a Tris-based buffer with 50% glycerol, optimized for protein stability . For short-term storage, the protein can be stored at -20°C, while for extended storage, -20°C or -80°C is recommended . The shelf life of the liquid form is approximately 6 months at -20°C/-80°C, while the lyophilized form can remain stable for up to 12 months at the same temperatures . When working with the protein, it's advisable to avoid repeated freezing and thawing cycles, as this can degrade the protein structure and function. Working aliquots can be stored at 4°C for up to one week .

What secondary metabolites are produced by B. spizizenii?

B. spizizenii produces a distinctive set of secondary metabolites that differentiates it from other B. subtilis subspecies. These include mycosubtilin, bacillaene, and 3,30-neotrehalosadiamine . Many B. spizizenii strains are characterized by their ability to produce lanthipeptides of the subtilin family . The subtilin-producing B. spizizenii strains (including DSM 618, 6405, HS, and N5) differ from the entianin-producing type strain TU-B-10 T . Beyond lanthipeptides, B. spizizenii can also produce sactipeptides like subtilosin and lipopeptides like surfactin . This ability to produce more than two dozen antibiotics with diverse structures makes B. spizizenii particularly interesting for antimicrobial research .

How can genomic analysis be used to distinguish B. spizizenii from other Bacillus species?

Genomic analysis provides powerful tools for distinguishing B. spizizenii from other Bacillus species and understanding its unique characteristics. Comparative genomics can reveal genes unique to each subspecies, which can then guide the discovery of distinguishing phenotypes . For instance, 16S rRNA gene sequencing has been instrumental in classifying field-collected Bacillus strains as B. spizizenii . The subtilin-encoding spaS gene has also been used for taxonomic classification, with sequences from various B. spizizenii strains deposited in GenBank (accession numbers DQ452514, DQ452515, DQ452516, and DQ452517) .

Additionally, GC content calculation and GC profiling can help differentiate B. spizizenii from related species. The comparative analysis of antibiotic profiles, particularly the ability to produce specific lanthipeptides, lipopeptides, and sactipeptides, further supports the taxonomic classification . The presence of specific enzymes, such as the subtilin cyclase SpaC (verified by immunoblotting), can also provide evidence for species identification .

What are the structural characteristics of qoxA protein in B. spizizenii?

The qoxA protein in B. spizizenii is characterized by a specific amino acid sequence that contributes to its function as a quinol oxidase subunit. The full amino acid sequence is available and begins with CSNASVLDPKGPVAEQQSDLILLSIGF . The protein is expressed in the region spanning amino acids 26-321 of the full-length sequence . The protein contains transmembrane domains, as indicated by the hydrophobic regions in its sequence, which are critical for its integration into the cell membrane and function in the respiratory chain .

As a quinol oxidase subunit, qoxA likely contains binding sites for heme groups and other cofactors necessary for electron transfer, though specific details on the three-dimensional structure are not explicitly provided in the available search results. The protein's structural characteristics are optimized for its role in transferring electrons from quinol to oxygen during aerobic respiration, making it an integral part of the organism's energy metabolism machinery.

What methods can be used to study the function of recombinant qoxA in respiratory metabolism?

To study the function of recombinant qoxA in respiratory metabolism, researchers can employ several methodological approaches:

• Oxygen Consumption Assays: Measuring oxygen consumption rates in the presence of the recombinant qoxA protein and quinol substrates can provide direct evidence of its oxidase activity.

• Membrane Reconstitution Studies: Incorporating the purified recombinant qoxA into artificial membrane systems or proteoliposomes allows for the study of its function in a controlled environment that mimics its native membrane context.

• Spectroscopic Analysis: UV-visible spectroscopy and electron paramagnetic resonance (EPR) can be used to characterize the redox states of heme groups and other cofactors associated with qoxA during electron transfer.

• Site-Directed Mutagenesis: Creating specific mutations in the recombinant qoxA protein can help identify critical amino acid residues involved in substrate binding, catalysis, or interaction with other subunits of the oxidase complex.

• Protein-Protein Interaction Studies: Techniques such as co-immunoprecipitation, pull-down assays, or crosslinking experiments can reveal interactions between qoxA and other components of the respiratory chain.

These methods collectively provide a comprehensive understanding of how qoxA contributes to respiratory metabolism in B. spizizenii.

How does qoxA contribute to the antibiotic resistance profile of B. spizizenii?

