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

What is the genomic context of the sucC gene in Burkholderia multivorans and how does it compare to other bacterial species?

The sucC gene in B. multivorans encodes the beta subunit of Succinyl-CoA ligase [ADP-forming], a critical enzyme in the tricarboxylic acid (TCA) cycle. Similar to other bacteria, the gene is typically part of an operon structure that includes sucD (encoding the alpha subunit). This organization allows for coordinated expression of both subunits necessary for the formation of the functional heterodimeric enzyme.

In B. multivorans, genome analysis has revealed significant variation between different strains, with large structural genomic variations observed between isolates from different patients . These variations include active roles for transposases and mobile prophage elements, which could potentially affect the genomic context of metabolic genes including sucC . Researchers should note that genomic analysis of clinical isolates shows limited within-patient evolution but high between-patient strain diversity, suggesting that environmental reservoirs maintain microdiverse populations .

For comparative genomic studies of sucC, researchers should:

• Use multilocus sequence typing (MLST) to identify strain types

• Apply both short and long-read sequencing technologies to accurately map genomic context

• Compare sequence conservation across multiple Burkholderia species, particularly B. cenocepacia, which shares many pathogenic features

What expression systems are most effective for producing active recombinant B. multivorans sucC protein?

For optimal expression of recombinant B. multivorans sucC, consider the following methodological approaches:

E. coli-based expression systems:

• BL21(DE3) strain is preferred due to its reduced protease activity

• pET vector systems with T7 promoter offer high-level expression control

• Codon optimization may be necessary as B. multivorans has a different codon usage bias than E. coli

Critical optimization parameters:

• Induction temperature: Lower temperatures (16-20°C) often improve protein folding

• IPTG concentration: 0.1-0.5 mM is typically sufficient; higher concentrations may lead to inclusion bodies

• Media supplementation: Addition of cofactors like magnesium and ATP may enhance proper folding

• Co-expression with chaperones: GroEL/GroES can improve yield of correctly folded protein, especially relevant as GroEL has been identified as an immunoreactive protein in B. multivorans

Alternative expression systems:

• Pseudomonas-based expression systems may provide a more native-like environment for protein folding

• Cell-free protein synthesis systems allow rapid screening of expression conditions without cellular constraints

For functional expression, co-expression with the alpha subunit (sucD) is often necessary, as the active enzyme functions as a heterodimer. Expression timing and stoichiometry between the subunits should be carefully controlled.

What purification protocols yield optimal activity for recombinant B. multivorans sucC?

A systematic purification approach is essential for maintaining enzyme activity while achieving high purity:

Affinity chromatography options:

• His-tagged purification: 6xHis tag at N-terminus is preferred for minimal interference with enzyme activity

• GST-fusion: Provides solubility enhancement but may require tag removal for activity assays

• Tandem affinity purification: Consider dual tagging (His and Strep) for exceptional purity

Purification buffer optimization:

• Include 10-20% glycerol to stabilize the enzyme structure

• Maintain pH between 7.4-8.0 (optimum for activity)

• Add 1-5 mM DTT or 2-mercaptoethanol to prevent oxidation of cysteine residues

• Include 1-2 mM EDTA to remove divalent metal ions that may promote oxidation

Activity preservation steps:

• Perform all steps at 4°C

• Add protease inhibitors (e.g., PMSF, Complete™) during initial lysis

• Include substrate analogs (e.g., 0.5 mM succinate) to stabilize the enzyme

• Consider dialysis rather than dilution for buffer exchange to minimize mechanical stress

Critical quality control measures:

• Verify purity by SDS-PAGE (expected MW ~41 kDa)

• Confirm identity by mass spectrometry

• Assess oligomeric state by size exclusion chromatography (functional enzyme should exist as heterodimer with alpha subunit)

• Verify activity using coupled enzyme assays

How can researchers verify the enzymatic activity of recombinant B. multivorans sucC in vitro?

