Recombinant Listeria innocua serovar 6a ATP synthase subunit b (atpF)

What is ATP synthase subunit b (atpF) in Listeria innocua and what are its synonyms in scientific literature?

ATP synthase subunit b (atpF) in Listeria innocua serovar 6a is a critical component of the F-type ATP synthase complex, which is responsible for ATP production through oxidative phosphorylation. In scientific literature, this protein is known by several synonyms, including ATP synthase F(0) sector subunit b, ATPase subunit I, and F-type ATPase subunit b . The gene encoding this protein is primarily referred to as atpF, with the alternative designation lin2677 in some genomic annotations . This nomenclature consistency is important for accurate literature searches and cross-referencing in research publications.

How does the structure of Listeria innocua ATP synthase subunit b compare to other bacterial species?

The ATP synthase subunit b from Listeria innocua exhibits structural characteristics common to other bacterial species, particularly the formation of dimers that are critical for the proper functioning of the ATP synthase complex. Research indicates that the b subunit dimer is extremely elongated, with a frictional ratio of 1.60, a maximal dimension of 95 Å, and a radius of gyration of 27 Å . These measurements are consistent with an alpha-helical coiled-coil structure that has been observed in other bacterial species. Crystal structure analysis reveals that the isolated dimerization domain (residues 62-122) forms a monomeric alpha helix with a length of approximately 90 Å . This structural conservation suggests evolutionary importance of this domain for ATP synthase function across bacterial species.

What is the amino acid sequence of the Listeria innocua ATP synthase subunit b, and how does it influence protein function?

While the complete amino acid sequence of ATP synthase subunit b (atpF) is not fully detailed in the provided literature, related ATP synthase components in Listeria innocua, such as subunit a (atpB), have been characterized. The partial recombinant form of ATP synthase subunit b (atpF) is available for research purposes , suggesting segmental expression for functional studies. The amino acid composition and sequence determine the protein's structural properties, including its ability to form the characteristic alpha-helical structure and to dimerize correctly. These structural features are essential for the protein's role in anchoring the F1 portion of ATP synthase and preventing its rotation relative to the F0 sector, thereby allowing the enzyme to function properly in ATP synthesis .

How does the dimerization domain of ATP synthase subunit b contribute to protein function in Listeria innocua?

The dimerization domain of ATP synthase subunit b, comprising residues 62-122, plays a crucial role in the proper assembly and function of the ATP synthase complex in Listeria innocua. This domain mediates the formation of b-subunit dimers, which are essential for the structural integrity of the ATP synthase complex . The extremely elongated nature of the b subunit dimer, with its distinctive alpha-helical coiled-coil structure, suggests that it serves as a structural scaffold connecting the membrane-embedded F0 sector with the catalytic F1 sector of ATP synthase . This connection prevents rotation of the F1 sector relative to F0, which is necessary for the rotary catalysis mechanism of ATP synthesis. The precise arrangement of amino acids within this domain facilitates the specific protein-protein interactions required for dimerization and subsequent interaction with other ATP synthase components.

What evolutionary insights can be gained from comparing ATP synthase subunit b across Listeria species and other bacteria?

Evolutionary analysis of ATP synthase subunit b across Listeria species and other bacteria provides valuable insights into bacterial adaptation and energy metabolism. While specific phylogenetic analysis of atpF is not detailed in the provided literature, related evolutionary studies on Listeria species reveal interesting patterns. For instance, analysis of LIPI-4 (Listeria Pathogenicity Island-4) across Listeria innocua and Listeria monocytogenes shows species-specific clustering with longer evolutionary distances in L. innocua, suggesting a more extended evolutionary history . By analogy, comparative analysis of ATP synthase components like atpF could reveal similar evolutionary patterns relevant to energy metabolism adaptation. Such comparisons could identify conserved regions essential for function versus variable regions that might confer species-specific advantages, potentially correlating with habitat adaptation or pathogenicity potential.

What are the optimal conditions for expressing and purifying recombinant Listeria innocua ATP synthase subunit b?

For optimal expression and purification of recombinant Listeria innocua ATP synthase subunit b (atpF), researchers typically use E. coli expression systems due to their efficiency and scalability . Based on protocols for similar proteins, the following methodological approach is recommended:

• Vector Selection: Use vectors with strong inducible promoters (such as T7) and appropriate tags (typically His-tag) for purification.

• Expression Conditions:

  • Host strain: BL21(DE3) or derivatives

  • Induction: 0.5-1 mM IPTG at OD600 of 0.6-0.8

  • Post-induction growth: 16-18°C for 16-20 hours (to minimize inclusion body formation)

• Purification Protocol:

  • Lysis in buffer containing 50 mM Tris pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM PMSF

  • Nickel affinity chromatography for His-tagged proteins

  • Size exclusion chromatography for final purification

• Storage: Store in buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, with 6% Trehalose as a stabilizer

For long-term storage, addition of 5-50% glycerol and aliquoting for storage at -20°C/-80°C is recommended to prevent repeated freeze-thaw cycles, which can damage protein structure .

