Recombinant Bacillus cereus Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is Bacillus cereus Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) and what is its primary function?

Bacillus cereus Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) is a critical component of the Krebs cycle (TCA cycle). This enzyme, also known as BCQ_3621, EC 6.2.1.5, or Succinyl-CoA synthetase subunit beta (SCS-beta), catalyzes the reversible conversion of succinyl-CoA to succinate with concomitant synthesis of ATP via substrate-level phosphorylation .

What expression systems are commonly used for recombinant production of Bacillus cereus sucC?

Multiple expression systems have been validated for recombinant production of Bacillus cereus sucC, each with distinct advantages:

For optimal experimental design, researchers should select the expression system based on their specific downstream applications. For basic enzymatic studies, E. coli-derived protein is typically sufficient, while interaction studies may benefit from mammalian expression systems .

How can researchers verify the purity and activity of recombinant Bacillus cereus sucC?

Methodological approach for quality assessment:

• Purity verification: SDS-PAGE analysis with Coomassie staining (target >85% purity). Western blot analysis using anti-sucC antibodies provides specificity confirmation.

• Activity assessment: Measure enzymatic activity through:

  • Spectrophotometric assay tracking the conversion of succinyl-CoA to succinate

  • Coupling with pyruvate kinase and lactate dehydrogenase to monitor ADP production through NADH oxidation

  • Standard assay conditions: 50 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 0.1 mM succinyl-CoA, 0.5 mM ADP at 37°C

• Structural integrity: Circular dichroism spectroscopy to assess secondary structure integrity compared to native enzyme.

What controls are essential when studying recombinant Bacillus cereus sucC functions?

When designing experiments to investigate sucC functions, implement these methodological controls:

• Enzymatic activity controls:

  • Positive control: Commercially validated succinyl-CoA ligase

  • Negative control: Heat-inactivated enzyme preparation

  • Substrate specificity control: Test with alternative CoA derivatives

• Expression system controls:

  • Empty vector expression in parallel with sucC-expressing vector

  • Wild-type enzyme versus site-directed mutants of catalytic residues

• Functional studies controls:

  • Parallel assessment of alpha subunit (SUCLG1) to differentiate subunit-specific effects

  • Complementation studies in sucC-deficient bacterial strains

  • When studying non-canonical functions, include mitochondrial fractionation controls

These controls enable researchers to differentiate between specific sucC-mediated effects and non-specific experimental artifacts.

How should researchers design experiments to study potential non-canonical functions of Bacillus cereus sucC?

Based on recent findings that SUCLA2 (human homolog of sucC) demonstrates functions beyond the TCA cycle, researchers should consider these methodological approaches:

• Subcellular localization studies:

  • Fluorescently-tagged sucC protein tracking under different cellular stress conditions

  • Subcellular fractionation followed by Western blot analysis

  • Co-localization studies with known stress granule markers

• Protein interaction screening:

  • Pull-down assays with recombinant sucC as bait

  • Yeast two-hybrid or mammalian two-hybrid screening

  • Co-immunoprecipitation studies followed by mass spectrometry

• Functional dissection:

  • Site-directed mutagenesis to separate catalytic function from potential protein-interaction domains

  • Domain swapping experiments between sucC and related proteins

  • Complementation studies in eukaryotic cells using the Bacillus cereus protein

Recent research indicates that SUCLA2 can relocate from mitochondria to cytosol upon cellular stress and may interact with stress granules, suggesting similar potential non-canonical roles for bacterial sucC that warrant investigation .

How can researchers effectively study the role of Bacillus cereus sucC in redox homeostasis?

Recent studies indicate that SUCLA2 (human homolog) plays a crucial role in redox homeostasis independent of its TCA cycle function . To investigate whether Bacillus cereus sucC has similar properties:

• Redox state measurement methodologies:

  • Fluorescent redox sensors (e.g., roGFP) in recombinant Bacillus cereus strains with sucC variants

  • Direct measurement of ROS using fluorescent probes (DCF-DA, MitoSOX)

  • Quantification of reduced/oxidized glutathione ratios in wild-type vs. sucC-modified strains

• Stress response experimental design:

  • Challenge cultures with oxidative stressors (H₂O₂, paraquat) at sublethal concentrations

  • Assess survival rates and recovery kinetics between wild-type and sucC-modified strains

  • Measure expression of antioxidant enzymes (catalase, superoxide dismutase) in response to sucC manipulation

• Proteomic analysis:

  • Compare the stress granule proteome between wild-type and sucC-mutant strains

  • Identify potential stress-responsive proteins that interact with sucC

  • Quantify changes in antioxidant enzyme expression and activity

This methodological framework allows researchers to systematically explore potential redox-related functions of Bacillus cereus sucC beyond its canonical metabolic role .

What approaches can resolve contradictory data regarding sucC localization and function?

When faced with contradictory data regarding sucC localization or function, implement these methodological solutions:

• Spatiotemporal analysis:

  • Use time-course experiments with fine temporal resolution to capture transient localizations

  • Employ super-resolution microscopy (STORM, PALM) for precise subcellular localization

  • Correlate localization changes with specific cellular stress phases

• Mutational analysis framework:

  • Generate a panel of sucC mutants with targeted modifications to functional domains

  • Test each mutant for both canonical (TCA cycle) and potential non-canonical functions

  • Create chimeric proteins to identify domains responsible for specific localizations or functions

• Heterologous expression system comparison:

  • Express Bacillus cereus sucC in diverse cellular backgrounds (bacterial, yeast, mammalian)

  • Compare localization and function across systems to identify context-dependent behaviors

  • Perform complementation studies with sucC homologs from related species

When analyzing contradictory data, researchers should systematically evaluate experimental conditions, including buffer compositions, cellular state (exponential vs. stationary phase), and stress conditions that might reveal condition-specific functions .

