Recombinant Legionella pneumophila Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) in Legionella pneumophila?

Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) is a critical metabolic enzyme encoded in the genome of Legionella pneumophila. It functions as the beta subunit of the Succinyl-CoA synthetase complex (EC 6.2.1.5), which plays an essential role in the tricarboxylic acid (TCA) cycle. The recombinant form of this protein consists of 387 amino acids with a sequence beginning with "MNLHEYQAKQ LFASYGLPVP..." and is typically expressed in E. coli expression systems for research purposes . The protein participates in energy metabolism by catalyzing the reversible reaction that converts succinyl-CoA to succinate while generating a molecule of ATP.

How does sucC differ from other similar proteins in bacterial pathogens?

Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) in Legionella pneumophila shares structural similarities with homologs in other bacterial species but contains unique sequence characteristics that may influence its specific function in Legionella metabolism and pathogenicity. Unlike some other bacterial pathogens, L. pneumophila's metabolic adaptations are specialized for both environmental persistence and intracellular replication within host cells. The protein contains specific domains including substrate binding sites and catalytic regions that may differ from those in non-pathogenic bacteria. While the core enzymatic function remains conserved, these subtle differences may contribute to L. pneumophila's ability to thrive in diverse environments including natural aquatic habitats and human-made water systems .

What are the optimal storage conditions for recombinant sucC protein?

For optimal stability and activity preservation of recombinant Legionella pneumophila Succinyl-CoA ligase [ADP-forming] subunit beta (sucC), researchers should store the protein at -20°C for regular storage, or at -80°C for extended preservation. The protein's shelf life varies depending on formulation - liquid preparations typically maintain viability for approximately 6 months, while lyophilized forms can remain stable for up to 12 months when stored properly at -20°C/-80°C .

To minimize protein degradation, it is recommended to:

• Avoid repeated freeze-thaw cycles that can compromise structural integrity

• Store working aliquots at 4°C for no longer than one week

• When reconstituting lyophilized protein, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% being optimal) before aliquoting for long-term storage

These storage parameters ensure maximum retention of enzymatic activity and structural integrity for experimental applications.

What is the recommended protocol for reconstituting lyophilized sucC protein?

For optimal reconstitution of lyophilized Legionella pneumophila Succinyl-CoA ligase [ADP-forming] subunit beta (sucC), follow this detailed protocol:

• Centrifuge the vial briefly (30 seconds at 10,000 × g) prior to opening to ensure all material is collected at the bottom

• Reconstitute the protein using deionized sterile water to achieve a final concentration between 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 50% to enhance stability

• Mix gently by inversion rather than vortexing to prevent protein denaturation

• Divide into small working aliquots (20-50 μL) to minimize freeze-thaw cycles

• Store aliquots at -20°C for regular use or -80°C for extended storage

• Working aliquots can be kept at 4°C for up to one week

This reconstitution protocol ensures maximum retention of protein structure and enzymatic activity for downstream applications including enzymatic assays, antibody production, and structural studies.

How can researchers generate sucC gene deletions in Legionella pneumophila?

Creating precise sucC gene deletions in Legionella pneumophila requires a methodical approach using allelic exchange. The following protocol details the process:

• Design and Construction of Deletion Vector:

  • Use an allelic exchange plasmid (such as pLAW344) containing chloramphenicol and ampicillin selection markers and the counter-selection gene SacB

  • Clone approximately 1 kb homologous regions flanking the upstream and downstream sequences of the sucC gene into this vector

  • Verify the construct integrity through restriction digestion and sequencing

• Transformation and Selection:

  • Introduce the constructed plasmid into L. pneumophila via electroporation

  • Select transformants on media containing chloramphenicol

  • Verify plasmid integration into the chromosome using colony PCR

• Counter-selection and Deletion Verification:

  • Grow integrated transformants on media containing sucrose to counter-select against SacB

  • Screen resulting colonies for loss of the plasmid backbone

  • Confirm clean gene deletion through PCR amplification and Sanger sequencing across the deletion junction

• Complementation Analysis:

  • For functional validation, clone the wildtype sucC coding region into an expression plasmid (such as pMMB207c) downstream of an inducible promoter (e.g., ptac)

  • Transform this complementation construct into the deletion strain

  • Induce expression with 1 mM IPTG when required for phenotypic rescue experiments

This genetic engineering approach enables precise functional analysis of sucC in L. pneumophila metabolism and pathogenesis.

What assays can be used to measure Succinyl-CoA ligase activity in vitro?

