Recombinant Shigella flexneri serotype 5b ATP synthase subunit b (atpF)

What is the role of ATP synthase subunit b (atpF) in Shigella flexneri serotype 5b?

ATP synthase subunit b (atpF) is a critical component of the F0 sector of the ATP synthase complex in Shigella flexneri serotype 5b. This protein functions as part of the membrane-embedded stator stalk that connects the F1 and F0 portions of ATP synthase. The subunit plays a crucial role in maintaining the structural integrity of the complex and facilitating the conversion of the proton gradient across the membrane into mechanical energy that drives ATP synthesis. The protein is particularly important during anaerobic growth conditions when S. flexneri must efficiently manage energy production in the absence of oxygen . Methodologically, researchers investigating the function of atpF typically employ gene knockout studies followed by growth curve analysis under different oxygen conditions to assess the impact on bacterial fitness.

How does the amino acid sequence of S. flexneri serotype 5b atpF compare to other Shigella species?

While the specific sequence for S. flexneri serotype 5b atpF is not directly provided in the search results, we can make comparisons based on related information. The ATP synthase subunit b protein is generally highly conserved among closely related bacterial species, with specific variations that may reflect adaptations to different ecological niches. For example, Shigella sonnei atpF consists of 156 amino acids and contains characteristic membrane-spanning domains in its N-terminal region and a more hydrophilic C-terminal domain .

What are the optimal conditions for expressing recombinant S. flexneri serotype 5b atpF protein?

For optimal expression of recombinant S. flexneri serotype 5b atpF protein, researchers typically use E. coli as an expression system, as demonstrated with related proteins like S. sonnei atpF . The methodology involves:

• Cloning the atpF gene with an N-terminal His-tag into an appropriate expression vector

• Transforming the construct into a suitable E. coli strain (commonly BL21(DE3))

• Growing cultures at 37°C until mid-log phase

• Inducing protein expression with IPTG (0.5-1.0 mM) at reduced temperature (16-25°C) to enhance proper folding

• Harvesting cells after 4-6 hours of induction

For membrane proteins like atpF, expression conditions must be carefully optimized to prevent protein aggregation. Addition of glycerol (5-10%) to the growth medium and lowering the induction temperature to 16°C often improves solubility. Purification is typically performed using Ni-NTA affinity chromatography under native or mild denaturing conditions, followed by size exclusion chromatography to obtain pure protein .

What methods are most effective for studying atpF function in the context of S. flexneri metabolism?

To effectively study atpF function in S. flexneri metabolism, researchers employ multiple complementary approaches:

• Gene knockout and complementation studies: Creating atpF deletion mutants followed by phenotypic characterization under different growth conditions (aerobic vs. anaerobic). Complementation with plasmid-expressed atpF confirms phenotype specificity.

• RNA-seq analysis: This approach reveals transcriptomic changes in response to different environmental conditions. For example, RNA-seq has been used to study how anaerobiosis affects gene expression in S. flexneri, including genes involved in energy metabolism .

• Metabolomic profiling: Comparing metabolite levels between wild-type and atpF mutants provides insights into altered metabolic pathways.

• Growth studies under variable conditions: Testing growth in media with different carbon sources and oxygen availability helps define the metabolic role of atpF. S. flexneri strains exhibit significant differences in carbon metabolism and oxygen sensitivity despite high genetic similarity .

• Protein-protein interaction studies: Techniques such as bacterial two-hybrid systems or co-immunoprecipitation can identify interaction partners of atpF, elucidating its role in larger metabolic networks.

A comprehensive understanding requires integration of multiple datasets to place atpF function within the broader context of S. flexneri metabolism and adaptation to environmental changes.

How has the atpF gene evolved across different S. flexneri serotypes?

The evolution of the atpF gene across S. flexneri serotypes reflects both conservation of essential function and adaptive changes. Comparative genomic analysis reveals:

To study atpF evolution, researchers typically conduct whole-genome sequencing of multiple S. flexneri isolates, followed by comparative genomic analysis and calculation of dN/dS ratios to assess selective pressure. When comparing S. flexneri 5b (Sf8401) with S. flexneri 2a (Sf301), researchers found high structural and functional conservation between serotypes, with differences reflecting diverse selection pressures during evolution .

What genomic context surrounds the atpF gene in S. flexneri serotype 5b, and how does this differ from other serotypes?

The genomic context of atpF in S. flexneri is characterized by its location within the highly conserved ATP synthase operon. While specific details for serotype 5b are not directly provided in the search results, general patterns can be inferred:

• Operon structure: The atpF gene is typically part of the atpBEFHAGDC operon, which encodes the components of ATP synthase.

