Recombinant Burkholderia cenocepacia Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is the fundamental role of sucC in B. cenocepacia metabolism?

The sucC gene in B. cenocepacia encodes the beta subunit of Succinyl-CoA ligase [ADP-forming], an essential enzyme in the tricarboxylic acid (TCA) cycle. This enzyme catalyzes the reversible conversion of succinyl-CoA to succinate, coupled with the synthesis of ATP from ADP and inorganic phosphate. The reaction represents a critical step in energy metabolism where a high-energy thioester bond is converted to a high-energy phosphate bond.

In metabolic pathways such as the phenylacetic acid (PAA) degradation pathway in B. cenocepacia, the final products include succinyl-CoA and acetyl-CoA that feed into the TCA cycle . This demonstrates the integration of various catabolic pathways with central metabolism, where sucC plays a pivotal role in energy production and carbon flux regulation.

How does B. cenocepacia Succinyl-CoA ligase compare structurally and functionally to homologous enzymes in other organisms?

B. cenocepacia Succinyl-CoA ligase shares functional characteristics with homologous enzymes found in other organisms, though with species-specific adaptations. For comparison, in humans, the SUCLA2 gene encodes the beta subunit of Succinyl-CoA ligase that is most active in high-energy demanding tissues like the brain and muscles . Both bacterial and human enzymes participate in the citric acid cycle and play critical roles in energy production.

What experimental approaches are most effective for confirming the essentiality of sucC in B. cenocepacia?

Determining gene essentiality in B. cenocepacia requires sophisticated genetic manipulation techniques. The most effective approach involves creating conditional knock-down mutants by replacing the native promoter of the sucC gene with a controllable promoter system, such as the rhamnose-inducible promoter described in previous studies .

The experimental workflow involves:

• Amplifying approximately 300 bp fragments of the sucC gene starting at the start codon

• Cloning these fragments into a vector containing a rhamnose-inducible promoter (such as pSC200)

• Introducing the recombinant plasmid into B. cenocepacia via triparental mating

• Selecting conditional mutants on media supplemented with rhamnose and appropriate antibiotics

• Testing essentiality by comparing growth in permissive (rhamnose-containing) versus non-permissive (glucose-containing) conditions

If the gene is essential, growth will occur only under permissive conditions. Complementation experiments, where the wild-type gene is provided in trans on a separate plasmid, should restore growth under non-permissive conditions, confirming that growth defects are specifically due to the targeted gene disruption .

What are the optimal conditions for expressing recombinant B. cenocepacia sucC protein in heterologous systems?

For optimal expression of recombinant B. cenocepacia sucC protein, the following methodological approach is recommended:

Expression System Selection:

• E. coli BL21(DE3) or similar strains designed for high-level protein expression

• Vector systems containing strong inducible promoters (T7 or tac)

• Incorporation of affinity tags (His6 or GST) at either N- or C-terminus for purification

Expression Conditions:

• Initial induction testing at various temperatures (16°C, 25°C, 30°C, 37°C)

• IPTG concentration optimization (typically 0.1-1.0 mM)

• Extended expression periods (4-16 hours) at lower temperatures (16-25°C) often yield higher amounts of soluble protein

• Supplementation with cofactors such as ATP and divalent cations (Mg²⁺) may enhance proper folding

Solubility Enhancement:

• Co-expression with molecular chaperones (GroEL/GroES) can improve folding efficiency

• Expression as fusion proteins with solubility-enhancing partners like thioredoxin or NusA

• Addition of low concentrations of detergents (0.05-0.1% Triton X-100) to lysis buffers

Monitoring of expression levels should be performed via SDS-PAGE and Western blotting using antibodies against the affinity tag or the protein itself. Optimization of these conditions is essential as Succinyl-CoA ligase is an enzyme with complex quaternary structure requiring proper assembly for activity.

What methods can be used for assessing Succinyl-CoA ligase activity in B. cenocepacia?