Researchers investigating Succinyl-CoA ligase [ADP-forming] activity can employ several complementary biochemical assays:

• Spectrophotometric Coupled Assay:

  • Measure ADP formation by coupling to pyruvate kinase and lactate dehydrogenase

  • Monitor NADH oxidation at 340 nm as an indirect measure of enzyme activity

  • Reaction components: succinyl-CoA, ADP, Pi, MgCl₂, NADH, phosphoenolpyruvate, pyruvate kinase, and lactate dehydrogenase in appropriate buffer

  • Calculate activity from the linear decrease in absorbance using the NADH extinction coefficient

• Radioisotope-Based Assay:

  • Use [¹⁴C]-labeled succinyl-CoA or [³²P]-labeled ADP as substrates

  • Separate reaction products by thin-layer chromatography

  • Quantify radioactive products using phosphorimaging or scintillation counting

  • Particularly useful for kinetic analysis and inhibitor screening

• Malachite Green Phosphate Detection:

  • Measure inorganic phosphate released during the reaction

  • Add malachite green reagent to form colorimetric complex

  • Measure absorbance at 620-640 nm

  • Suitable for high-throughput screening applications

Each method provides distinct advantages, with the coupled enzyme assay offering real-time monitoring, radioisotope methods providing high sensitivity, and the malachite green approach enabling high-throughput applications.

How does sucC contribute to Legionella pneumophila pathogenesis?

Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) plays multifaceted roles in Legionella pneumophila pathogenesis through its contributions to bacterial metabolism and virulence:

• Energy Metabolism Adaptation:

  • As a critical TCA cycle enzyme, sucC enables efficient energy production crucial for intracellular replication

  • In nutrient-limited host environments, the ability to maintain ATP generation through efficient metabolism provides a competitive advantage

  • Metabolic flexibility conferred by functional sucC may allow L. pneumophila to adapt to changing conditions within host cells

• Relationship to Virulence Systems:

  • Metabolic functions of sucC are interconnected with energy requirements for type IV secretion systems, including the Dot/Icm system that secretes over 300 different effector proteins essential for L. pneumophila virulence

  • Energy derived from reactions involving sucC likely supports the substantial metabolic demand of maintaining the Dot/Icm complex, which constitutes approximately 10% of the L. pneumophila proteome

• Potential Metabolic Reprogramming:

  • During infection, metabolic adaptations involving enzymes like sucC may contribute to bacterial survival within host-derived vacuoles

  • Modified metabolic pathways could potentially influence the composition of the Legionella-containing vacuole

While direct experimental evidence linking sucC gene function to specific virulence phenotypes remains limited, its central role in core metabolism suggests it likely contributes indirectly to multiple aspects of L. pneumophila pathogenesis.

What is known about the interaction between sucC and host cellular components?

• Metabolic Interface:

  • Unlike secreted virulence factors that directly interact with host components, sucC primarily functions within bacterial metabolism

  • The enzyme's activity may indirectly influence host-pathogen interactions by supporting energy requirements for virulence factor production

  • L. pneumophila is known to modify host cell processes using its extensive repertoire of effector proteins secreted through the Dot/Icm system

• Potential Immunological Recognition:

  • As a conserved bacterial protein, sucC may potentially serve as a pathogen-associated molecular pattern (PAMP) if released during bacterial lysis

  • Released bacterial metabolic enzymes can sometimes trigger host immune responses, though this has not been specifically demonstrated for sucC

• Relationship to Competitive Advantage:

  • L. pneumophila exhibits competitive advantages against other bacterial species, including L. micdadei, through secreted inhibitory molecules

  • Efficient metabolism supported by enzymes like sucC may provide energy for producing these inhibitory factors

The precise role of sucC in direct host interactions remains an open area for investigation, with experimental approaches needed to elucidate any specific interactions with host cell components.

How do CRISPR-Cas systems in Legionella pneumophila relate to sucC function?

While direct functional relationships between CRISPR-Cas systems and sucC in Legionella pneumophila have not been explicitly established in the literature, several potential connections can be hypothesized based on their roles in bacterial physiology:

• Metabolic Support for CRISPR-Cas Function:

  • CRISPR-Cas systems are prevalent in L. pneumophila strains, with type I-C systems being particularly common

  • The energy-intensive processes of CRISPR-Cas surveillance and defense mechanisms require ATP

  • As a component of central metabolism, sucC contributes to the cell's energy budget, potentially supporting CRISPR-Cas functionality

• Co-regulation Possibilities:

  • Metabolic status of the cell can influence gene expression patterns

  • Under certain environmental conditions, there may be coordinated regulation between metabolic genes like sucC and defense systems including CRISPR-Cas

  • Such co-regulation could potentially optimize resource allocation during environmental stresses

• Evolutionary Considerations:

  • Both metabolic enzymes and CRISPR-Cas systems contribute to bacterial fitness and adaptation

  • The presence of CRISPR-Cas systems may help protect L. pneumophila genome stability, indirectly preserving the integrity of metabolic genes including sucC

The intersection of metabolism and bacterial defense systems represents an intriguing area for future research, potentially revealing integrated networks that collectively enhance L. pneumophila survival and pathogenesis.

