Recombinant Lactobacillus sakei subsp. sakei ATP synthase subunit b (atpF)

How is the ATP synthase operon organized in Lactobacillus sakei compared to other bacteria?

The ATP synthase (atp) operon in Lactobacillus sakei follows a gene arrangement that is conserved among many bacterial species. The operon typically consists of nine genes arranged in the order atpI(i), atpB(a), atpE(c), atpF(b), atpH(δ), atpA(α), atpG(γ), atpD(β), and atpC(ε) . This organization is identical to that found in many other bacteria including Escherichia coli.

The atpF gene encoding subunit b is positioned in the middle of the operon, following atpE that encodes subunit c. This arrangement is functionally significant as these gene products form part of the membrane-embedded Fo portion of the ATP synthase complex .

While many bacteria follow this arrangement, some variations exist. For example, the anaerobic acetogenic bacterium Acetobacterium woodii has 11 genes in its atp operon, including two additional copies of atpE . The Lactobacillus sakei operon lacks these additional genes, reflecting its different evolutionary adaptation and energy metabolism requirements.

What expression systems are optimal for producing recombinant L. sakei atpF protein?

Based on published research, E. coli expression systems are most commonly used for heterologous expression of L. sakei proteins including atpF. The search results indicate that for the commercial production of recombinant L. sakei subsp. sakei ATP synthase subunit b, E. coli was the expression host of choice .

For optimal expression of membrane proteins like atpF, the following methodological considerations are important:

• Vector selection: pBAD-based expression vectors have proven effective for L. sakei proteins, as they allow for arabinose-inducible expression with fine control over expression levels .

• Expression conditions:

  • Induction at lower temperatures (30°C vs. 37°C) improves proper folding

  • Addition of osmolytes (like 0.3 M sorbitol) to the growth medium enhances stability

  • For membrane proteins like atpF, expression at lower inducer concentrations (0.1 mM arabinose) helps prevent aggregation

• Purification tags: N-terminal His-tags are commonly used, as demonstrated in commercial preparations where the full-length atpF (1-173) was fused to an N-terminal His tag .

• Strain selection: E. coli TOP10 and BL21(DE3) strains are suitable hosts for L. sakei membrane protein expression .

What are the challenges in purifying functional recombinant L. sakei ATP synthase subunit b?

Purification of functional recombinant L. sakei ATP synthase subunit b presents several methodological challenges:

• Membrane protein solubilization: As a membrane-associated protein, atpF requires proper detergent extraction. Typical approaches include:

  • Initial extraction with buffer containing EDTA, which has been successfully used for ATP synthase components from Clostridium pasteurianum and can be applied to L. sakei

  • Use of mild detergents such as DDM (n-dodecyl β-D-maltoside) or CHAPS to maintain native structure

• Maintaining stability: The protein requires specific storage conditions:

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

  • Addition of 5-50% glycerol for long-term storage

  • Storage at -20°C/-80°C with avoidance of repeated freeze-thaw cycles

• Reconstitution process: For functional studies, the lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Protein-lipid interactions: Since atpF is a membrane protein, its function depends on proper interaction with lipids, requiring careful consideration during purification and subsequent functional assays.

• Verification of purity: SDS-PAGE analysis is typically used to confirm purity, with commercial preparations achieving >90% purity .

How do carbon source and energy limitation affect ATP synthase expression in L. sakei?

The expression of ATP synthase genes including atpF in L. sakei is significantly influenced by carbon source availability and energy limitation, as revealed by transcriptome and proteome analyses.

When comparing growth on different carbon sources (glucose versus ribose), significant changes in global gene expression are observed. Under glucose-limited conditions at different growth rates, L. sakei demonstrates a shift from homolactic to mixed acid fermentation, with corresponding changes in energy metabolism .

Key observations include:

• Growth rate effects: Different dilution rates (D = 0.357 h^-1 vs. D = 0.045 h^-1) in glucose-limited chemostats result in distinct metabolic profiles and gene expression patterns .

• Strain-specific responses: L. sakei strains (23K vs. LS25) show different degrees of response to the same energy restriction, suggesting strain-specific regulation of energy metabolism genes .