Several complementary methods can be employed to assess Succinyl-CoA ligase activity:

Spectrophotometric Coupled Assays:

• Forward reaction (Succinyl-CoA → Succinate): Couple ADP production to pyruvate kinase and lactate dehydrogenase reactions, monitoring NADH oxidation at 340 nm

• Reverse reaction (Succinate → Succinyl-CoA): Couple CoA incorporation with thiol-reactive dyes such as DTNB (5,5'-dithiobis-(2-nitrobenzoic acid))

Radiometric Assays:

• Use of ¹⁴C-labeled substrates (succinate or succinyl-CoA) followed by separation of products by thin-layer chromatography or HPLC

• Incorporation of ³²P from labeled ATP into ADP during the reaction

Mass Spectrometry-Based Analysis:

• Direct quantification of substrate consumption and product formation using LC-MS/MS

• Metabolic flux analysis through stable isotope labeling (¹³C-labeled substrates)

Real-Time Activity Monitoring:

• Bioluminescence assays coupling ATP production to luciferase reaction

• Fluorescence resonance energy transfer (FRET)-based sensors designed to detect conformational changes during catalysis

When comparing enzyme activity across experimental conditions, standardization is crucial. Typical reaction buffers contain Tris-HCl (pH 7.4-8.0), MgCl₂ (5-10 mM), KCl (50-100 mM), and substrates at saturating concentrations determined through preliminary kinetic analyses.

How can researchers effectively design genetic manipulation studies to investigate sucC function in B. cenocepacia?

Effective genetic manipulation strategies for investigating sucC function include:

Conditional Expression Systems:

• Rhamnose-inducible promoter replacement: Clone a fragment (~300 bp) of the sucC gene into a vector like pSC200 containing the rhamnose-inducible promoter

• Introduce the construct into B. cenocepacia through triparental mating

• Select recombinants where the native promoter is replaced with the rhamnose-inducible promoter

• Analyze phenotypes under varying rhamnose concentrations to achieve different expression levels

Site-Directed Mutagenesis:

• Design mutations targeting catalytic residues, substrate binding sites, or regulatory domains

• Introduce mutant alleles either chromosomally (using allelic exchange) or on complementation plasmids

• Evaluate the effect of mutations on enzyme activity, protein stability, and metabolic function

Interactome Analysis:

• Construct tagged versions of SucC for pull-down assays to identify protein interaction partners

• Use bacterial two-hybrid systems for targeted interaction studies

• Apply cross-linking approaches followed by mass spectrometry for unbiased interactome mapping

Complementation Studies:

• Generate broad-host-range complementation plasmids (e.g., pBBRMCS2 derivatives) expressing wild-type or mutant sucC under native or constitutive promoters

• Introduce complementation constructs into conditional mutants to verify phenotype rescue

• Cross-species complementation with sucC homologs from related organisms can provide insights into functional conservation

For all genetic manipulations, verification of construct integrity by sequencing and confirmation of expression levels by RT-qPCR or Western blotting are essential quality control steps.

How does sucC function integrate with other metabolic pathways in B. cenocepacia?

Succinyl-CoA ligase functions as a critical node connecting multiple metabolic pathways in B. cenocepacia:

TCA Cycle Integration:
The enzyme catalyzes the conversion of succinyl-CoA to succinate with concomitant ATP production, representing a key energy-conserving step in the TCA cycle.

Aromatic Compound Degradation:
B. cenocepacia can utilize the phenylacetic acid (PAA) degradation pathway to catabolize diverse aromatic compounds, including phenylalanine. This pathway culminates in the production of succinyl-CoA and acetyl-CoA that feed directly into the TCA cycle . The degradation pathway involves:

• Conversion of PAA to PAA-CoA by phenylacetyl-CoA ligase (PaaK)

• Epoxidation of PAA-CoA by multicomponent monooxygenase (PaaABCDE)

• Further degradation to yield succinyl-CoA and acetyl-CoA

Amino Acid Metabolism:
Catabolism of several amino acids (methionine, isoleucine, valine, threonine) generates propionyl-CoA, which is carboxylated and rearranged to form succinyl-CoA via the methylmalonyl-CoA pathway.

Heme Biosynthesis:
Succinyl-CoA serves as a precursor for δ-aminolevulinic acid synthesis, the first committed step in heme biosynthesis.