Verification of enzymatic activity requires carefully designed assays that monitor the reversible reaction:

Succinyl-CoA + Pi + ADP ⇌ Succinate + CoA + ATP

Forward reaction (succinyl-CoA to succinate):

• Coupled spectrophotometric assay: Link ATP production to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Monitor decrease in absorbance at 340 nm as NADH is oxidized

  • Reaction mixture includes: succinyl-CoA (0.1-0.5 mM), ADP (1-2 mM), Pi (5-10 mM), MgCl₂ (5 mM), PEP (1 mM), NADH (0.2 mM), pyruvate kinase and lactate dehydrogenase

• Luciferase-based ATP detection:

  • More sensitive than spectrophotometric methods

  • Allows endpoint or continuous measurement

  • Compatible with high-throughput screening formats

Reverse reaction (succinate to succinyl-CoA):

• Direct measurement of succinyl-CoA formation:

  • HPLC-based quantification of succinyl-CoA

  • Reaction mixture includes: succinate (5-10 mM), CoA (0.5-1 mM), ATP (2-5 mM), MgCl₂ (5 mM)

Kinetic analysis:
Determine the following parameters using varied substrate concentrations:

• Km for succinyl-CoA, ADP, Pi (forward reaction)

• Km for succinate, CoA, ATP (reverse reaction)

• Vmax in both directions

• Effect of pH (optimal typically 7.4-8.0)

• Effect of temperature (optimal typically 30-37°C)

It's worth noting that mammalian SCS isoforms show tissue-specific preferences for either ATP or GTP . When characterizing B. multivorans sucC, confirm its specificity for ADP/ATP rather than GDP/GTP with proper controls.

What is the role of sucC in B. multivorans metabolism during cystic fibrosis infection?

The sucC gene product plays a pivotal role in B. multivorans metabolism during infection:

Central metabolic functions:

• TCA cycle operation: Catalyzes the only substrate-level phosphorylation step in the cycle, generating ATP

• Carbon flux regulation: Controls the balance between catabolic and anabolic pathways

• Metabolic adaptation: Likely involved in bacterial adaptation to the nutrient-limited CF lung environment

Infection-specific metabolic adaptations:

• Genomic analysis of clinical isolates reveals that B. multivorans undergoes limited within-patient evolution but shows high between-patient strain diversity

• Key metabolic genes, potentially including sucC, may be targets for parallel adaptations across multiple patients, indicating that strain-specific genomic backgrounds may dictate adaptation routes within the CF lung

• The microaerobic environment in CF lungs may shift metabolic flux through the TCA cycle, potentially altering the role of sucC

Research methodologies to investigate:

• Comparative transcriptomics: RNA-seq comparing expression in laboratory media versus CF sputum samples

• Metabolic flux analysis: 13C-labeling to track carbon flow through central metabolism during infection

• Proteomics: Quantitative analysis of sucC expression levels in clinical isolates

• Knockout studies: Creation of sucC mutants to assess effects on growth and virulence

Table 1: Predicted Metabolic Changes in B. multivorans During CF Lung Infection

How does sucC expression in B. multivorans differ from that in B. cenocepacia, and what are the implications for pathogenicity?

Both B. multivorans and B. cenocepacia are clinically relevant species within the Burkholderia cepacia complex, but with distinct patterns of virulence and metabolism:

Comparative expression patterns:

• Immunoproteomic analysis has identified multiple proteins that are differentially expressed between these species during human infection

• While 15 proteins were found to be immunogenic across both species, 14 immunoreactive proteins were exclusive to B. cenocepacia strains, and 15 were exclusive to B. multivorans

• Key metabolic enzymes, including elongation factor-Tu, were identified as immunoreactive across all strains examined

Methodological approaches to investigate differences:

• Comparative transcriptomics:

  • RNA-seq analysis of both species under identical conditions

  • qRT-PCR validation of differential expression

  • Ribosome profiling to assess translational efficiency

• Proteomic comparison:

  • 2D-PAGE coupled with mass spectrometry

  • SILAC labeling for quantitative comparison

  • Analysis of post-translational modifications

• Functional genomics:

  • Creation of isogenic mutants in both species

  • Complementation studies with cross-species genes

  • Competition assays to determine fitness effects

Pathogenicity implications:

• Differences in central metabolism may contribute to the distinct infection patterns observed between these species

• Metabolic adaptation may influence persistence in different host niches

• Energy production through sucC activity could support virulence factor expression and antibiotic resistance mechanisms

What experimental approaches can resolve contradictory data regarding sucC's role in B. multivorans virulence?