• Redox balance regulation: The transcriptional regulator Rex and NADH oxidase show differential expression under energy limitation, indicating that maintenance of the cell redox balance (NADH/NAD+ ratio) is a key process during metabolic adaptation .

• Carbon catabolite repression (CCR): The expression of ATP synthase genes appears to be subject to catabolite control protein A (CcpA)-mediated CCR, with putative catabolite-responsive element (cre) sites found in proximity to promoters of several genes affected by carbon source changes .

These findings indicate that ATP synthase components, including atpF, are part of a sophisticated regulatory network that responds to energy availability in L. sakei.

What experimental approaches can be used to study the assembly and function of recombinant L. sakei atpF in membrane complexes?

Several advanced experimental approaches can be employed to study the assembly and function of recombinant L. sakei atpF in membrane complexes:

• Cryo-electron microscopy (cryo-EM) has been successfully used to determine the structure of bacterial ATP synthases in different rotational states, providing insights into the arrangement of subunits including atpF. This technique can reveal:

  • The position of the b subunit relative to other components

  • Structural changes during the catalytic cycle

  • Interaction interfaces between subunits

• Site-directed mutagenesis of key residues in atpF can provide information about:

  • Residues critical for interaction with subunit a

  • Regions important for assembly with the F1 portion

  • Domains involved in the stability of the peripheral stalk

• Cross-linking experiments combined with mass spectrometry can identify:

  • Proximity relationships between atpF and other subunits

  • Conformational changes under different physiological conditions

  • Unexpected interactions with other cellular components

• Reconstitution of ATP synthase in liposomes containing:

  • Purified recombinant atpF and other ATP synthase components

  • A controllable proton gradient

  • ATP synthesis/hydrolysis measurement capabilities

• Fluorescence resonance energy transfer (FRET) using labeled subunits to study:

  • Dynamic interactions during catalysis

  • Conformational changes in response to different conditions

  • Assembly processes in real-time

These methods can be combined to provide a comprehensive understanding of atpF function within the ATP synthase complex.

How does L. sakei ATP synthase function in relation to its ecological niche in meat fermentation?

L. sakei has evolved specialized adaptations for its ecological niche in meat fermentation, and its ATP synthase, including the atpF subunit, plays important roles in this adaptation:

• Heme interaction: L. sakei thrives in heme-rich environments such as meat products. While it doesn't require iron or heme for growth, it possesses:

  • A heme-dependent catalase

  • The ability to incorporate iron from myoglobin and hemoglobin

  • Specific transport systems for heme uptake

• Energy metabolism flexibility: In the meat environment where glucose is limited, L. sakei can:

  • Shift from homolactic to mixed acid fermentation

  • Utilize alternative carbon sources like ribose

  • Catabolize nucleosides and arginine, which are abundant in meat

• Low-oxygen adaptation: ATP synthase operation in L. sakei is adapted to the low-oxygen conditions of meat:

  • The F-type ATP synthase may function in reverse under certain conditions, hydrolyzing ATP to maintain membrane potential

  • The peripheral stalk containing atpF is crucial for stabilizing the complex during these adaptive changes

• Cold adaptation: Since meat fermentation often occurs at refrigeration temperatures, L. sakei proteins, including ATP synthase components, maintain functionality at lower temperatures compared to many other bacteria .

This ecological specialization makes L. sakei ATP synthase an interesting model for studying how energy-generating machinery adapts to specific environmental niches.

What is the role of atpF in ATP synthase assembly and how can it be studied using recombinant protein?