This metabolic interconnectivity positions sucC as a potential regulator of carbon flux between catabolic pathways and energy generation, making it particularly important during infection when the bacterium must adapt to changing nutrient availability.

How does the ADP-forming Succinyl-CoA ligase differ functionally from the GDP-forming variant in Burkholderia species?

B. cenocepacia, like many bacteria, possesses succinyl-CoA ligase variants with different nucleotide specificities. The ADP-forming (encoded by sucC and sucD) and GDP-forming variants exhibit distinct functional characteristics:

Comparative Characteristics:

Functional Significance:
The presence of isoforms with different nucleotide specificities provides metabolic flexibility, allowing the bacterium to maintain TCA cycle function while balancing the cellular adenylate and guanylate pools. This adaptation is particularly advantageous during environmental transitions or stress conditions where the ATP/GTP ratio may fluctuate.

Evolutionary Perspective:
The distribution of these variants across bacterial species suggests that nucleotide specificity evolved in response to the metabolic demands of different ecological niches. For pathogens like B. cenocepacia, maintaining both variants may contribute to metabolic robustness during infection.

Further research using precise biochemical characterization of both enzyme forms, coupled with metabolomics and fluxomics approaches, would provide deeper insights into their complementary roles in bacterial metabolism.

How might sucC contribute to B. cenocepacia pathogenesis during infection?

While sucC is primarily known for its metabolic function, several mechanisms potentially link it to B. cenocepacia pathogenesis:

Metabolic Adaptation During Infection:
Succinyl-CoA ligase enables efficient carbon utilization from host-derived compounds. During infection, B. cenocepacia may rely on alternative carbon sources such as amino acids or fatty acids that feed into the TCA cycle at the level of succinyl-CoA. The PAA degradation pathway, which generates succinyl-CoA as an end product, allows the bacterium to catabolize aromatic compounds that may be available in the host environment .

Energy Production for Virulence Factor Expression:
The energy generated through sucC activity supports the production and secretion of virulence factors. B. cenocepacia produces several virulence determinants, including the BC2L-C lectin which has been shown to trigger IL-8 production in airway epithelial cells . The expression and secretion of such factors require substantial energy input, which depends partly on efficient TCA cycle operation.

Survival Within Host Cells:
B. cenocepacia can survive within macrophages and epithelial cells, environments where the bacterium faces nutrient limitation and oxidative stress. TCA cycle activity, including sucC function, may be critical for maintaining bacterial viability under these challenging conditions.

Connection to Quorum Sensing:
The PAA degradation pathway in B. cenocepacia has been linked to quorum sensing regulation through the CepIR system . Disruption of this pathway leads to the accumulation of metabolic intermediates that affect virulence trait expression. Given that succinyl-CoA is a product of this pathway, sucC activity may indirectly influence quorum sensing-regulated virulence.

Biofilm Formation:
The metabolic activity supported by sucC may contribute to the energy requirements for biofilm formation, a key virulence trait that enhances B. cenocepacia persistence in the host.

Experimental approaches to investigate these potential roles could include comparative transcriptomics of wild-type and sucC conditional mutants during infection, metabolic profiling during host cell interaction, and virulence testing in appropriate model systems.

What experimental models are most appropriate for studying the role of sucC in B. cenocepacia infection?

A multi-level approach using complementary experimental models provides the most comprehensive assessment of sucC's role in infection:

Cell Culture Models:

• Human bronchial epithelial cells (16HBE14o-, CFBE41o-): Evaluate bacterial adhesion, invasion, intracellular survival, and inflammatory response

• Macrophage models (THP-1, primary human macrophages): Assess phagocytosis efficiency, intracellular replication, and macrophage activation

• 3D airway epithelial models: Examine bacterial interaction with differentiated respiratory epithelium, including mucociliary clearance effects

Simple Organism Models:

• Caenorhabditis elegans: Offers a rapid virulence assessment platform where B. cenocepacia pathogenicity has been previously characterized

• Galleria mellonella (wax moth larva): Provides a simple infection model with a primitive innate immune system