While the direct contribution of qoxA to antibiotic resistance in B. spizizenii is not explicitly detailed in the search results, we can infer potential connections based on its role in respiratory metabolism and the known antibiotic properties of B. spizizenii strains.

The respiratory chain, of which qoxA is a component, is a common target for antibiotics. Alterations in respiratory proteins like qoxA could potentially modify sensitivity to antibiotics that target cellular respiration. B. spizizenii produces numerous antibiotics with diverse structures, including lanthipeptides like subtilin, sactipeptides like subtilosin, and lipopeptides like surfactin . These compounds can provide competitive advantages against other microorganisms.

The antibiotic production capacity of B. spizizenii has been identified as one of the distinguishing characteristics that supports its promotion to species status . Understanding how respiratory proteins like qoxA interact with or influence the production of these antibiotic compounds could provide insights into both the organism's antibiotic resistance and its ability to produce antibiotics that target other species.

What is the relationship between qoxA expression and the production of secondary metabolites in B. spizizenii?

The relationship between qoxA expression and secondary metabolite production in B. spizizenii represents an interesting area for research, although the search results don't directly address this connection. Energy metabolism, which involves the respiratory chain containing qoxA, is intricately linked to secondary metabolite production in bacteria.

B. spizizenii produces distinct secondary metabolites including mycosubtilin, bacillaene, and 3,30-neotrehalosadiamine , as well as lanthipeptides like subtilin, sactipeptides like subtilosin, and lipopeptides like surfactin . The production of these complex molecules requires significant energy input, which is generated through respiratory metabolism involving proteins like qoxA.

Changes in respiratory metabolism, potentially through altered qoxA expression or activity, could affect the energy availability for secondary metabolite production. Additionally, both respiratory metabolism and secondary metabolite production respond to environmental conditions, suggesting potential regulatory connections. Research examining the correlation between qoxA expression levels and secondary metabolite profiles under various growth conditions could reveal important insights into these relationships.

What are the optimal conditions for expressing recombinant B. spizizenii qoxA?

Based on the available information, recombinant B. spizizenii qoxA can be successfully expressed in mammalian cell systems . While specific optimization parameters are not detailed in the search results, the following general considerations would apply to maximizing expression:

• Expression System Selection: The choice of mammalian cell line should be optimized for membrane protein expression, as qoxA is a membrane-associated respiratory protein.

• Vector Design: The expression vector should include appropriate promoters for the chosen cell system, along with tags for detection and purification that don't interfere with protein function.

• Expression Region: The expression construct should focus on the functional region of qoxA, which spans amino acids 26-321 of the full-length sequence .

• Culture Conditions: Temperature, pH, media composition, and induction timing should be optimized to maximize protein yield while ensuring proper folding and membrane integration.

• Post-translational Modifications: Consideration should be given to any required post-translational modifications that might be necessary for proper qoxA function.

The resulting recombinant protein should achieve a purity of >85% as assessed by SDS-PAGE , and be stored in a Tris-based buffer with 50% glycerol at -20°C or -80°C for optimal stability .

How can researchers verify the identity and activity of purified recombinant qoxA?

Researchers can employ several methods to verify both the identity and activity of purified recombinant qoxA:

• SDS-PAGE and Western Blotting: These techniques can confirm the protein's molecular weight and identity. Western blotting using specific antibodies against qoxA or any included tags can further verify identity . The search results mention using 10% tris-glycine gels for SDS-PAGE analysis .

• Mass Spectrometry: MALDI-TOF or LC-MS/MS analysis can provide peptide mass fingerprinting to confirm the protein's identity and sequence.

• Oxygen Consumption Assays: Since qoxA functions as part of a quinol oxidase complex, measuring oxygen consumption in the presence of the purified protein and appropriate quinol substrates can confirm its functional activity.

• Spectroscopic Analysis: UV-visible spectroscopy can detect characteristic absorption spectra of heme groups within the active quinol oxidase complex.

• Reconstitution Experiments: Incorporating the purified protein into artificial membrane systems or proteoliposomes and measuring electron transfer activities can verify functional integration.

These complementary approaches provide comprehensive verification of both the molecular identity and functional activity of the purified recombinant qoxA protein.

What methods can be used to study qoxA in its native membrane environment?

