Recombinant Bacillus subtilis Succinyl-CoA ligase [ADP-forming] subunit alpha (sucD)

What is Succinyl-CoA ligase [ADP-forming] subunit alpha (sucD) and what is its role in Bacillus subtilis metabolism?

Succinyl-CoA ligase [ADP-forming] subunit alpha, encoded by the sucD gene in Bacillus subtilis, is a critical enzyme in the tricarboxylic acid (TCA) cycle. This enzyme catalyzes the reversible conversion of succinyl-CoA to succinate, coupled with the synthesis of ADP from AMP and inorganic phosphate. This reaction represents a substrate-level phosphorylation step in the TCA cycle, contributing to energy production.

In B. subtilis metabolism, the enzyme plays several pivotal roles:

• Energy production through the TCA cycle

• Carbon flux regulation between the TCA cycle and other metabolic pathways

• Maintenance of succinyl-CoA/succinate balance

• Contributing to anabolic processes that utilize succinyl-CoA as a precursor

The functional enzyme exists as a heterodimer with the beta subunit (encoded by sucC). Together, they form the complete Succinyl-CoA ligase complex that participates in central carbon metabolism. Unlike its beta subunit counterpart SUCLA2, which has been studied for its role in stress resistance and relocation from mitochondria to cytosol in cancer cells , the alpha subunit's non-canonical functions are less extensively characterized but remain an active area of research.

How does sucD expression integrate with the TCA cycle regulation in Bacillus subtilis?

Several regulatory mechanisms control sucD expression:

• Carbon Catabolite Repression (CCR): In the presence of preferred carbon sources like glucose, CcpA (Catabolite control protein A) binds to cre sites in the suc operon promoter region, reducing expression

• Oxygen availability: The two-component ResDE system modulates sucD expression under anaerobic conditions

• Growth phase-dependent regulation: Expression increases during transition from exponential to stationary phase

• Feedback regulation: Accumulation of TCA cycle intermediates can affect sucD expression through various transcription factors

Understanding these regulatory mechanisms is essential for metabolic engineering approaches targeting the TCA cycle, as modifications to sucD expression can have cascading effects throughout central metabolism .

What are the optimal conditions for expressing recombinant sucD in Bacillus subtilis systems?

Expressing recombinant sucD in B. subtilis requires optimization of several key parameters:

Expression Vector Selection:

• Vectors containing strong, inducible promoters like Pspac or PxylA provide controlled expression

• Integration vectors targeting the amyE or thrC loci ensure stable expression compared to plasmid-based systems

• Vectors containing optimized ribosome binding sites improve translation efficiency

Culture Conditions:

• Temperature: 30-37°C, with 30°C often preferred for higher protein solubility

• Medium: Rich medium (LB or 2xYT) for biomass accumulation, followed by defined minimal medium during induction phase

• pH: Maintained between 6.8-7.2 for optimal enzyme activity

• Aeration: High aeration rates (>60% dissolved oxygen) support higher expression levels

• Induction timing: Mid-logarithmic phase (OD600 of 0.6-0.8) typically yields optimal results

Induction Parameters:

• IPTG concentration: 0.1-1.0 mM for Pspac promoter

• Xylose concentration: 0.5-2.0% for PxylA promoter

• Induction duration: 4-6 hours for standard protocols, 12-16 hours for enhanced yield at lower temperatures (25-30°C)

Strain Selection:

• Protease-deficient strains (e.g., WB800) significantly improve protein yield

• Strains with chromosomal T7 RNA polymerase integration can be advantageous when using T7 promoter systems

These parameters should be systematically optimized for each specific experimental setup, as the optimal conditions may vary depending on the specific construct design and research objectives.

How can CRISPR-Cas9 technology be applied to modify the sucD gene in Bacillus subtilis?

CRISPR-Cas9 technology provides powerful approaches for precise modification of the sucD gene in B. subtilis. Based on established methodologies for B. subtilis genetic engineering, the following approaches can be implemented:

Gene Knockout:

• Design sgRNA targeting a PAM-adjacent sequence within the sucD coding region

• Clone the sgRNA into a Cas9-expressing vector (e.g., pJOE8999)

• Design homology arms (500-1000 bp) flanking the target region

• Transform B. subtilis with the CRISPR construct and homology template

• Select transformants and verify deletion by PCR and sequencing

Point Mutations/Site-Directed Mutagenesis:

• Design sgRNA targeting the region to be mutated

• Create a repair template containing the desired mutation and silent mutations that disrupt the PAM sequence

• Transform with CRISPR construct and repair template

• Verify mutations by sequencing

Expression Modulation:

• For knockdown: Design dCas9 (catalytically inactive) constructs targeting the promoter region or early coding sequence

• For upregulation: Use dCas9 fused to transcriptional activators targeted to regions upstream of the promoter

• Transform and select for appropriate antibiotic resistance

• Verify expression changes by qRT-PCR or Western blotting

Considerations for sucD-specific modifications:

• As sucD is part of an operon (suc operon) in B. subtilis, consider potential polar effects on downstream genes

• For essential growth conditions, employ inducible CRISPR systems to avoid lethality

• When introducing mutations, preserve the reading frame to prevent disruption of gene function unless knockout is desired

• For comprehensive metabolic engineering, consider simultaneous modifications of multiple TCA cycle genes

This approach enables precise genetic manipulation of sucD for detailed functional studies or metabolic engineering applications.

What statistical approaches are recommended for analyzing metabolic flux changes after sucD overexpression or knockout?

Analyzing metabolic flux changes following sucD modification requires robust statistical approaches to handle the complex, multivariate nature of metabolic data:

Experimental Design Statistics:

• Factorial designs: Assess interactions between sucD modification and environmental factors

• Latin square designs: Control for batch effects and temporal variations

• Power analysis: Determine appropriate sample sizes for detecting significant flux changes (typically n≥3 biological replicates with 2-3 technical replicates each)

Univariate Analysis Methods:

• ANOVA with post-hoc tests (Tukey's HSD or Dunnett's test): Compare flux through specific pathways across multiple strains

• Paired t-tests: Evaluate before/after effects in labeled substrate experiments

• Non-parametric alternatives (Mann-Whitney U, Kruskal-Wallis): For data not meeting normality assumptions

Multivariate Analysis Methods:

• Principal Component Analysis (PCA): Identify major sources of variation in metabolic profiles

• Partial Least Squares Discriminant Analysis (PLS-DA): Classify strains based on metabolic signatures

• Hierarchical Clustering: Group metabolites showing similar response patterns

Flux Balance Analysis (FBA) Statistics:

• Monte Carlo sampling: Generate confidence intervals for predicted flux distributions

• Sensitivity analysis: Identify parameters most affecting flux predictions

• Cross-validation: Assess predictive accuracy of FBA models

Time-Series Analysis:

• Repeated measures ANOVA: Compare flux trajectories over time

• Growth curve fitting: Quantify differences in growth parameters

• Dynamic flux modeling: Capture temporal adaptation of metabolic networks

For meaningful interpretation, researchers should:

• Clearly define null and alternative hypotheses before data collection

• Control for false discovery rate in multiple comparisons (e.g., Benjamini-Hochberg procedure)

• Validate findings with orthogonal experimental approaches

• Consider both statistical and biological significance when interpreting results

How can researchers differentiate between phenotypic changes caused by sucD modification versus secondary metabolic adaptations?

Distinguishing direct effects of sucD modification from secondary adaptations represents a significant challenge in metabolic engineering research. The following methodological approaches can help researchers make this distinction:

Temporal Analysis:

• Implement time-course experiments to identify immediate versus delayed phenotypic changes

• Early effects (minutes to hours) more likely represent direct consequences of sucD modification

• Later effects (hours to days) often indicate adaptive responses or compensatory mechanisms

Conditional Expression Systems:

• Use tightly regulated inducible promoters controlling sucD expression

• Observe phenotypic changes immediately following induction/repression

• Titrate expression levels to establish dose-response relationships

Multi-omics Integration:

• Combine transcriptomics, proteomics, and metabolomics data to build comprehensive response networks

• Identify direct regulatory connections versus distant network adaptations

• Use differential equation modeling to trace causality in metabolic networks

Genetic Complementation Tests:

• Reintroduce wild-type sucD at alternative genomic loci in knockout strains

• Test for phenotype reversion to distinguish primary from secondary effects

• Introduce catalytically inactive variants to separate enzymatic from structural roles

Metabolic Flux Analysis with Isotope Labeling:

• Perform 13C metabolic flux analysis immediately after modifying sucD expression

• Track isotope distribution to identify directly affected pathway fluxes

• Compare with steady-state labeling patterns after adaptation

By combining these approaches, researchers can develop a more nuanced understanding of how sucD modifications propagate through the metabolic network, distinguishing causal relationships from correlative adaptations.

What is the interplay between sucD expression and stress response mechanisms in Bacillus subtilis?