How can recombinant sucC be used in structural biology studies?

Recombinant Legionella pneumophila Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) presents valuable opportunities for structural biology investigations using the following methodologies:

• X-ray Crystallography:

  • The high purity (>85% by SDS-PAGE) of commercially available recombinant sucC provides suitable starting material for crystallization trials

  • Optimization protocol:

    • Further purify using size exclusion chromatography to achieve >95% homogeneity

    • Screen various buffer conditions (pH 6.5-8.5) with different precipitants

    • Co-crystallize with substrates (succinyl-CoA, ADP) or inhibitors to capture functional states

    • Cryoprotect crystals using 20-30% glycerol before X-ray diffraction data collection

• Cryo-Electron Microscopy (Cryo-EM):

  • For studying the complete Succinyl-CoA synthetase complex:

    • Express and purify both alpha and beta (sucC) subunits

    • Reconstitute the complete enzyme complex in vitro

    • Optimize sample concentration (typically 0.5-5 mg/mL) and grid preparation

    • Collect data using state-of-the-art cryo-electron microscopes

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • For studying protein dynamics and ligand interactions:

    • Express isotopically labeled sucC using minimal media supplemented with ¹⁵N-ammonium chloride and/or ¹³C-glucose

    • Optimize buffer conditions for NMR experiments (typically phosphate buffers with reduced salt)

    • Perform ¹H-¹⁵N HSQC experiments to analyze protein folding and stability

    • Conduct ligand titration experiments to map binding interfaces

These structural biology approaches can reveal atomic-level details of sucC function, substrate specificity, and potential for targeted inhibition in the context of antimicrobial development.

What are the challenges in developing specific inhibitors targeting Legionella pneumophila sucC?

Developing specific inhibitors targeting Legionella pneumophila Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) presents several significant challenges:

• Structural Conservation Issues:

  • Succinyl-CoA ligase is highly conserved across bacterial species and has homologs in eukaryotes

  • The active site architecture shares similarities with human mitochondrial Succinyl-CoA ligase

  • This conservation creates potential for cross-reactivity, requiring sophisticated approaches to achieve L. pneumophila specificity:

    • Focus on subtle structural differences in substrate binding pockets

    • Target unique surface features present in L. pneumophila sucC

    • Develop allosteric inhibitors that exploit conformational differences

• Technical Challenges in Assay Development:

  • Enzymatic assays require:

    • Purification of both alpha and beta subunits to reconstitute active enzyme

    • Optimization of coupled enzyme systems for high-throughput screening

    • Development of specific detection methods for various reaction components

  • Proposed solutions include:

    • Fluorescence-based assays using labeled substrates

    • Surface plasmon resonance for direct binding measurements

    • Fragment-based screening approaches

• Pharmacokinetic Considerations:

  • Inhibitors must penetrate both host cell membranes and bacterial cell walls

  • L. pneumophila replicates within specialized vacuoles, creating additional barrier challenges

  • Potential approaches include:

    • Lipid-based delivery systems to enhance membrane penetration

    • Prodrug strategies activated by bacterial enzymes

    • Conjugation with cell-penetrating peptides

• Validation Challenges:

  • Confirming on-target activity requires genetic validation approaches

  • Methods should include:

    • Generation of sucC point mutants with altered inhibitor sensitivity

    • Complementation studies with heterologous sucC variants

    • Cellular infection models to confirm intracellular efficacy

These multifaceted challenges require integrated approaches combining structural biology, medicinal chemistry, and sophisticated biological validation systems.

How does recombinant sucC compare to the native protein in functional studies?

Understanding the similarities and differences between recombinant and native Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) is crucial for interpreting experimental results:

For most biochemical characterizations, recombinant sucC provides a reliable proxy for the native enzyme, especially for:

• Basic enzymatic activity measurements

• Structural studies

• Antibody production

• Inhibitor screening

What genetic engineering applications involving sucC are currently being explored?