The atpF gene product (ATP synthase subunit b) plays a critical role in the assembly and structural integrity of the ATP synthase complex. Based on research with bacterial ATP synthases, we can outline several approaches to study this using recombinant L. sakei atpF:

• Assembly studies using knockout and complementation:

  • Create an L. sakei atpF deletion mutant

  • Complement with recombinant wildtype or modified atpF

  • Assess ATP synthase assembly, localization, and function

• Interaction mapping:

  • The N-terminal membrane-embedded α-helix of subunit b forms different interactions with subunit a

  • One surface interacts with transmembrane α-helices 1, 2, 3, and 4 of subunit a

  • The other interacts with α-helices 5 and 6 and the loop between α-helices 3 and 4

• Structural studies:

  • Recombinant expression of the cytoplasmic domain for structural analysis

  • Investigation of dimerization properties of the b subunits

  • Analysis of interactions with the δ subunit of the F1 portion

• In vitro reconstitution experiments:

  • Stepwise assembly of F1Fo complex using purified recombinant components

  • Analysis of assembly intermediates

  • Identification of critical regions using truncated or mutated atpF variants

These approaches can provide valuable insights into the specific role of atpF in L. sakei ATP synthase assembly and function, which may differ in subtle ways from other well-studied bacterial ATP synthases.

How can recombinant L. sakei atpF be used to study proton translocation mechanisms in bacterial ATP synthases?

Recombinant L. sakei atpF can serve as a valuable tool for investigating proton translocation mechanisms in bacterial ATP synthases through several sophisticated experimental approaches:

• Reconstitution in proteoliposomes:

  • Incorporation of purified recombinant atpF along with other ATP synthase components into artificial membrane vesicles

  • Generation of a proton gradient using ionophores or light-driven pumps

  • Measurement of ATP synthesis rates in relation to proton translocation efficiency

• Site-directed mutagenesis of key residues:

  • Identification of conserved residues at the interface between atpF and the a subunit

  • Mutation of these residues to alter proton channel characteristics

  • Analysis of effects on proton conductance and ATP synthesis

• Accessibility studies:

  • Similar to experiments with E. coli ATP synthase, where residues were mutated to cysteines and tested for Ag+ accessibility

  • Determination of water-accessible regions in the transmembrane domain of atpF

  • Mapping of potential proton pathways

• Biophysical analyses:

  • Solid-state NMR to study dynamics of the reconstituted complex

  • Fluorescence spectroscopy with pH-sensitive probes

  • Electrophysiological measurements in reconstituted membranes

• Cross-species chimeric proteins:

  • Creation of hybrid proteins combining regions of L. sakei atpF with corresponding regions from other bacterial species

  • Analysis of functional consequences in terms of proton translocation efficiency

  • Identification of species-specific adaptations in the proton translocation machinery

These approaches can help elucidate the specific contributions of the b subunit to proton translocation, which appears to involve interactions between the transmembrane helices of subunits a, b, and the c-ring.

What is known about the regulation of ATP synthase expression in L. sakei and how does atpF fit into this regulatory network?

The regulation of ATP synthase expression in L. sakei, including the atpF gene, involves sophisticated mechanisms responding to environmental conditions:

• Carbon source regulation:

  • Transcriptomic studies have shown differential regulation of metabolic genes including those in the ATP synthase operon when L. sakei is grown on different carbon sources (glucose vs. ribose)

  • The presence of putative catabolite-responsive element (cre) sites near promoters suggests regulation by a catabolite control protein A (CcpA)-mediated carbon catabolite repression mechanism

• Growth phase-dependent regulation:

  • ATP synthase expression appears to be linked to growth phase

  • The HPr kinase/phosphatase (HprK) plays a major role in this regulation by controlling the phosphorylation state of the phosphocarrier protein HPr

• Energy status sensing:

  • The NADH/NAD+ ratio affects expression of energy metabolism genes through the Rex transcriptional regulator

  • During energy limitation, differential expression of Rex and NADH oxidase indicates a key role in maintaining redox balance

• Coordinated regulation of the atp operon:

  • The genes in the atp operon, including atpF, appear to be co-regulated as part of a single transcriptional unit

  • This ensures stoichiometric production of ATP synthase components

• Strain-specific regulation:

  • Different L. sakei strains (23K vs. LS25) show distinct regulatory responses to the same energy limitation conditions

  • This suggests strain-specific adaptation mechanisms affecting ATP synthase expression

Understanding these regulatory networks provides insights into how L. sakei adapts its energy metabolism to different environmental conditions, which is crucial for its survival in meat fermentation environments.