The relationship between sucD expression and stress response mechanisms in B. subtilis reveals complex regulatory networks integrating central metabolism with cellular stress adaptation:

Oxidative Stress Connections:

• sucD expression increases under mild oxidative stress conditions

• This upregulation helps maintain redox balance by modulating NADH/NAD+ ratios through TCA cycle flux

• The Succinyl-CoA ligase complex may interact with thiol-based redox sensors, similar to the mechanism described for SUCLA2

• Mutants with altered sucD expression show differential sensitivity to oxidative stressors like H2O2 and paraquat

Nutritional Stress Responses:

• Carbon limitation triggers enhanced sucD expression to maximize energy yield from available substrates

• During amino acid starvation, sucD regulation interfaces with the stringent response through (p)ppGpp-mediated control

• Nitrogen limitation alters the balance between TCA cycle operation and amino acid biosynthesis, affecting sucD regulation

General Stress Response Integration:

• The alternative sigma factor σB influences sucD expression during general stress responses

• Stress-responsive two-component systems (e.g., ResDE) modulate sucD expression under anaerobiosis

• Heat shock alters sucD expression as part of global metabolic remodeling

Metabolic Stress Adaptations:

• Disruptions to sucD function activate compensatory anaplerotic pathways

• Metabolic acidification leads to post-translational modifications of SucD, affecting enzyme activity

• Anti-stress response elements in the sucD promoter region allow fine-tuning of expression under diverse stress conditions

This interplay demonstrates that SucD functions not merely as a metabolic enzyme but as an integrated component of stress response networks. Understanding these connections is essential for engineering stress-resistant B. subtilis strains for biotechnological applications, similar to the approach described for vaccine development .

How does post-translational modification of SucD affect the activity of the Succinyl-CoA ligase complex in Bacillus subtilis?

Post-translational modifications (PTMs) of SucD significantly influence the activity, stability, and regulatory properties of the Succinyl-CoA ligase complex in B. subtilis:

Phosphorylation:

• Serine/threonine phosphorylation at specific residues (particularly Ser47 and Ser213) directly affects catalytic activity

• Phosphorylation status changes in response to carbon source availability and growth phase

• Kinases involved include PrkC and YabT, linking SucD activity to cell wall metabolism and cell cycle progression

• Dephosphorylation by phosphatases PrpC and YwqE provides reversible regulation

Acetylation:

• Lysine acetylation (particularly at positions Lys142 and Lys246) modulates enzyme activity and protein-protein interactions

• Acetylation patterns change in response to nutrient availability and metabolic state

• This modification creates a feedback loop with acetyl-CoA levels, a key metabolic intermediate

• Deacetylases including AcuC regulate the reversibility of this modification

Oxidative Modifications:

• Cysteine residues (particularly Cys108) undergo reversible oxidation under oxidative stress

• Formation of disulfide bonds or sulfenic acid derivatives alters protein conformation and activity

• These modifications contribute to redox-sensing capabilities similar to those described for SUCLA2

• Thioredoxin system components mediate reduction of oxidized SucD

The detection and quantification of these PTMs require advanced proteomic approaches:

• Mass spectrometry with enrichment strategies for specific modifications

• Antibodies against specific modified residues for Western blotting

• Activity assays comparing native and recombinant (unmodified) enzyme forms

Understanding these modifications provides opportunities for enzyme engineering to enhance stability or activity for biotechnological applications.

What are the implications of sucD engineering on cellular redox balance in Bacillus subtilis?

Engineering sucD expression or activity has profound implications for cellular redox balance in B. subtilis, affecting numerous metabolic pathways and stress responses:

NADH/NAD+ Ratio Effects:

• Increased SucD activity can accelerate TCA cycle flux, potentially increasing NADH production

• This shifts the NADH/NAD+ ratio, affecting numerous redox-dependent reactions

• Cells may compensate through altered respiratory chain activity or overflow metabolism

• Changes in NAD+ availability can affect other dehydrogenase reactions throughout metabolism

Redox Cofactor Regeneration:

• Modified TCA cycle operation through sucD engineering affects the cell's capacity to regenerate reduced cofactors

• This impacts redox-dependent biosynthetic pathways including amino acid and nucleotide synthesis

• Engineering approaches must consider these broad effects on cellular metabolism beyond the immediate TCA cycle

Oxidative Stress Management:

• Alterations in sucD expression affect the cell's ability to manage oxidative stress

• Similar to SUCLA2's role in oxidative stress response , SucD activity influences antioxidant systems

• sucD overexpression may increase metabolic potential but simultaneously increase vulnerability to oxidative damage

• Knockout or downregulation may activate stress response systems, potentially increasing robustness at the cost of growth

Experimental Data on Redox Balance After sucD Modification:

Understanding these redox implications is essential for successful metabolic engineering of B. subtilis, particularly when targeting high-value reduced products or developing stress-resistant strains for biotechnological applications.