• Conservation across serotypes: The ATP synthase operon structure is generally conserved across S. flexneri serotypes due to its essential function.

• Nearby genomic features: Unlike some metabolic genes that might be affected by nearby mobile genetic elements, the ATP synthase operon typically remains intact during genomic rearrangements.

• Chromosomal position: The relative position of the ATP synthase operon is generally stable in the S. flexneri genome, though large-scale chromosomal inversions may occur between serotypes, as observed between S. flexneri 5b and 2a .

Methodologically, researchers map the atpF gene and its surrounding regions using genome sequencing and annotation tools, followed by comparative analysis using programs like Mauve or ACT (Artemis Comparison Tool) to visualize synteny and rearrangements between serotypes.

How is atpF expression regulated under anaerobic conditions in S. flexneri serotype 5b?

The regulation of atpF expression under anaerobic conditions involves several mechanisms:

• FNR-dependent regulation: The Fumarate and Nitrate Reduction (FNR) regulator plays a crucial role in modulating gene expression during anaerobiosis in S. flexneri. FNR recognizes specific binding sites and can activate or repress transcription based on oxygen availability .

• Global metabolic adjustments: Under anaerobic conditions, S. flexneri undergoes global transcriptional changes affecting metabolic pathways, with ATP synthase components potentially regulated as part of this response .

• Energy conservation strategies: When oxygen is limiting, ATP generation through oxidative phosphorylation is compromised, potentially leading to compensatory regulation of ATP synthase components.

• Small RNA regulation: Anaerobic conditions induce expression of small RNAs like csrB and csrC in an FNR-independent manner in S. flexneri, which may indirectly affect atpF expression through post-transcriptional mechanisms .

Research methodologies to study anaerobic regulation include:

• RNA-seq analysis comparing aerobic vs. anaerobic growth conditions

• Chromatin immunoprecipitation (ChIP) to identify direct FNR binding to regulatory regions

• Reporter gene assays using atpF promoter fusions to quantify expression under different oxygen conditions

• Western blotting to monitor protein levels across growth conditions

What role does atpF play in the metabolic rewiring observed between different S. flexneri strains?

ATP synthase subunit b (atpF) contributes to the metabolic differences observed between S. flexneri strains, particularly in energy metabolism:

• Energy production efficiency: Differences in ATP synthase efficiency may contribute to strain-specific metabolic profiles, as even closely related S. flexneri strains (98.9% genetic identity) can exhibit drastically different metabolic phenotypes .

• Adaptation to oxygen availability: S. flexneri strains vary in oxygen sensitivity, with some strains better adapted to anaerobic conditions than others. The ATP synthase complex is central to energy conservation during transitions between aerobic and anaerobic metabolism .

• Carbon source utilization: Different S. flexneri strains show variation in carbon metabolism, which affects energy production pathways. For example, some strains utilize the pyruvate oxidase/acetyl-CoA synthetase (PoxB/Acs) pathway while others cannot due to mutations .

• Integration with alternative energy pathways: When ATP production via oxidative phosphorylation is limited, alternative pathways like substrate-level phosphorylation become important. The efficiency of ATP synthase, influenced by its subunits including atpF, affects the balance between these pathways.

Methodologically, researchers investigate these effects through:

• Growth rate comparisons on different carbon sources

• Oxygen consumption measurements

• Metabolic flux analysis using isotope labeling

• ATP production quantification under varying conditions

How does atpF function contribute to S. flexneri serotype 5b virulence and host adaptation?

The ATP synthase subunit b (atpF) contributes to S. flexneri virulence and host adaptation through several mechanisms:

• Energy provision during infection: ATP synthase ensures sufficient energy production during the different stages of infection, particularly important during intracellular growth and spread.

• Adaptation to the intestinal environment: The gastrointestinal tract presents varying oxygen concentrations, from aerobic to microaerobic to anaerobic. ATP synthase function, including atpF, is critical for adaptation to these changing conditions .

• Support for virulence factor expression: Energy metabolism and virulence are interconnected. Metabolites like formate, produced during anaerobic metabolism, can induce virulence genes like icsA and ipaJ in S. flexneri, affecting intracellular growth and intercellular movement .

• Survival within macrophages: Following phagocytosis, bacteria face oxygen-limited conditions within macrophages. Efficient ATP production via ATP synthase may be crucial for survival in this environment.