Studying qoxA in its native membrane environment requires specialized techniques that preserve the membrane context while allowing functional and structural investigations:

• Membrane Isolation: Careful isolation of membrane fractions from B. spizizenii preserving respiratory complexes intact is the first step. Differential centrifugation and density gradient methods can be used to purify membrane fractions.

• Blue Native PAGE: This technique allows separation of intact membrane protein complexes and can reveal qoxA's association with other respiratory components.

• Cryo-Electron Microscopy: This advanced imaging technique can visualize membrane proteins in their native lipid environment, potentially revealing structural details of qoxA within the membrane.

• Solid-State NMR: This specialized form of nuclear magnetic resonance spectroscopy can provide structural information about membrane proteins like qoxA within lipid bilayers.

• Fluorescence Microscopy: Using fluorescently labeled antibodies against qoxA or fusion of qoxA with fluorescent proteins can help visualize its distribution and dynamics within bacterial membranes.

• Membrane Protein Crosslinking: Chemical crosslinking followed by mass spectrometric analysis can identify proteins that interact with qoxA in the membrane.

These approaches collectively provide insights into how qoxA functions within its native membrane context in B. spizizenii.

What quantitative assays can be used to measure quinol oxidase activity?

Several quantitative assays can be employed to measure the quinol oxidase activity associated with qoxA:

• Oxygen Electrode Measurements: Clark-type oxygen electrodes can directly measure oxygen consumption rates in the presence of quinol substrates and purified quinol oxidase complexes containing qoxA.

• Spectrophotometric Assays: These assays monitor the oxidation of reduced quinol substrates by following absorbance changes at specific wavelengths. The rate of absorbance change correlates with enzyme activity.

• HPLC Analysis: High-performance liquid chromatography can be used to quantitatively measure the conversion of quinol substrates to their oxidized forms. The search results mention using RP-HPLC with an analytical ODS Hypersil column (particle size: 5 μm, width and length: 2 × 250 mm) for quantitative analysis of other molecules produced by B. spizizenii , and similar approaches could be adapted for quinol oxidase activity measurements.

• Electron Transfer Assays: These assays use artificial electron acceptors like cytochrome c or artificial dyes that change color upon reduction to measure electron transfer rates from quinol through the oxidase complex.

• Membrane Potential Measurements: Since quinol oxidase activity contributes to the generation of proton motive force, measuring changes in membrane potential or pH gradients can indirectly assess enzyme activity.

These quantitative approaches provide complementary data on the enzymatic activity of quinol oxidase complexes containing qoxA.

How can gene knockout or mutation studies be designed to investigate qoxA function?

Designing gene knockout or mutation studies to investigate qoxA function requires careful consideration of several factors:

• Knockout Strategy: Complete deletion of the qoxA gene (BSUW23_18875) can be achieved using homologous recombination techniques specific for Bacillus species. This involves creating a construct with antibiotic resistance markers flanked by sequences homologous to regions surrounding the qoxA gene.

• Conditional Knockout Systems: Since qoxA is involved in respiration and may be essential under certain conditions, conditional knockout systems (like inducible promoters controlling qoxA expression) might be necessary.

• Site-Directed Mutagenesis: Rather than complete knockout, specific amino acid residues predicted to be important for qoxA function can be mutated. The detailed amino acid sequence provided can guide the selection of residues for mutation based on conservation or predicted functional importance.

• Complementation Studies: To confirm phenotypes observed in knockout strains are specifically due to qoxA deletion, complementation with wild-type qoxA or various mutants can be performed.

• Phenotypic Analysis: The effects of qoxA knockout or mutation should be assessed through multiple phenotypic analyses, including growth rates under various conditions, oxygen consumption, membrane potential measurements, and secondary metabolite production profiles.

• Genome-Wide Transcriptional Analysis: RNA-seq or microarray analysis comparing wild-type and qoxA mutant strains can reveal compensatory changes and regulatory networks connected to qoxA function.

These approaches collectively provide a comprehensive understanding of qoxA's role in B. spizizenii physiology.

How does the potential reclassification of B. subtilis subspecies impact qoxA research?

The potential reclassification of Bacillus subtilis subspecies to species status has significant implications for qoxA research. Recent genomic comparisons have suggested that B. subtilis subsp. spizizenii, along with other subspecies, should be promoted to species status . This taxonomic change would affect how researchers approach qoxA studies in several ways:

• Comparative Genomics: The reclassification highlights the genetic distinctiveness of B. spizizenii, suggesting that qoxA might have unique characteristics or functions compared to homologous proteins in related species. This encourages comparative studies of qoxA across the newly defined species.