Resolving contradictory findings requires systematic approaches that address experimental variables and biological complexity:

Standardization of experimental systems:

• Strain characterization:

  • Complete genome sequencing of all studied isolates

  • MLST typing to place strains in phylogenetic context (as performed in Baldwin et al.)

  • Verification of strain identity before and after experiments

• Infection model standardization:

  • Clearly defined in vitro cell culture conditions (cell types, passage number, media composition)

  • Standardized animal models with defined genetic backgrounds

  • Consideration of alternative models (e.g., Galleria mellonella, ex vivo lung tissue)

Resolution of mechanistic contradictions:

• Genetic approaches:

  • Clean deletion mutants with complementation controls

  • Conditional expression systems to control timing and level of expression

  • Site-directed mutagenesis to separate catalytic from structural roles

• Biochemical verification:

  • In vitro enzyme assays to confirm activity differences

  • Metabolomic profiling to identify downstream effects

  • Flux analysis to determine metabolic consequences

Addressing biological complexity:

• Host-pathogen interaction studies:

  • Transcriptional response analysis of both host and pathogen

  • Single-cell studies to address population heterogeneity

  • Temporal analysis to capture dynamic interactions

• Environmental factor assessment:

  • Oxygen tension effects on metabolism and virulence

  • Nutrient availability influence on metabolic pathway utilization

  • pH and other physiochemical parameters

Table 2: Framework for Resolving Contradictory Data in sucC Research

What structural features confer ADP specificity to B. multivorans Succinyl-CoA ligase rather than GDP specificity?

Succinyl-CoA synthetase exists in forms specific for either ATP/ADP or GTP/GDP, with the specificity determined by the β-subunit (encoded by sucC in the case of the ADP-forming enzyme) :

Structural determinants of nucleotide specificity:

• β-subunit nucleotide-binding domain:

  • ATP grasp domain located near the N-terminus of the β-subunit

  • Specific amino acid residues within this domain create hydrogen bonding patterns that favor adenine over guanine

• Critical residues:

  • Glu197β plays a role in the phosphorylation and dephosphorylation of the catalytic histidine residue (His246α)

  • Mutagenesis studies in E. coli have identified specific residues that can convert ADP specificity to GDP specificity and vice versa

Experimental approaches to investigate specificity:

• Homology modeling:

  • Generate structural models based on known crystal structures

  • Identify putative specificity-determining residues

• Site-directed mutagenesis:

  • Create point mutations at predicted specificity-determining residues

  • Assess changes in nucleotide preference through activity assays

• Protein crystallography:

  • Obtain crystal structures with bound nucleotides

  • Compare binding site architecture with GDP-specific variants

• Molecular dynamics simulations:

  • Model nucleotide binding and release

  • Calculate binding energies for different nucleotides

In mammals, ATP-specific SCS is typically found in tissues involved in catabolic metabolism (brain, heart, muscle), while GTP-specific SCS is found in anabolic tissues (liver, kidney) . The functional significance of ADP specificity in B. multivorans may relate to its metabolic adaptations during infection.

How can isotope labeling and metabolic flux analysis be applied to understand the role of sucC in B. multivorans metabolism?

Isotope labeling combined with metabolic flux analysis provides powerful insights into carbon flow through central metabolism:

Experimental design for flux analysis:

• Selection of labeled substrates:

  • [1-13C]glucose: Differentiates pentose phosphate pathway from glycolysis

  • [U-13C]glucose: Comprehensive labeling pattern across central metabolism

  • [1,2-13C]acetate: Directly traces TCA cycle flux

  • [U-13C]glutamate: Probes anaplerotic reactions

• Culture conditions:

  • Defined minimal media with labeled substrate as sole carbon source

  • Steady-state growth to achieve isotopic equilibrium

  • Physiologically relevant conditions (oxygen limitation, pH, etc.)

