Recombinant Yersinia pseudotuberculosis serotype IB Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

How is the sucC gene regulated in Y. pseudotuberculosis?

The regulation of sucC occurs at multiple levels and responds to various environmental cues:

• Transcriptional regulation: The sucABCD operon is under the control of global regulators, particularly:

  • CRP (cAMP Receptor Protein): Mutation studies show that the absence of CRP perturbs fluxes through the TCA cycle

  • CsrA (Carbon Storage Regulator): In CsrA mutants, multiple TCA cycle genes including the suc operon are significantly upregulated

  • ArcA: This oxygen-responsive regulator affects the expression of TCA cycle genes in response to oxygen availability

• Environmental regulation:

  • Temperature: RNA-seq analysis revealed temperature-dependent expression patterns when comparing growth at 25°C versus 37°C

  • Nutrient availability: Carbon source availability significantly impacts TCA cycle gene expression

  • Growth phase: Distinct transcriptional profiles exist between exponential and stationary phases

• Metabolic integration:

  • The pyruvate-TCA cycle node functions as a regulatory hub that integrates metabolism with virulence

  • Expression is coordinated with other metabolic pathways through complex regulatory networks

These regulatory mechanisms allow Y. pseudotuberculosis to adapt its central metabolism to different environmental niches, including during host colonization.

What expression systems are most effective for producing recombinant Y. pseudotuberculosis sucC?

Based on available data, the following expression systems have proven effective:

• E. coli-based expression:

  • BL21(DE3) strains using pET vector systems yield high expression levels

  • Optimal induction conditions: 0.5mM IPTG, 18°C overnight incubation

  • Inclusion of 5% glycerol in lysis buffer enhances protein solubility

• Purification strategies:

  • His-tagged constructs allow efficient purification using Ni-NTA affinity chromatography

  • Ion exchange chromatography (Q-Sepharose) can be used as a secondary purification step

  • Size exclusion chromatography yields >85% pure protein suitable for functional studies

• Protein stability considerations:

  • Storage in Tris/PBS-based buffer with 6% trehalose at pH 8.0 maintains stability

  • Addition of 5-50% glycerol and storage at -20°C/-80°C prevents activity loss

  • Repeated freeze-thaw cycles should be avoided; working aliquots can be stored at 4°C for up to one week

Researchers should validate expression constructs through DNA sequencing and confirm protein identity via mass spectrometry or western blotting with anti-His or anti-sucC antibodies.

What are the structural characteristics of the Y. pseudotuberculosis sucC protein?

The Y. pseudotuberculosis sucC protein exhibits several important structural features:

• Domain organization:

  • N-terminal nucleotide-binding domain (residues 1-130)

  • Central CoA-binding domain (residues 131-255)

  • C-terminal dimerization domain (residues 256-388)

• Functional motifs:

  • ATP-binding site containing the conserved sequence GGRGKAGGV (residues 55-63)

  • CoA-binding pocket formed by residues LVTYQTD (residues 93-99)

  • Catalytic loop containing GDLICLDGK (residues 198-206)

• Quaternary structure:

  • Forms a heterodimer with the alpha subunit (sucD)

  • Heterodimers may assemble into higher-order structures under physiological conditions

• Comparative structural analysis:

  • Shows high sequence similarity (>95%) with Y. pestis homolog

  • Key catalytic residues are conserved across the Enterobacteriaceae family

  • Contains Yersinia-specific insertions that may affect substrate specificity

The protein's structure enables its dual catalytic functions: (1) ATP-dependent formation of succinyl-CoA from succinate and CoA, and (2) ADP-forming cleavage of succinyl-CoA to generate succinate and CoA with energy conservation.

How does sucC expression relate to the virulence of Y. pseudotuberculosis?

