Recombinant Salmonella schwarzengrund ATP synthase subunit c (atpE)

What is the fundamental role of ATP synthase in Salmonella metabolism and survival?

ATP synthase in Salmonella serves as the primary machinery for energy production through oxidative phosphorylation. This enzyme complex couples proton translocation to ATP synthesis/hydrolysis, which is essential for cellular energy homeostasis . The proper functioning of ATP synthase is critical for:

• Maintaining physiological ATP levels required for cellular processes

• Regulating cytoplasmic pH, which affects numerous metabolic pathways

• Enabling adaptation to different environmental conditions, including those encountered during infection

Research has demonstrated that F1F0 ATP synthase activity directly affects Salmonella's ability to survive within host cells and establish infection. The complex not only produces ATP but also plays a regulatory role in controlling bacterial metabolism in response to environmental challenges .

How does ATP synthase subunit c contribute to the virulence mechanisms of Salmonella?

ATP synthase subunit c plays a multifaceted role in Salmonella virulence through several interconnected mechanisms:

• Energy metabolism regulation: The c-subunit ring is integral to ATP production, which powers virulence processes .

• Interaction with virulence proteins: Studies have demonstrated that the bacterial virulence protein MgtC interacts directly with the a-subunit of the F1F0 ATP synthase, hindering ATP-driven proton translocation and NADH-driven ATP synthesis . This interaction is crucial for Salmonella's ability to survive within macrophages.

• Modulation of biofilm formation: By lowering ATP levels through inhibition of ATP synthase, MgtC prevents the rise in cyclic diguanylate (c-di-GMP), a second messenger that promotes biofilm formation . This mechanism represses cellulose biosynthesis, which would otherwise interfere with Salmonella's replication inside macrophages and virulence in mice.

• Maintenance of cytoplasmic pH: ATP synthase activity affects intracellular pH, with mutants lacking proper regulatory mechanisms displaying an acidic cytoplasm that impairs virulence .

Research has shown that a mutation in MgtC that attenuates Salmonella pathogenicity prevents the protein from interacting with and inhibiting the F1F0 ATP synthase, suggesting that MgtC's virulence role is primarily due to its action on ATP synthase .

What methodologies are effective for analyzing ATP synthase activity in Salmonella under different experimental conditions?

Several methodological approaches have proven effective for studying ATP synthase activity in Salmonella:

• Inverted membrane vesicle assays: These can be used to measure:

  • ATP-driven proton translocation using fluorescent probes

  • NADH-driven ATP synthesis

  • ATP hydrolysis by measuring phosphate release

• ATP level quantification: Comparative measurements between wild-type strains and mutants (such as mgtC null mutants) can reveal differences in ATP homeostasis .

• Cytoplasmic pH measurement: This helps evaluate the physiological impact of ATP synthase activity on cellular environment .

• Molecular interaction studies: Techniques such as bacterial two-hybrid assays and co-immunoprecipitation can detect interactions between ATP synthase components and regulatory proteins like MgtC .

• Genetic approaches: Creating targeted mutations in ATP synthase components or regulatory genes can identify functional domains and interaction sites.

For example, researchers have demonstrated that vesicles prepared from wild-type Salmonella release less phosphate than those from isogenic mgtC mutant strains, indicating lower ATP hydrolysis activity. This effect was shown to be dependent on the F0 a subunit, as no difference in phosphate release was observed in atpB single mutants .

How does the interplay between ATP synthase and cellulose production impact Salmonella virulence?

Research has established a clear relationship between ATP synthase activity, cellulose production, and virulence in Salmonella:

• ATP-dependent regulation of cellulose synthesis: Inactivation of the mgtC gene results in a sevenfold increase in bcsA mRNA (encoding the catalytic subunit of cellulose synthase) . This increase is due to heightened cytosolic ATP levels, as demonstrated by experiments where expression of the α, β, and γ components of the F1 subunit of ATP synthase prevented cellulose production in the mgtC mutant .