Current genetic engineering applications involving Legionella pneumophila Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) span several innovative research directions:

• Gene Deletion and Complementation Systems:

  • RecA-independent recombination methods are being applied for creating precise sucC deletions

  • These approaches allow for unmarked gene deletions that facilitate studies of L. pneumophila multi-gene systems

  • Complementation systems using site-specific Flp recombination coupled with phage recombination provide sophisticated tools for functional analysis

• Reporter Fusion Applications:

  • Construction of sucC-reporter gene fusions enables:

    • Monitoring of gene expression under different environmental conditions

    • Analysis of protein localization within bacterial cells

    • Tracking of promoter activity during different growth phases

• Oligo Mutagenesis Approaches:

  • Novel "oligo mutagenesis" techniques utilizing short homologous sequences are being developed

  • These methods enable precise engineering of L. pneumophila genes including sucC

  • This approach represents a potentially conserved mechanism across bacteria that could provide genetic tools for previously challenging organisms

• Metabolic Engineering Potential:

  • Modification of sucC and related genes could potentially:

    • Alter carbon flux through central metabolism

    • Create attenuated strains for vaccine development

    • Develop reporter strains for environmental monitoring of Legionella

These genetic engineering approaches are expanding the molecular toolkit available for studying L. pneumophila, potentially leading to new applications in both basic science and applied research contexts.

How might sucC be involved in Legionella pneumophila's competitive advantage in microbial communities?

Emerging research suggests potential mechanisms by which Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) could contribute to Legionella pneumophila's competitive advantage:

• Energy Production for Antagonistic Mechanisms:

  • L. pneumophila demonstrates strong inhibitory effects against neighboring bacteria, including other Legionella species

  • Growth inhibition experiments show L. pneumophila can cause up to 10,000-fold reduction in neighboring L. micdadei growth

  • As a central metabolic enzyme, sucC contributes to energy production potentially supporting:

    • Synthesis of secreted inhibitory molecules

    • Maintenance of secretion systems required for competitive interactions

    • Production of antimicrobial compounds distinct from known surfactants

• Metabolic Flexibility in Resource-Limited Environments:

  • Efficient operation of the TCA cycle through enzymes like sucC may provide competitive advantages in environments with fluctuating nutrient availability

  • This metabolic efficiency could be particularly important in:

    • Water distribution systems with low nutrient levels

    • Competitive biofilm communities

    • Intracellular environments during host infection

• Potential Role in Environmental Persistence:

  • L. pneumophila strains show expanding geographic distribution patterns

  • Metabolic adaptations involving central carbon metabolism enzymes like sucC might contribute to:

    • Survival under environmental stresses

    • Adaptation to different water chemistry conditions

    • Persistence in the face of disinfection treatments

What are the most promising research gaps to address in sucC studies?

Several high-priority research areas present opportunities for significant advances in understanding Legionella pneumophila Succinyl-CoA ligase [ADP-forming] subunit beta (sucC):

• Structure-Function Relationships:

  • Determine high-resolution crystal structures of L. pneumophila sucC alone and in complex with the alpha subunit

  • Map substrate binding sites and catalytic residues through site-directed mutagenesis

  • Compare structural features with homologs from other bacteria to identify unique characteristics

  • Research question: How do structural features of L. pneumophila sucC influence its catalytic properties and potential as a therapeutic target?

• Metabolic Integration and Regulation:

  • Characterize how sucC activity is regulated in response to environmental conditions

  • Investigate potential post-translational modifications affecting enzyme function

  • Examine metabolic flux through the TCA cycle during different growth phases and infection stages

  • Research question: How does L. pneumophila modulate sucC activity to optimize metabolism during environmental persistence versus intracellular replication?

• Host-Pathogen Interface:

  • Determine if sucC-dependent metabolic activities influence virulence factor expression

  • Investigate potential connections between central metabolism and secretion system function

  • Examine how host cell metabolites might influence bacterial sucC activity

  • Research question: Does metabolic adaptation involving sucC contribute to L. pneumophila's ability to evade host defense mechanisms?

• Inhibitor Development:

  • Screen for small molecule inhibitors with specificity for L. pneumophila sucC

  • Develop structure-based drug design approaches targeting unique features

  • Evaluate inhibitor efficacy in cellular infection models

  • Research question: Can specific inhibitors of L. pneumophila sucC be developed as potential therapeutic agents for treating Legionnaires' disease?

Addressing these research gaps would significantly advance our understanding of this important metabolic enzyme and potentially lead to new therapeutic strategies against L. pneumophila infections.