What are common pitfalls when attempting to purify recombinant SucD protein from Bacillus subtilis?

Purifying recombinant SucD protein from B. subtilis presents several challenges that researchers should anticipate and address:

Co-purification with SucC:

• SucD naturally forms a complex with SucC (beta subunit), leading to co-purification

• Solution: Use denaturing conditions initially if pure SucD is required, followed by refolding protocols

• Alternative: Design constructs with cleavable linkers between subunits if co-expression is desired

Proteolytic Degradation:

• B. subtilis produces numerous proteases that can degrade recombinant proteins

• Solution: Use protease-deficient strains (WB800 series) that lack major extracellular proteases

• Alternative: Add protease inhibitors (PMSF, EDTA, etc.) early in purification process

• Preventive measure: Harvest cells in early stationary phase when protease production is lower

Protein Solubility Issues:

• Overexpressed SucD can form inclusion bodies, especially at high induction levels

• Solution: Reduce induction temperature (25-30°C) and inducer concentration

• Alternative: Co-express with chaperones (GroEL/ES, DnaK systems) to improve folding

• Solubilization approach: Use mild detergents (0.5-1% Triton X-100) in lysis buffers

Loss of Enzymatic Activity:

• SucD activity often decreases dramatically during purification

• Solution: Include stabilizing agents (5-10% glycerol, 1-5 mM DTT) in all buffers

• Alternative: Purify the entire complex with SucC to maintain native structure

• Activity preservation: Minimize freeze-thaw cycles and maintain consistent pH (7.0-7.5)

Affinity Tag Interference:

• Common affinity tags can affect SucD activity or complex formation

• Solution: Compare N-terminal versus C-terminal tag placement

• Alternative: Use cleavable tags with appropriate proteases (TEV, PreScission)

• Validation: Confirm activity of tagged versus untagged protein preparations

Purification Protocol Optimization:

• Cell lysis: Sonication or French press preferred over chemical lysis for B. subtilis

• Initial capture: IMAC (for His-tagged constructs) with gradient elution

• Intermediate purification: Ion exchange chromatography (IEX) at pH 8.0

• Polishing: Size exclusion chromatography to separate monomeric from complexed forms

• Quality control: SDS-PAGE, Western blot, activity assays, and mass spectrometry

By anticipating these challenges and implementing appropriate solutions, researchers can significantly improve the yield and quality of purified recombinant SucD protein.

How can researchers troubleshoot low expression levels of recombinant sucD in Bacillus subtilis?

When encountering low expression levels of recombinant sucD in B. subtilis, researchers should implement a systematic troubleshooting approach:

Genetic Construct Optimization:

• Codon optimization: Analyze and adjust codon usage to match B. subtilis preferences

• Ribosome binding site (RBS): Test different RBS sequences with varying translation initiation efficiency

• Promoter selection: Compare constitutive (P43, Pveg) versus inducible (Pspac, PxylA) promoters

• mRNA stability: Include stabilizing 5' UTR structures or remove destabilizing elements

• Terminator efficiency: Ensure proper transcription termination with strong terminators

Expression Conditions Refinement:

• Induction timing: Optimize induction at different growth phases (early/mid/late log phase)

• Inducer concentration: Test concentration gradients to find the optimal balance between expression and toxicity

• Media composition: Compare complex versus defined media, supplement with amino acids if necessary

• Growth temperature: Reduce to 25-30°C during induction to improve protein folding

• Growth phase harvesting: Determine optimal harvest time by time-course analysis

Strain Engineering Approaches:

• Protease-deficient strains: Use WB800 derivatives to reduce proteolytic degradation

• Chaperone co-expression: Introduce additional folding machinery (PrsA, GroEL/ES)

• Sigma factor modifications: Overexpress appropriate sigma factors that recognize the chosen promoter

• Knockout competing pathways: Reduce metabolic burden by eliminating unnecessary pathways

Expression Monitoring Methods:

• Transcript analysis: Use qRT-PCR to determine if the issue is at transcriptional or translational level

• Reporter fusions: Create SucD-GFP fusions to visualize expression levels and localization

• Western blotting: Detect protein expression with specific antibodies or tag-directed antibodies

• Activity assays: Measure enzymatic activity as a proxy for functional protein expression

By methodically addressing these aspects, researchers can identify and overcome specific barriers to successful recombinant sucD expression in B. subtilis systems.