• Cellulose as a virulence inhibitor: Cellulose production interferes with Salmonella's ability to replicate inside macrophages and reduces virulence in mice . When cellulose production was artificially induced in Salmonella by expressing the c-di-GMP synthase AdrA from the phoP promoter, the engineered strain:

  • Produced cellulose in response to low Mg²⁺ signals

  • Was defective for replication inside macrophages

  • Recovered replication capability when the bcsA gene was deleted

• Metabolic balance: The regulation of ATP levels through ATP synthase activity represents a critical balance point where Salmonella must maintain sufficient energy for virulence functions while preventing excessive ATP production that would trigger cellulose synthesis .

This research indicates that virulence genes like mgtC can function specifically to repress traits that would otherwise interfere with pathogenicity, demonstrating a sophisticated regulatory mechanism in bacterial virulence.

What experimental approaches can be used to study the c-subunit ring permeability and its functional significance?

The c-subunit ring of ATP synthase has been identified as containing a regulated channel that influences membrane permeability. Several experimental approaches can be employed to study this feature:

• Proteoliposome reconstitution: Purified recombinant ATP synthase c-subunit can be reconstituted into artificial lipid bilayers to study channel properties under controlled conditions.

• Electrophysiological measurements: Patch-clamp techniques applied to membrane preparations containing the c-subunit can measure conductance properties and response to regulatory factors.

• Fluorescent probe studies: Using membrane-impermeable fluorescent dyes to detect ion/molecule passage through the c-subunit ring under different conditions.

• Mutagenesis approaches: Systematic mutation of specific residues in the c-subunit can identify amino acids critical for channel formation and regulation.

• Structural analysis: Techniques such as cryo-electron microscopy and X-ray crystallography can reveal conformational changes in the c-subunit ring under different physiological conditions.

Research has indicated that the c-subunit ring may form or significantly contribute to the cyclosporin A-regulated mitochondrial permeability transition pore (mPTP) in eukaryotic systems, suggesting evolutionary conservation of this function . Studies have shown that aberrant activity of this channel is associated with pathological conditions, as demonstrated by the finding that mPTP is abnormally active in Fmr1 mitochondria .

How can researchers design experiments to study the interaction between virulence proteins and ATP synthase in Salmonella?

To effectively study interactions between virulence proteins (such as MgtC) and ATP synthase components in Salmonella, researchers can employ multiple complementary approaches:

• Genetic screening methods:

  • Bacterial two-hybrid systems to identify protein-protein interactions

  • Suppressor mutation analysis to identify compensatory mutations that restore function

  • Construction of domain-swapping chimeras to identify interaction regions

• Biochemical approaches:

  • Co-immunoprecipitation assays using antibodies against ATP synthase components or virulence proteins

  • Pull-down assays using purified recombinant proteins

  • Cross-linking experiments followed by mass spectrometry to identify interaction interfaces

• Functional assays:

  • ATP synthesis/hydrolysis measurements in membrane vesicles from wild-type and mutant strains

  • Proton translocation assays using pH-sensitive fluorescent probes

  • Intracellular ATP level measurements using luminescence-based assays

• Structural studies:

  • Cryo-EM analysis of ATP synthase complexes with and without bound virulence factors

  • NMR studies of isolated subunits and their interactions with regulatory proteins

Research has demonstrated that MgtC interacts with the a subunit of the F1F0 ATP synthase, and that a single amino acid substitution in MgtC that attenuates Salmonella pathogenicity prevents this interaction . This provides a model for how similar experiments might be designed to study other virulence factor interactions with ATP synthase.

How can recombinant Salmonella schwarzengrund ATP synthase subunit c be used in studying bacterial resistance mechanisms?