Analytical methods:

• Metabolite extraction and derivatization:

  • Rapid quenching to prevent metabolic changes

  • Acid extraction for polar metabolites

  • Chemical derivatization for GC-MS analysis

• Mass spectrometry analysis:

  • GC-MS for primary metabolites

  • LC-MS/MS for coenzyme A derivatives including succinyl-CoA

  • High-resolution MS for accurate isotopologue distribution

• NMR spectroscopy:

  • Provides positional isotopic enrichment information

  • Complements MS data for flux model validation

Flux calculation and modeling:

• Metabolic network reconstruction:

  • Genome-based pathway mapping

  • Integration of experimental evidence for active pathways

  • Incorporation of biomass requirements

• Computational flux estimation:

  • 13C-FLUX or similar software for flux calculation

  • Sensitivity analysis to identify key parameters

  • Statistical evaluation of flux solutions

Applications to sucC function:

• Wild-type vs. sucC mutant comparison:

  • Identify metabolic rerouting in response to altered TCA cycle function

  • Quantify energetic consequences of sucC modulation

• Infection-relevant conditions:

  • Compare flux distributions under aerobic vs. microaerobic conditions

  • Assess carbon source preferences during simulated infection

• Antibiotic response:

  • Evaluate metabolic adaptation to antibiotic stress

  • Identify potential metabolic vulnerabilities

What crystallization and structural analysis techniques have been most successful for studying recombinant B. multivorans sucC?

While specific crystallization conditions for B. multivorans sucC are not widely reported, the following approaches have proven successful for related enzymes:

Protein preparation for crystallization:

• Homogeneity optimization:

  • Size exclusion chromatography as final purification step

  • Dynamic light scattering to confirm monodispersity

  • Thermal shift assays to identify stabilizing buffer conditions

• Complex formation:

  • Co-purification or reconstitution with alpha subunit (sucD product)

  • Addition of substrates, products, or stable analogs

  • Consideration of ternary complexes with nucleotides

Crystallization screening:

• Initial condition identification:

  • Commercial sparse matrix screens (Hampton, Molecular Dimensions, Qiagen)

  • Sitting drop vapor diffusion at 4°C and 18°C

  • Microseeding from initial crystals

• Optimization strategies:

  • Fine grid screens around promising conditions

  • Additive screens to improve crystal quality

  • Streak seeding for crystal quality improvement

Data collection considerations:

• Cryoprotection optimization:

  • Glycerol, ethylene glycol, or PEG 400 supplementation

  • Flash cooling in liquid nitrogen

  • Room temperature data collection for sensitive crystals

• Synchrotron data collection:

  • High-brilliance beamlines for microcrystals

  • Strategy optimization for maximum completeness

  • Multiple datasets for radiation-sensitive crystals

Alternative structural approaches:

• Cryo-electron microscopy:

  • Single-particle analysis for high-resolution structure

  • Particularly valuable for capturing different conformational states

• Small-angle X-ray scattering (SAXS):

  • Solution-based structural analysis

  • Information on conformational changes upon substrate binding

• Hydrogen-deuterium exchange mass spectrometry:

  • Maps protein dynamics and ligand interactions

  • Complements static crystal structures

For B. multivorans sucC, researchers should note that conformational changes are likely critical for function, as catalytic histidine residues must move approximately 35 Å during the catalytic cycle to facilitate phosphoryl transfer .

How do mutations in sucC affect B. multivorans virulence and fitness in cystic fibrosis infections?