• Drosophila melanogaster: Enables genetic manipulation of host factors for interaction studies

Mammalian Models:

• Chronic lung infection models in mice or rats: Mimic the persistent infection characteristic of cystic fibrosis patients

• Acute pneumonia models: Assess virulence during rapid pulmonary infection

• Zebrafish embryo model: Offers transparency for real-time imaging of infection progression

Experimental Design Considerations:

• Use conditional mutants where sucC expression can be modulated during different infection stages

• Include appropriate complemented strains to confirm phenotype specificity

• Employ metabolomics approaches to track changes in metabolic profiles during infection

• Combine with transcriptomics to identify host responses specific to sucC manipulation

• Consider mixed infection experiments (wild-type vs. sucC mutant) to assess competitive fitness

For interpretation of results, it's essential to distinguish between direct effects of sucC disruption and indirect consequences from general growth impairment. Time-course analyses and careful metabolic profiling can help separate these factors.

How does the metabolic function of sucC potentially interact with host immune responses?

The metabolic function of sucC may interact with host immune responses through several interconnected mechanisms:

Metabolite-Mediated Immunomodulation:

• Succinate, the product of the reaction catalyzed by Succinyl-CoA ligase, can function as a signaling molecule that modulates immune cell function

• Accumulation or depletion of TCA cycle intermediates due to altered sucC activity could potentially affect host cell metabolism and signaling

• These metabolic changes may influence inflammatory cytokine production by host cells

Energy-Dependent Immune Evasion:

• Efficient energy production through sucC activity enables B. cenocepacia to produce factors that modulate host immune responses

• The BC2L-C lectin from B. cenocepacia has been shown to trigger IL-8 production in airway epithelial cells in a carbohydrate-independent manner

• This proinflammatory response contributes to the dysregulated inflammation observed in B. cenocepacia infections

Survival Under Immune Attack:

• sucC function supports bacterial adaptation to the metabolic stresses imposed by immune defenses

• Within phagocytes, bacteria face nutrient limitation and oxidative stress, conditions where TCA cycle functionality becomes crucial

• Metabolic flexibility enabled by sucC allows the bacterium to utilize alternative carbon sources available in immune cells

Connection to Quorum Sensing and Collective Behavior:

• B. cenocepacia uses quorum sensing systems like CepIR to coordinate virulence gene expression

• Metabolic pathways that generate succinyl-CoA, such as the PAA degradation pathway, have been linked to altered quorum sensing activity

• Through these connections, sucC activity may indirectly influence the expression of virulence factors that interact with host immunity

Experimental Approach to Study These Interactions:

• Transcriptomics of host cells exposed to wild-type vs. sucC conditional mutants

• Metabolomic profiling of the host-pathogen interface during infection

• Analysis of cytokine production and immune cell activation in response to bacteria with altered sucC expression

• Fluorescence microscopy to track bacterial metabolism within host cells using reporter systems linked to metabolic activity

These investigations would provide valuable insights into how bacterial central metabolism influences host-pathogen interactions during B. cenocepacia infection.

What structural biology approaches would be most informative for understanding B. cenocepacia Succinyl-CoA ligase function?

Several complementary structural biology approaches would provide comprehensive insights into B. cenocepacia Succinyl-CoA ligase function:

X-ray Crystallography:

• High-resolution structure determination of the SucC/SucD heterodimer in various functional states

• Co-crystallization with substrates (succinyl-CoA, ADP/ATP), products, and inhibitors

• Analysis of enzyme-ligand interactions through difference electron density maps

• Resolution target: 1.5-2.5 Å for detailed mechanistic insights

Cryo-Electron Microscopy (Cryo-EM):

Nuclear Magnetic Resonance (NMR) Spectroscopy:

• Dynamics studies of specific domains using isotopically labeled proteins

• Investigation of substrate binding and conformational changes in solution

• Chemical shift perturbation experiments to map interaction surfaces

• Best suited for individual domains or subunits rather than the entire complex

Small-Angle X-ray Scattering (SAXS):

• Low-resolution envelope determination in solution conditions

• Analysis of conformational flexibility and oligomeric states

• Complementary to crystallography for validating physiological assemblies

• Can be conducted under various solution conditions to assess stability

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

• Mapping of solvent accessibility and conformational dynamics

• Identification of regions undergoing structural changes upon substrate binding

• Comparison of wild-type and mutant proteins to understand allosteric regulation

Integrative Structural Biology Workflow:

Specific challenges for B. cenocepacia Succinyl-CoA ligase include potential flexibility between domains and the transient nature of certain catalytic states. Using a combination of techniques allows researchers to overcome limitations of individual methods.