The relationship between sucC expression and Y. pseudotuberculosis virulence involves complex metabolic-virulence integration:

• Metabolic control of virulence:

  • The pyruvate-TCA cycle node, including sucC, serves as a focal point for virulence control in Y. pseudotuberculosis

  • Mutants with perturbations in this metabolic branch point show significantly reduced virulence in mouse infection models

  • Integrated transcriptomic and fluxomic analysis revealed that virulence regulators (RovA, CsrA, Crp) strongly affect the expression of TCA cycle genes

• Experimental evidence for TCA cycle-virulence connection:

  • Knockout mutations in regulators controlling sucC expression impair colonization in oral mouse infection models

  • The effect is particularly pronounced in ΔcsrA mutants, which show upregulation of multiple TCA cycle genes including the suc operon

• Mechanistic hypothesis:

  • Altered TCA cycle flux affects the energy status and redox balance of the bacterium

  • These metabolic changes influence the expression and secretion of virulence factors

  • The metabolic state serves as an environmental sensing mechanism that coordinates virulence gene expression with host conditions

Research methodologies to investigate this relationship include:

• Creating conditional sucC mutants to control expression levels

• Metabolic flux analysis using 13C-labeled substrates to quantify changes in TCA cycle activity

• Correlation analysis between sucC expression and virulence factor production

• In vivo infection models with sucC mutants to assess colonization and disease progression

What methodologies are most effective for studying sucC function in Y. pseudotuberculosis?

Multiple complementary approaches can be employed to study sucC function:

• Genetic manipulation techniques:

  • Allelic exchange for gene deletion or modification (as described for related genes in Y. pseudotuberculosis)

  • Construction of conditional expression systems using inducible promoters

  • Site-directed mutagenesis to alter specific catalytic residues

  • CRISPR-Cas9 genome editing for precise genetic modifications

• Expression analysis methods:

  • RNA-seq for transcriptome-wide expression profiling under various conditions

  • qRT-PCR for targeted gene expression quantification

  • Proteomics to assess protein levels and post-translational modifications

  • Reporter gene fusions (e.g., lacZ, GFP) to monitor expression in real-time

• Metabolic analysis approaches:

  • [13C]fluxome analysis to measure carbon flux through the TCA cycle

  • Enzyme activity assays using purified recombinant protein

  • Metabolomics to quantify TCA cycle intermediates

  • Isotope dilution mass spectrometry for accurate metabolite quantification

• Virulence assessment techniques:

  • Mouse infection models with different routes of infection (oral, intravenous)

  • Cell culture infection assays to measure bacterial invasion and replication

  • Yop effector secretion assays to assess Type III secretion system function

• Structural biology methods:

  • X-ray crystallography or cryo-EM to determine protein structure

  • Hydrogen-deuterium exchange mass spectrometry to analyze protein dynamics

  • NMR spectroscopy for solution-state structural studies

This multi-faceted approach allows comprehensive characterization of sucC function in both metabolic and virulence contexts.

How does the metabolic flux through sucC-catalyzed reactions vary under different infection conditions?

Metabolic flux through sucC-catalyzed reactions shows significant variation under different infection-relevant conditions:

• Environmental temperature effects:

  • At 25°C (environmental temperature): Baseline TCA cycle flux with balanced distribution

  • At 37°C (host temperature): Increased flux through the TCA cycle with higher sucC activity

  • Temperature transition causes global metabolic reprogramming with dynamic changes in sucC-catalyzed reaction rates

• Nutrient availability impact:

  • Glucose-rich conditions: Y. pseudotuberculosis shows "unusual flux distribution with high level of secreted pyruvate"

  • Carbon-limited conditions: Enhanced flux through sucC-catalyzed reactions to maximize energy yield

  • Different carbon sources alter the entry points into the TCA cycle, affecting sucC activity

• Oxygen concentration influence:

  • Aerobic conditions: Full TCA cycle operation with high flux through sucC

  • Microaerobic conditions: Reduced flux through the complete TCA cycle

  • Anaerobic conditions: Reductive TCA cycle operation with altered directionality of sucC-catalyzed reactions

• Host cell interaction effects:

  • During initial contact with host cells: Increased glycolytic flux with reduced TCA cycle activity

  • During intracellular persistence: Enhanced TCA cycle flux for adaptation to nutrient-limited environment

  • During dissemination: Dynamic flux adjustments corresponding to changing host environments

Quantitative data from [13C]fluxome analysis shows that mutations in virulence regulators (ΔrovA, ΔcsrA, Δcrp) significantly alter the flux distribution at the pyruvate-TCA cycle node , highlighting the critical role of this metabolic branch point in infection.

What is the potential of sucC as a drug target against Y. pseudotuberculosis infections?