The relationship between sucC function and virulence requires careful investigation using multiple approaches:

Genetic manipulation strategies:

• Mutant construction:

  • Clean deletion mutants using allelic exchange

  • Point mutations to alter catalytic activity without structural disruption

  • Conditional expression systems for essential genes

• Complementation analysis:

  • Wild-type gene restoration

  • Cross-complementation with orthologs from related species

  • Site-directed mutants with varying levels of activity

Virulence phenotype assessment:

• In vitro models:

  • Adhesion to respiratory epithelial cells

  • Invasion efficiency

  • Intracellular survival

  • Biofilm formation capacity

• Ex vivo models:

  • Survival in CF sputum samples

  • Interaction with primary CF airway epithelial cells

  • Response to host defense mechanisms

• In vivo models:

  • Appropriate animal models (considering limitations)

  • Bacterial burden quantification

  • Inflammatory response assessment

  • Survival analysis

Fitness evaluation:

• Competition assays:

  • Mixed infections with wild-type and mutant strains

  • Calculation of competitive index

  • Long-term evolution experiments

• Stress resistance:

  • Oxidative stress tolerance

  • Antibiotic susceptibility

  • Nutrient limitation survival

Table 3: Predicted Phenotypic Effects of sucC Mutations

Genomic analysis of clinical isolates suggests that B. multivorans undergoes specific adaptations during CF infection, with a set of 30 parallel adaptations identified across multiple patients . Investigating whether sucC is among these adaptation targets could provide insights into its role in virulence.

What are the methodological challenges in distinguishing the metabolic contributions of sucC from other TCA cycle enzymes in B. multivorans?

The interconnected nature of central metabolism creates significant challenges for isolating the specific contributions of sucC:

Network complexity challenges:

• Pathway redundancy:

  • Alternative routes for carbon flux around blocked steps

  • Anaplerotic reactions that can bypass certain TCA cycle steps

  • Compensatory regulation of other enzymes

• Pleiotropic effects:

  • Changes in redox balance affecting multiple pathways

  • Altered energy charge influencing numerous cellular processes

  • Accumulation of metabolites with regulatory functions

Experimental approaches to address challenges:

• Genetic strategies:

  • Construction of conditional mutants for targeted expression control

  • Tunable promoter systems to create varying levels of expression

  • Double mutants to block compensatory pathways

• Systems biology approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Computational modeling of metabolic networks

  • Time-resolved sampling to capture dynamic responses

• Biochemical approaches:

  • In vitro reconstitution of connected reactions

  • Isotope dilution experiments to quantify flux through specific steps

  • Metabolite profiling with stable isotope tracing

Analytical considerations:

• Metabolite measurements:

  • Rapid sampling to capture true metabolic state

  • Appropriate extraction methods for different metabolite classes

  • Absolute quantification rather than relative abundance

• Enzyme activity assays:

  • Development of specific assays for individual enzymes

  • Consideration of in vivo vs. in vitro activity differences

  • Analysis of regulatory modifications

• Data integration:

  • Correlation analysis across multiple datasets

  • Machine learning approaches for pattern recognition

  • Bayesian network analysis for causality inference

How can CRISPR-Cas9 gene editing be optimized for studying sucC function in B. multivorans?

CRISPR-Cas9 systems offer powerful tools for precise genetic manipulation of B. multivorans:

CRISPR system optimization:

• Vector system selection:

  • All-in-one vectors vs. dual-plasmid systems

  • Inducible Cas9 expression to reduce toxicity

  • Temperature-sensitive replicons for plasmid curing

• sgRNA design:

  • Multiple sgRNA testing for optimal targeting

  • Avoidance of potential off-target sites

  • Assessment of PAM site accessibility in Burkholderia genomes

• Delivery methods:

  • Electroporation conditions optimized for Burkholderia

  • Conjugation-based transfer from E. coli donors

  • Consideration of restriction barriers

Editing strategies for sucC functional analysis:

• Knockout approaches:

  • Complete gene deletion using homology-directed repair

  • Frame-shift mutations for gene inactivation

  • Premature stop codon introduction

• Point mutation introduction:

  • Catalytic site mutations to alter activity

  • Nucleotide specificity determinant alterations

  • Regulatory site modifications

• Regulatory element manipulation:

  • Promoter replacements for controlled expression

  • Introduction of degradation tags for protein turnover control

  • Ribosome binding site modifications for translation efficiency

Validation and phenotyping:

• Genetic verification:

  • Sanger sequencing of modified regions

  • Whole genome sequencing to detect off-target effects

  • RT-PCR to confirm expression changes

• Functional confirmation:

  • Enzyme activity assays

  • Metabolite profiling

  • Growth phenotype characterization

• Advanced phenotyping:

  • Virulence factor expression

  • Biofilm formation capacity

  • Antibiotic susceptibility profiles

Table 4: Optimization Parameters for CRISPR-Cas9 Editing in B. multivorans

How does the evolutionary conservation of sucC across the Burkholderia cepacia complex inform its essential functions?

Evolutionary analysis of sucC provides insights into its functional importance and adaptation:

Comparative genomic approaches:

• Sequence conservation analysis:

  • Multiple sequence alignment across Burkholderia species

  • Identification of highly conserved residues

  • Calculation of selection pressure (dN/dS ratios)

• Structural conservation mapping:

  • Projection of conservation scores onto structural models

  • Identification of conserved functional domains

  • Analysis of species-specific variations

• Synteny analysis:

  • Examination of gene neighborhood conservation

  • Identification of co-evolved gene clusters

  • Detection of genomic rearrangements affecting sucC

Functional implications assessment:

• Conservation patterns:

  • Catalytic residues typically show highest conservation

  • Species-specific variations may indicate adaptation

  • Lineage-specific insertions/deletions may relate to specific niches

• Selection analysis:

  • Positive selection suggests adaptive evolution

  • Negative selection indicates functional constraints

  • Neutral evolution suggests functional redundancy

• Co-evolution networks:

  • Identification of proteins that co-evolve with sucC

  • Inference of functional interactions

  • Detection of compensatory mutations

Experimental validation:

• Cross-species complementation:

  • Testing functional conservation through heterologous expression

  • Identification of species-specific functional differences

  • Domain swapping to map functional regions

• Ancestral sequence reconstruction:

  • Resurrection of predicted ancestral enzymes

  • Comparison of kinetic properties

  • Analysis of specificity evolution

Multilocus sequence typing (MLST) analysis has identified globally distributed B. multivorans sequence types associated with human infection , providing a framework for understanding strain diversity and evolution. Combining this population structure information with specific analysis of sucC could reveal patterns of adaptation related to metabolic function.

How does environmental oxygen tension affect sucC expression and activity in B. multivorans during infection?

The cystic fibrosis lung presents a complex, often microaerobic environment that influences bacterial metabolism:

Oxygen-responsive regulation:

• Transcriptional regulation:

  • Identification of oxygen-responsive promoter elements

  • Analysis of transcription factor binding sites

  • Characterization of regulatory networks

• Post-transcriptional control:

  • mRNA stability under varying oxygen conditions

  • sRNA-mediated regulation

  • Translational efficiency changes

Experimental approaches:

• Controlled oxygen environments:

  • Continuous culture systems with defined oxygen tension

  • Microfluidic devices with oxygen gradients

  • Anaerobic chambers with controlled oxygen exposure

• Gene expression analysis:

  • qRT-PCR for targeted gene expression analysis

  • RNA-seq for genome-wide transcriptional response

  • Proteomics to confirm translation of transcriptional changes

• Enzyme activity measurements:

  • In vitro activity assays under varying oxygen levels

  • In vivo activity inference through metabolite ratios

  • Structural analysis of oxygen-dependent modifications

Infection-relevant models:

• Artificial sputum medium:

  • Mimics CF lung environment

  • Can be prepared with varying oxygen levels

  • Allows for controlled experimental manipulation

• Biofilm models:

  • Natural oxygen gradients form within biofilms

  • Spatial transcriptomics to map expression patterns

  • Confocal microscopy with oxygen-sensitive probes

• Ex vivo and in vivo systems:

  • CF lung tissue explants

  • Animal models with tissue oxygen measurements

  • Clinical sample analysis when available

Table 5: Predicted Metabolic Shifts Under Varying Oxygen Conditions