• Maintenance of membrane potential: Beyond ATP production, the proton-pumping function of ATP synthase helps maintain membrane potential, which is important for various cellular processes including resistance to antimicrobial peptides.

Research approaches to study these connections include:

• Tissue culture infection models comparing wild-type and atpF mutants

• Animal infection studies

• Transcriptomic analysis during different stages of infection

• Measurement of ATP levels and membrane potential during host cell interaction

Can mutations in atpF affect antibiotic susceptibility in S. flexneri serotype 5b?

Mutations in atpF can potentially affect antibiotic susceptibility in S. flexneri through several mechanisms:

• Energy-dependent drug efflux: Many antibiotic efflux pumps require energy (ATP or proton motive force) to function. Alterations in ATP synthase efficiency may affect the activity of these pumps, changing susceptibility to various antibiotics.

• Membrane potential alterations: ATP synthase contributes to maintaining membrane potential, which affects the uptake of certain antibiotics, particularly positively charged ones.

• Metabolic state influence: Changes in energy metabolism due to atpF mutations can alter bacterial growth rate and metabolic state, which in turn affects susceptibility to antibiotics targeting active processes.

• Persister cell formation: ATP depletion has been linked to persister cell formation, a phenotypic state associated with antibiotic tolerance. Mutations affecting ATP synthase function could potentially influence persister formation.

• Compensatory metabolic changes: Alterations in ATP synthase function may trigger compensatory changes in metabolism that indirectly affect antibiotic susceptibility.

Research methodologies to investigate these effects include:

• Minimum inhibitory concentration (MIC) determinations for various antibiotics comparing wild-type and atpF mutants

• Time-kill kinetics under different metabolic conditions

• Membrane potential measurements using fluorescent dyes

• Combination treatments with ATP synthase inhibitors and conventional antibiotics

• Transcriptomic analysis to identify compensatory changes in response to atpF mutation

What novel approaches can be used to study the structure-function relationship of recombinant S. flexneri serotype 5b atpF?

Advanced techniques for studying structure-function relationships of recombinant S. flexneri serotype 5b atpF include:

• Cryo-electron microscopy (Cryo-EM): This technique can resolve the structure of the entire ATP synthase complex, including the position and interactions of the b subunit, at near-atomic resolution without crystallization.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This method identifies regions of proteins that are exposed to solvent versus those buried in the structure, providing dynamic information about protein conformation and interactions.

• Site-directed spin labeling combined with electron paramagnetic resonance (EPR): This approach can measure distances between specific residues in atpF, providing constraints for structural modeling.

• Molecular dynamics simulations: Based on available structural data, simulations can predict how the protein behaves in a membrane environment and how mutations might affect its function.

• Single-molecule FRET (Förster Resonance Energy Transfer): This technique can measure conformational changes in the protein during the catalytic cycle in real-time.

• Native mass spectrometry: This can determine the stoichiometry and stability of protein complexes, including how atpF interacts with other ATP synthase subunits.

• Cross-linking mass spectrometry (XL-MS): This identifies proximity relationships between different parts of the protein or between atpF and other subunits.

Implementation of these techniques requires careful protein expression and purification, often with appropriate tags or labels, while maintaining native-like conditions to preserve structural integrity.

How can systems biology approaches integrate atpF function into broader metabolic networks in S. flexneri?

Systems biology approaches offer powerful frameworks for integrating atpF function into broader metabolic networks:

• Multi-omics data integration: Combining transcriptomics, proteomics, and metabolomics data allows researchers to track how changes in atpF expression propagate through the system. For example, RNA-seq analysis of S. flexneri under anaerobic conditions revealed extensive metabolic rewiring , which could be further integrated with proteomic and metabolomic data.

• Protein-protein interaction networks: Mapping the interactions between atpF and other proteins using techniques such as affinity purification-mass spectrometry or bacterial two-hybrid screening places atpF in a broader cellular context.

• Metabolic control analysis: This approach quantifies how control of metabolic fluxes is distributed among different enzymes, including ATP synthase, providing insights into the relative importance of atpF in different metabolic contexts.

• In silico knockouts and synthetic lethality predictions: Computational simulations of atpF deletion or modification can predict synthetic lethal interactions, guiding experimental design for functional studies.

• Agent-based modeling: For infection dynamics, agent-based models can incorporate metabolic models of individual bacteria to simulate how atpF-dependent energy production affects population behavior during infection.

The integration of these approaches enables researchers to move beyond studying atpF in isolation and understand its role in the context of cellular adaptation to changing environments, particularly the anaerobic conditions encountered during infection .