How can genetic engineering approaches be used to study sucC function in Bacillus cereus?

Advanced genetic manipulation techniques for studying sucC function:

• Gene deletion and complementation strategy:

  • Create a sucC deletion strain using homologous recombination

  • Complement with wild-type or mutant versions under native or inducible promoters

  • Analyze phenotypic changes in growth, stress resistance, and metabolic profiles

• Promoter manipulation approach:

  • Replace native sucC promoter with regulatable promoters (e.g., xylose-inducible)

  • Fine-tune expression levels to identify threshold effects

  • Study the impact of regulatory systems similar to the scoC regulatory pathway described for other Bacillus enzymes

• Fusion protein strategy:

  • Create translational fusions with reporter proteins (GFP, luciferase)

  • Engineer split protein complementation systems to detect protein-protein interactions

  • Develop FRET-based sensors to monitor conformational changes during enzymatic activity

As demonstrated in related research with Bacillus species, manipulation of regulatory genes (like scoC) can significantly impact enzyme expression and activity. For example, deletion of the scoC regulatory gene in Bacillus cereus led to a 58% increase in protease activity, suggesting similar approaches might reveal regulatory mechanisms for sucC expression .

How should researchers address unexpected data that contradicts existing models of sucC function?

When experimental data contradicts established models of sucC function, apply this structured approach:

• Data validation protocol:

  • Verify experimental reproducibility with increased biological and technical replicates

  • Employ alternative methodologies to confirm observations

  • Perform statistical power analysis to ensure adequate sample size

• Model reconciliation framework:

  • Consider context-dependent functions (stress conditions, growth phase, nutrient availability)

  • Develop integrative models incorporating both canonical and non-canonical functions

  • Explore potential moonlighting functions under specific conditions

• Comparative analysis:

  • Examine whether contradictions exist in homologs from other species

  • Consider evolutionary context that might explain functional divergence

  • Review literature for similar contradictions in related enzymes

As noted in research literature, unexpected findings can lead to new discoveries. For example, the discovery that SUCLA2 functions outside the TCA cycle in stress response was initially contradictory to established models but led to new understanding of cancer cell survival mechanisms .

What methodologies can help differentiate between canonical and non-canonical functions of Bacillus cereus sucC?

To rigorously differentiate between canonical TCA cycle functions and potential non-canonical roles:

• Catalytic site mutation approach:

  • Generate catalytically inactive sucC mutants (targeting known active site residues)

  • Test for persistence of non-canonical functions despite loss of enzymatic activity

  • Compare phenotypes between wild-type, knockout, and catalytically inactive mutants

• Metabolic bypass strategy:

  • Create alternative metabolic routes to bypass the need for sucC's canonical function

  • Assess whether non-canonical functions persist when the canonical role is circumvented

  • Measure metabolic flux using isotope labeling to confirm effective bypass

• Targeted subcellular expression:

  • Engineer sucC variants with compartment-specific targeting sequences

  • Restrict expression to specific cellular locations using orthogonal expression systems

  • Measure compartment-specific effects on both metabolic and non-metabolic processes

This methodological approach can help resolve the type of functional dichotomy observed with SUCLA2, which demonstrated redox-regulating functions independent of its role in the TCA cycle through stress granule interactions in the cytosol .

How can Bacillus cereus sucC research inform therapeutic approaches for infectious diseases?

Bacillus cereus is increasingly recognized as a serious human pathogen capable of causing severe infections beyond food poisoning, including neuroinvasive disease in immunocompromised patients . Studying sucC provides these potential therapeutic insights:

• Pathogenesis mechanism elucidation:

  • Investigate whether sucC contributes to virulence or stress resistance during infection

  • Determine if sucC is differentially expressed during host infection

  • Compare expression patterns between clinical and environmental isolates

• Drug target assessment:

  • Evaluate sucC as a potential antimicrobial target using in silico docking studies

  • Develop high-throughput screening assays for sucC inhibitors

  • Test species-specific inhibition to target bacterial sucC without affecting human homologs

• Biomarker development:

  • Assess whether anti-sucC antibodies are generated during Bacillus cereus infections

  • Explore serological detection methods based on sucC epitopes

  • Develop diagnostic tools for rapid detection of virulent strains based on sucC variants

Recent clinical research has identified Bacillus cereus as an emerging pathogen in neuroinvasive disease, particularly in immunocompromised patients with acute myeloid leukemia. Understanding the metabolic adaptations mediated by enzymes like sucC could provide insights into bacterial survival during infection .

What are the emerging research directions for studying potential roles of sucC in stress response?

Based on discoveries about the human homolog SUCLA2's role in stress granule formation and redox homeostasis , these emerging research directions warrant investigation:

• Stress granule interaction studies:

  • Determine whether bacterial sucC interacts with RNA or RNA-binding proteins under stress

  • Identify potential bacterial analogs to eukaryotic stress granules

  • Investigate how sucC might influence protein synthesis during stress conditions

• Metabolic adaptation mapping:

  • Profile metabolic shifts in wild-type versus sucC-modified strains under various stressors

  • Measure energy charge (ATP/ADP/AMP ratios) correlation with sucC activity

  • Develop metabolic flux models integrating both canonical and stress-response functions

• Evolutionary conservation analysis:

  • Compare sucC functions across diverse bacterial species and in archaeal homologs

  • Identify conserved domains potentially responsible for non-canonical functions

  • Trace the evolutionary history of dual functionality in the sucC protein family

This emerging field connects metabolism with stress response mechanisms and may reveal new paradigms in bacterial adaptation to environmental challenges, potentially informing both basic science and applied research into bacterial survival strategies .