What analytical techniques are most effective for studying the structure-function relationship of ATP synthase subunit b?

Multiple complementary analytical techniques are recommended for comprehensive structure-function analysis of ATP synthase subunit b:

The integration of these techniques provides a comprehensive understanding of how the protein's structure determines its function within the ATP synthase complex.

How can proteomic approaches be applied to study ATP synthase subunit b in Listeria innocua?

Proteomic approaches offer powerful tools for studying ATP synthase subunit b in its cellular context. Based on methodologies applied to related Listeria proteins, the following proteomic strategies are recommended:

• Sample Preparation:

  • Careful cell lysis preserving membrane proteins

  • Fractionation to enrich membrane proteins

  • Protein digestion with multiple proteases for maximal coverage

• LC-MS/MS Analysis:

  • Liquid chromatography coupled to tandem mass spectrometry using high-resolution instruments like the LTQ Orbitrap Velos

  • Data-dependent acquisition for discovery proteomics

  • Parallel reaction monitoring for targeted quantification

• Data Analysis:

  • Protein identification against Listeria innocua databases

  • Post-translational modification analysis

  • Protein-protein interaction networks

  • Comparative analysis under different growth conditions

• Functional Validation:

  • Correlation with phenotypic assays

  • Integration with transcriptomic data

  • Validation of key findings with targeted experiments

These approaches can reveal not only the abundance and modifications of ATP synthase subunit b but also its interactions and regulation under different physiological conditions.

How do genomic and proteomic data on ATP synthase subunit b contribute to understanding Listeria innocua virulence potential?

While Listeria innocua is generally considered non-pathogenic compared to L. monocytogenes, genomic and proteomic analysis of components like ATP synthase subunit b can provide insights into its potential virulence. The whole genome analysis of L. innocua reveals several important features that may relate to ATP synthase function and broader virulence potential:

• Virulence Gene Presence: L. innocua contains multiple virulence genes related to adherence (fbpA, lap), invasion (iap/cwhA, gtcA, lpeA), surface protein anchoring (lspA), and intracellular survival (lplA1, prsA2) . The energy required for expressing and operating these virulence systems depends on ATP produced by ATP synthase.

• LIPI-4 Presence: Unlike other pathogenicity islands, LIPI-4 (which encodes a cellobiose-family phosphotransferase system) is widespread among L. innocua . This system enhances invasion of the central nervous system and maternal-neonatal infection in L. monocytogenes . The energy demands of this system are supported by ATP synthase activity.

• Evolutionary Considerations: Phylogenetic analysis suggests L. innocua has a longer evolutionary history than L. monocytogenes , potentially affecting the evolution of energy metabolism systems including ATP synthase components.

• Horizontal Gene Transfer: L. innocua can act as a gene sink, collecting antimicrobial resistance determinants , which may affect energy requirements and thus ATP synthase regulation.

Understanding ATP synthase subunit b in this context helps elucidate how energy metabolism supports potential virulence mechanisms in this organism.

What does the presence or absence of specific domains in ATP synthase subunit b tell us about bacterial adaptation?

The presence or absence of specific domains in ATP synthase subunit b can reveal important insights about bacterial adaptation strategies:

• Dimerization Domain Analysis: The specific characteristics of the dimerization domain (residues 62-122) in ATP synthase subunit b, with its elongated alpha-helical structure , reflect adaptation for efficient energy conversion. Variations in this domain across bacterial species may indicate adaptation to different energetic requirements or environmental conditions.

• Conservation Patterns: Highly conserved regions likely represent essential functional domains that cannot tolerate variation, while variable regions may indicate adaptation to specific niches or metabolic requirements. For example, thermophilic bacteria often have adaptations in ATP synthase components that enhance stability at high temperatures.

• Regulatory Elements: The presence of regulatory domains or modification sites can indicate adaptation to environments requiring rapid metabolic adjustment. While specific regulatory elements in L. innocua ATP synthase subunit b are not detailed in the provided literature, such elements could be identified through comparative genomic analysis.

• Interaction Interfaces: Variation in regions that interact with other ATP synthase components may reflect co-evolution of the entire complex in response to selective pressures such as pH, temperature, or energy availability in the bacterium's typical habitat.

These domain-level insights provide a molecular basis for understanding how bacteria adapt their energy production machinery to diverse ecological niches.

How can apparent contradictions in experimental data on ATP synthase subunit b be resolved?

Resolving apparent contradictions in experimental data on ATP synthase subunit b requires a systematic approach:

By systematically addressing these factors, apparently contradictory data can often be reconciled into a more nuanced understanding of ATP synthase subunit b structure and function.