How can systems biology approaches be applied to understand the network effects of sucC manipulation in B. cenocepacia?

Systems biology approaches offer powerful frameworks for understanding the broader impact of sucC manipulation:

Multi-omics Integration:

Network Analysis Methods:

• Genome-scale metabolic modeling to predict flux redistributions upon sucC perturbation

• Network topology analysis to identify critical nodes connected to sucC function

• Bayesian network inference to discover causal relationships between observed changes

• Constraint-based modeling approaches such as Flux Balance Analysis (FBA) to predict growth phenotypes

Dynamic Modeling:

• Develop ordinary differential equation (ODE) models of the TCA cycle including sucC

• Incorporate regulatory mechanisms affecting sucC expression and activity

• Simulate time-course responses to environmental perturbations

• Validate model predictions with experimental time-series data

Experimental Design for Systems Approaches:

• Generate time-resolved data using sucC conditional mutants under varying expression levels

• Include multiple environmental conditions to capture context-dependent effects

• Perform perturbation experiments targeting connected pathways

• Validate key predictions using targeted genetic or biochemical approaches

Integration with Host-Pathogen Interaction Data:

• Dual RNA-seq to simultaneously capture bacterial and host transcriptomes during infection

• Host-pathogen protein interaction mapping to identify direct interfaces

• Metabolic exchanges between pathogen and host cells

These systems approaches would reveal how sucC functions within the broader metabolic and regulatory networks of B. cenocepacia, providing insights beyond what could be achieved through reductionist approaches alone.

What are the challenges and solutions for developing inhibitors targeting Succinyl-CoA ligase for potential therapeutic applications?

Developing effective inhibitors against bacterial Succinyl-CoA ligase presents both significant challenges and promising opportunities:

Key Challenges:

• Selectivity: Achieving sufficient selectivity for bacterial over human Succinyl-CoA ligase is difficult due to conserved catalytic mechanisms.

• Essential Nature: As a potential essential gene, strong inhibition of Succinyl-CoA ligase may exert high selective pressure for resistance development.

• Structural Complexity: The heterodimeric structure and conformational changes during catalysis complicate rational inhibitor design.

• Compound Access: B. cenocepacia has multiple efflux pumps and an impermeable outer membrane, limiting inhibitor entry.

• Metabolic Redundancy: Alternative metabolic pathways may compensate for partial inhibition.

Strategic Solutions:

• Structure-Based Design Approaches:

  • Target bacterial-specific structural features identified through comparative structural analysis

  • Design allosteric inhibitors that lock the enzyme in inactive conformations

  • Develop covalent inhibitors targeting non-conserved cysteine residues

• Chemical Biology Strategies:

  • Fragment-based drug discovery to identify initial binding scaffolds

  • Activity-based protein profiling to develop selective probes

  • Photoaffinity labeling to identify novel binding sites

• Innovative Screening Approaches:

  • Whole-cell screening with metabolomic readouts to identify compounds affecting TCA cycle function

  • Conditional mutant-based screening to identify compounds synergistic with sucC depletion

  • Thermal shift assays to identify compounds that destabilize the enzyme complex

• Delivery Solutions:

  • Siderophore-conjugated inhibitors to hijack iron uptake systems

  • Nanoparticle formulations to enhance compound delivery

  • Combination with outer membrane permeabilizers to increase access

• Resistance Mitigation:

  • Dual-targeting compounds affecting multiple steps in central metabolism

  • Development of multi-targeting inhibitors with activity against additional essential processes

  • Combination therapy approaches with existing antibiotics