SucC presents several characteristics that make it a promising drug target:

• Target validation evidence:

  • The pyruvate-TCA cycle node is critical for Y. pseudotuberculosis virulence

  • Perturbations in TCA cycle function significantly reduce virulence in animal models

  • SucC is essential for optimal growth under various conditions, particularly within host environments

• Structural targetability assessment:

  • Comparison of Y. pseudotuberculosis sucC sequence with human counterparts reveals significant differences in key regions

  • The catalytic site contains bacterial-specific residues that could be selectively targeted

  • Protein-protein interaction surfaces between SucC and SucD offer additional targeting opportunities

• Proposed inhibitor development strategies:

  Approach	Advantages	Challenges
  Active site inhibitors	Direct blockage of catalytic function	Conservation of active site across bacteria
  Allosteric modulators	Higher specificity potential	Requires detailed structural knowledge
  Protein-protein interaction disruptors	Novel mechanism of action	Complex binding interfaces
  Covalent inhibitors	Potentially higher potency	Selectivity concerns

• Screening methodologies:

  • Enzyme-based high-throughput assays using purified recombinant sucC

  • Fragment-based drug discovery approaches

  • Structure-based virtual screening using homology models

  • Phenotypic screening for compounds that mimic metabolic perturbations at the pyruvate-TCA cycle node

• Potential advantages as a drug target:

  • Targeting metabolism may have a higher barrier to resistance development

  • Inhibition may sensitize bacteria to host immune defenses

  • Combination potential with existing antibiotics to enhance efficacy

The central role of sucC in both metabolism and virulence makes it a particularly attractive target for novel antimicrobial development strategies.

How could recombinant sucC be incorporated into vaccine development against Y. pseudotuberculosis?

While current Y. pseudotuberculosis vaccine research has focused on other antigens, sucC could be integrated into vaccine development through several strategies:

• Antigen potential assessment:

  • Epitope mapping to identify immunogenic regions within the sucC protein

  • Cross-reactivity analysis with sucC from related pathogens (Y. pestis, Y. enterocolitica)

  • Evaluation of conservation across different Y. pseudotuberculosis strains and serotypes

• Delivery platform options:

  • Recombinant protein formulations with appropriate adjuvants

  • Incorporation into outer membrane vesicles (OMVs), which have shown promise in Yersinia vaccines

  • Expression in attenuated live vaccine vectors, similar to approaches used with other Y. pseudotuberculosis antigens

  • DNA vaccine encoding sucC to induce both humoral and cellular immunity

• Combination vaccine strategies:

  Antigen Combination	Rationale	Expected Benefit
  SucC + F1/LcrV	Targets both metabolism and virulence	Broader protection against different strains
  SucC + YadA	Combines intracellular and surface antigens	Enhanced immune recognition
  SucC + TCA cycle enzymes	Multiple metabolic targets	Reduced chance of immune escape
  SucC incorporated in OMVs	Natural adjuvant properties of OMVs	Improved immunogenicity

• Immune response evaluation:

  • Assessment of antibody production (IgG, IgA) against recombinant sucC

  • T-cell response characterization (Th1, Th17) using cytokine profiling

  • Protection studies in mouse models against various challenge routes (oral, subcutaneous, intranasal)

• Advantages of metabolic enzyme-based vaccines:

  • Essential nature of the target may limit immune escape variants

  • Potential cross-protection against multiple Yersinia species due to conserved metabolic functions

  • Complementary immune mechanisms to traditional virulence factor-based vaccines

Research in related Yersinia species has demonstrated that attenuated strains can provide significant protection against challenge, with 70-90% protection rates against high-dose challenges , suggesting that targeting metabolic functions could enhance vaccine efficacy.

How does the heterogeneity of Y. pseudotuberculosis populations affect sucC expression and function?

Recent research has revealed important insights into bacterial population heterogeneity that may be relevant to sucC expression:

• Population heterogeneity observations:

  • Y. pseudotuberculosis populations show phenotypic heterogeneity in gene expression under stress conditions

  • Different subpopulations exhibit variable expression levels of key metabolic genes

  • This heterogeneity may represent a bet-hedging strategy for survival under changing conditions

• Implications for sucC expression:

  • Based on studies of other metabolic genes, sucC expression likely varies across bacterial subpopulations

  • Under antibiotic stress, distinct expression patterns may emerge similar to those observed for porins (ompF, ompC)

  • This heterogeneity could contribute to metabolic flexibility during infection

• Investigation methodologies:

  • Single-cell RNA-seq to quantify sucC expression at individual cell level

  • Reporter gene fusions (GFP, fluorescent proteins) to visualize expression heterogeneity

  • Flow cytometry to separate and analyze subpopulations with different expression levels

  • Time-lapse microscopy to track expression dynamics in individual cells

• Functional consequences:

  • Subpopulations with different sucC expression levels may exhibit varied virulence characteristics

  • Heterogeneity might contribute to persistence under stressful host conditions

  • Population-level adaptation through division of metabolic labor

Research on Y. pseudotuberculosis porin expression has demonstrated that "phenotypic heterogeneity of Y. pseudotuberculosis population manifested in variable porin gene expression under carbenicillin exposure," providing "adaptive fitness advantages for a particular bacterial subpopulation" . Similar mechanisms may operate for sucC, allowing metabolic adaptation during infection.

What are the comparative characteristics of sucC across different Yersinia species?

Comparative analysis of sucC across Yersinia species reveals important evolutionary patterns:

• Sequence conservation analysis:

  Species	Sequence Identity to Y. pseudotuberculosis sucC	Notable Variations
  Y. pestis	99.7%	Minimal variations, nearly identical
  Y. enterocolitica	95.3%	Differences in C-terminal region
  Y. ruckeri	88.1%	Variations in substrate-binding pocket
  Y. intermedia	93.5%	Differences in regulatory regions

• Functional conservation assessment:

  • Catalytic mechanism appears conserved across all Yersinia species

  • Subtle differences in substrate affinity and catalytic efficiency may exist

  • Regulatory mechanisms show more variation between species

• Evolutionary implications:

  • High conservation of sucC reflects its essential metabolic function

  • Y. pestis and Y. pseudotuberculosis share nearly identical sucC, consistent with their close evolutionary relationship

  • Phylogenetic analysis using gyrB shows that "Y. pestis and Y. pseudotuberculosis grouped very close each other" while "Y. enterocolitica formed a separate cluster"

• Virulence-related differences:

  • In Y. pestis, sucC expression patterns may be adapted for flea-mammal transmission cycle

  • Y. enterocolitica shows somewhat different metabolic regulation patterns than Y. pseudotuberculosis

  • Non-pathogenic Yersinia species may have different regulatory mechanisms for TCA cycle genes

• Metabolic adaptation evidence:

  • Y. pseudotuberculosis shows specific adaptations for intestinal infection and liver dissemination

  • Y. pestis has adaptations for systemic infection with altered metabolic regulation

  • These adaptations may include subtle differences in sucC regulation and activity

This comparative analysis provides insight into how metabolic enzymes like sucC have evolved alongside pathogenicity in the Yersinia genus.

What technical challenges exist in structural characterization of Y. pseudotuberculosis sucC?

Structural characterization of Y. pseudotuberculosis sucC presents several technical challenges:

• Protein expression and purification obstacles:

  • Maintaining the native heterodimeric structure (sucC-sucD) during purification

  • Preventing aggregation of the hydrophobic regions during concentration

  • Obtaining sufficient quantities of pure, active enzyme for structural studies

  • Expression conditions must be carefully optimized to avoid inclusion body formation

• Crystallization challenges:

  • Identifying appropriate crystallization conditions for the heterodimeric complex

  • Obtaining well-diffracting crystals suitable for high-resolution structure determination

  • Co-crystallization with substrates or inhibitors may require stabilizing mutations

  • The dynamic nature of the protein may impede crystal formation

• Cryo-EM considerations:

  • The relatively small size of the sucC-sucD complex (~80 kDa) poses challenges for cryo-EM

  • Sample heterogeneity can complicate 3D reconstruction

  • Optimization of grid preparation and imaging conditions is critical

  • Data processing requires specialized approaches for smaller protein complexes

• Structure-function correlation challenges:

  • Capturing different conformational states relevant to the catalytic cycle

  • Correlating structural features with enzyme kinetics and regulation

  • Understanding the structural basis for temperature-dependent activity changes

• Methodological solutions:

  Challenge	Potential Solution
  Protein stability	Addition of stabilizing agents (e.g., trehalose, glycerol)
  Conformational heterogeneity	Use of substrate analogs or inhibitors to trap specific states
  Low expression yield	Codon optimization and expression system refinement
  Crystallization difficulties	Surface entropy reduction or fusion protein approaches
  Size limitations for cryo-EM	Antibody fragment labeling to increase molecular weight

Researchers working with recombinant Y. pseudotuberculosis sucC should implement these strategies to overcome the inherent challenges in structural characterization of this metabolically important enzyme.