Recombinant ATP synthase subunit c from Salmonella schwarzengrund offers several valuable applications for studying bacterial resistance mechanisms:

• Structural basis for antimicrobial resistance:

  • Using purified recombinant protein to study binding interactions with antimicrobial compounds

  • Crystallographic studies to determine structural changes associated with resistance mutations

• Development of ATP synthase inhibitors:

  • High-throughput screening assays using recombinant protein to identify novel inhibitory compounds

  • Structure-based drug design targeting specific regions of ATP synthase

• Cross-species comparative studies:

  • Analyzing structural and functional differences between ATP synthase subunits across bacterial species to identify selective targets

  • Development of broad-spectrum antimicrobials targeting conserved regions

• Resistance mechanism elucidation:

  • Using recombinant protein with introduced mutations to characterize how specific changes affect antimicrobial susceptibility

  • Development of assays to detect antimicrobial binding to ATP synthase components

The connection between ATP synthase and virulence mechanisms suggests that targeting this complex could provide novel approaches to combat bacterial infections, particularly for pathogens with emerging antimicrobial resistance.

What is the relationship between plasmid-encoded factors and ATP synthase function in Salmonella schwarzengrund?

Research on Salmonella schwarzengrund has revealed interesting relationships between plasmid-encoded factors and bacterial physiology, including potential effects on ATP synthase function:

• Plasmid diversity in S. schwarzengrund: Studies have identified various plasmids in S. schwarzengrund isolates, including an IncFIB-IncFIC(FII) fusion plasmid conferring streptomycin resistance in approximately 31% of isolates (17 out of 55 studied) .

• Plasmid distribution patterns: Among the 17 isolates carrying the fusion plasmid, 9 were food isolates primarily from poultry meat, and 8 were clinical isolates from stool, urine, and gallbladder . SNP-based phylogenetic analyses showed these isolates formed a subclade, indicating the plasmid was acquired and maintained by the lineage.

• Plasmid-encoded virulence factors: The IncFIB-IncFIC(FII) plasmids carried genes of the aerobactin operon (iucABCD and iutA) and traT, which may contribute to bacterial fitness and virulence .

• Conjugative transfer capability: These plasmids were shown to be self-conjugative, successfully transferring to E. coli J53 through conjugation experiments , suggesting potential for horizontal spread of resistance and virulence traits.

• Metabolic impacts: While direct effects on ATP synthase have not been explicitly demonstrated for these specific plasmids, the presence of plasmid-encoded virulence and metabolic factors could indirectly influence cellular energetics and ATP synthase function through altered metabolic demands or regulatory networks.

The relationship between plasmid-encoded factors and core metabolic functions like ATP synthesis represents an important area for further research, particularly in understanding how acquired genetic elements may influence bacterial fitness in both host and environmental settings.

What are the recommended storage and handling conditions for recombinant Salmonella schwarzengrund ATP synthase subunit c?

Based on product information for recombinant Salmonella schwarzengrund ATP synthase subunit c, the following storage and handling recommendations should be observed:

• Storage temperature:

  • Short-term storage: 4°C for up to one week (working aliquots)

  • Standard storage: -20°C

  • Extended storage: -20°C or -80°C

• Buffer conditions:

  • Typically supplied in Tris-based buffer with 50% glycerol, optimized for protein stability

• Handling precautions:

  • Repeated freezing and thawing is not recommended as it may lead to protein degradation or loss of activity

  • Working aliquots should be prepared to minimize freeze-thaw cycles

• Reconstitution considerations:

  • For functional studies, the highly hydrophobic nature of this membrane protein requires appropriate detergents or lipid environments for solubilization and activity

• Quality control:

  • Verification of protein integrity by SDS-PAGE before experimental use is recommended

  • Functional assays should be performed to confirm activity when incorporating the protein into experimental systems

Following these storage and handling guidelines will help maintain the structural integrity and functional properties of the recombinant protein for research applications.

What experimental systems can be used to study the functional properties of recombinant ATP synthase subunit c?

Several experimental systems have been developed to study the functional properties of recombinant ATP synthase subunit c:

Research has shown that the c-subunit can form channels with multiple conductance states and is regulated by various factors . These experimental systems provide complementary approaches to study different aspects of c-subunit function in isolation or in the context of the complete ATP synthase complex.