• Metabolic Context: Each Bacillus species produces a distinct set of secondary metabolites , creating unique metabolic contexts within which qoxA functions. Understanding these species-specific metabolic networks becomes crucial for interpreting qoxA function.

• Evolutionary Insights: The reclassification provides an opportunity to study the evolution of respiratory proteins like qoxA across closely related but distinct Bacillus species, potentially revealing adaptive changes related to different ecological niches.

• Nomenclature and Database Organization: Practical aspects of research, such as gene annotation, database organization, and literature searches, would need to adapt to the new taxonomic classification.

This reclassification ultimately encourages more precise and context-specific studies of qoxA in B. spizizenii rather than generalizing findings across the broader B. subtilis group.

What is the relationship between qoxA and the antibiotic production capabilities of B. spizizenii?

The relationship between qoxA, as a component of respiratory metabolism, and the distinctive antibiotic production capabilities of B. spizizenii represents an intriguing area for future research. B. spizizenii produces a unique profile of antibiotics, including mycosubtilin, bacillaene, and 3,30-neotrehalosadiamine , as well as lanthipeptides like subtilin .

Several potential connections exist:

• Energy Metabolism: The respiratory chain, including qoxA, generates the energy required for the biosynthesis of complex secondary metabolites. Changes in respiratory efficiency could impact antibiotic production capacity.

• Redox Balance: Respiratory metabolism maintains cellular redox balance, which can influence the activity of enzymes involved in secondary metabolite biosynthesis, many of which are redox-sensitive.

• Regulatory Networks: Shared regulatory networks might coordinate respiratory metabolism and secondary metabolite production in response to environmental conditions.

• Evolutionary Co-adaptation: The distinctive qoxA characteristics and antibiotic production profile of B. spizizenii might have co-evolved as adaptations to specific ecological niches.

Future research investigating correlations between qoxA expression or mutation and antibiotic production profiles could reveal important insights into these relationships and potentially lead to strategies for enhancing antibiotic production.

How might structural biology approaches advance our understanding of qoxA function?

Advanced structural biology approaches could significantly deepen our understanding of qoxA function in B. spizizenii:

• Cryo-Electron Microscopy (Cryo-EM): This technique could reveal the three-dimensional structure of qoxA within the complete quinol oxidase complex, providing insights into subunit interactions and the arrangement of electron transfer components.

• X-ray Crystallography: If crystals of purified qoxA or the entire oxidase complex can be obtained, X-ray crystallography could provide atomic-level structural details of the protein.

• NMR Spectroscopy: Solution or solid-state NMR could provide information about dynamic aspects of qoxA structure and interactions, particularly for regions that might be flexible or undergo conformational changes during catalysis.

• Molecular Dynamics Simulations: Computational approaches using structural data can simulate qoxA dynamics within a membrane environment, revealing potential conformational changes during the catalytic cycle.

• Structure-Guided Mutagenesis: Structural information can guide targeted mutagenesis experiments to test hypotheses about specific amino acid residues involved in substrate binding, catalysis, or subunit interactions.

These structural approaches, combined with functional assays, would provide a comprehensive understanding of how qoxA's structure enables its function in respiratory metabolism.

What are the potential biotechnological applications of recombinant qoxA?

Recombinant qoxA from B. spizizenii has several potential biotechnological applications:

• Biosensors: As a respiratory protein that interacts with quinols and oxygen, qoxA could be developed into biosensors for detecting specific quinone compounds or measuring oxygen levels in various environments.

• Biocatalysis: The oxidoreductase activity of qoxA might be harnessed for biotransformation of specific substrates in industrial processes, particularly those requiring selective oxidation reactions.

• Antimicrobial Discovery: Understanding qoxA function could inform the development of new antimicrobials targeting bacterial respiratory chains, especially given B. spizizenii's own production of diverse antibiotics .

• Protein Engineering: The qoxA protein could serve as a scaffold for protein engineering efforts aimed at creating novel oxidoreductases with desired substrate specificities or improved catalytic efficiencies.

• Membrane Protein Expression Systems: Protocols developed for successful expression and purification of recombinant qoxA could advance methodologies for producing other challenging membrane proteins.

These applications leverage the natural properties of qoxA while potentially extending them to new contexts through protein engineering and creative deployment in biotechnological systems.