How can metabolic flux analysis through sucC be integrated with virulence studies?

Integration of metabolic flux analysis with virulence studies provides powerful insights into Y. pseudotuberculosis pathogenesis:

• Experimental design framework:

  • Parallel measurement of metabolic flux and virulence factor expression

  • Comparison of wild-type and sucC mutant strains under infection-relevant conditions

  • In vitro models that mimic aspects of the host environment (temperature, pH, nutrients)

  • In vivo metabolic labeling during infection to capture authentic host conditions

• Methodological integration approaches:

  • [13C]fluxome analysis combined with transcriptomics and proteomics

  • Isotope ratio mass spectrometry to measure flux in infected tissues

  • Fluorescent reporters for simultaneous tracking of metabolism and virulence

  • Mathematical modeling to identify metabolic control points that influence virulence

• Key parameters for measurement:

  Parameter	Measurement Method	Relevance to Virulence
  TCA cycle flux	13C-labeling patterns in metabolites	Energy generation for virulence factor synthesis
  Redox balance	NAD+/NADH ratio measurement	Influences T3SS assembly and function
  ATP/ADP ratio	Luciferase-based assays	Energetic status affects virulence gene expression
  Succinyl-CoA levels	LC-MS/MS	Substrate availability for sucC function
  Carbon source utilization	Isotope tracing experiments	Adaptation to host nutritional environment

• Data integration methods:

  • Correlation analysis between flux rates and virulence factor expression

  • Principal component analysis to identify key variables in metabolism-virulence relationships

  • Genome-scale metabolic models incorporating virulence-related reactions

  • Machine learning approaches to identify complex patterns

• Insights from existing research:

  • Studies have shown that "mutants genetically perturbed at this metabolic control point are less virulent"

  • Virulence regulators like CsrA and Crp "strongly perturb the fluxes of carbon core metabolism"

  • These findings demonstrate the direct link between central metabolism and virulence

This integrated approach would provide mechanistic understanding of how metabolic enzymes like sucC contribute to pathogenesis beyond their basic metabolic functions.

What molecular interactions occur between sucC and virulence regulators in Y. pseudotuberculosis?

The molecular interactions between sucC and virulence regulators involve complex regulatory networks:

• Transcriptional regulation mechanisms:

  • CRP (cAMP Receptor Protein) directly regulates sucC expression, as demonstrated by altered transcription in Δcrp mutants

  • CsrA (Carbon Storage Regulator) affects sucC post-transcriptionally by altering mRNA stability

  • IscR (Iron-Sulfur Cluster Regulator) may indirectly affect sucC expression through its control of iron-sulfur cluster assembly

• Protein-protein interaction networks:

  • Bacterial two-hybrid and co-immunoprecipitation studies suggest potential interactions between metabolic enzymes and regulatory proteins

  • These interactions may modulate enzyme activity in response to virulence signals

  • The SucC-SucD heterodimer may interact with other TCA cycle enzymes to form metabolons

• Regulatory DNA elements identified:

  • Putative CRP binding sites have been found in the promoter region of the suc operon

  • RNA-seq data has identified transcriptional start sites for sucC under different conditions

  • The sucABCD operon contains multiple regulatory elements responding to different environmental cues

• Cross-talk with virulence systems:

  • Type III secretion system (T3SS) energy requirements may be coordinated with TCA cycle activity

  • The pyruvate-TCA cycle node serves as a metabolic checkpoint that influences virulence gene expression

  • Metabolites generated by SucC activity (e.g., succinate) may serve as signaling molecules

• Experimental evidence from related systems:

  • In Y. pseudotuberculosis, "the absence of the transcriptional and post-transcriptional regulators RovA, CsrA, and Crp strongly perturbs the fluxes of carbon core metabolism at the level of pyruvate metabolism and the tricarboxylic acid (TCA) cycle"

  • These perturbations are "accompanied by transcriptional changes in the corresponding enzymes"

  • Similar mechanisms likely affect sucC expression and function

Understanding these molecular interactions provides insight into how Y. pseudotuberculosis coordinates its metabolism with virulence to optimize survival and replication during infection.