Recombinant Bdellovibrio bacteriovorus ATP synthase subunit b (atpF), partial

What is the role of ATP synthase subunit b (atpF) in Bdellovibrio bacteriovorus predation cycle?

ATP synthase subunit b is a critical component of the F₀ sector of ATP synthase in B. bacteriovorus, forming part of the peripheral stalk that connects the F₁ catalytic domain to the membrane-embedded F₀ domain. This stalk is essential for preventing rotation of the α₃β₃ hexamer during ATP synthesis . In the predatory lifecycle, ATP synthase activity is particularly important during the intraperiplasmic growth phase, when the bacterium requires significant energy to digest prey contents and replicate . Recent studies indicate enhanced ATP synthase expression during the transition from attack phase to growth phase, suggesting its importance in energy production during prey invasion and consumption .

Methodologically, researchers investigating this can use:

• RT-PCR across the predatory life cycle to monitor temporal expression patterns

• Targeted gene knockouts to assess phenotypic changes in predation efficiency

• ATP measurements during different predatory stages to correlate with enzyme activity

How does the structure of B. bacteriovorus ATP synthase subunit b compare to other bacterial species?

B. bacteriovorus ATP synthase subunit b has a unique architecture compared to other bacterial species. Based on structural analyses, the N-terminal membrane-embedded α-helix in the b-subunit of bacterial ATP synthases forms different interactions with subunit a . In B. bacteriovorus, this interaction is particularly important given the extreme energy demands of predatory lifestyle.

Table 1: Comparison of ATP Synthase b-subunit Properties Across Bacterial Species

Most bacterial ATP synthases feature two copies of subunit b that form different interactions with subunit a. One surface typically interacts with transmembrane α-helices 1, 2, 3, and 4 of subunit a, while the other interacts with α-helices 5 and 6 . This arrangement is critical for proper assembly and function of the enzyme complex.

What experimental approaches are most effective for expressing and purifying recombinant B. bacteriovorus ATP synthase subunit b?

Expression and purification of recombinant B. bacteriovorus ATP synthase subunit b presents unique challenges due to its hydrophobic regions. Based on current methodologies with similar proteins:

Recommended Expression Systems:

• E. coli BL21(DE3) with C41/C43 derivatives for membrane proteins

• Yeast expression systems for proteins with problematic folding

Purification Protocol:

• Use His-tag or other affinity tags positioned to avoid interference with protein folding

• Employ mild detergents (DDM or LDAO) for solubilization

• Consider nanodiscs or amphipols for maintaining native-like environment

• Purify using a combination of affinity chromatography and size exclusion

Storage recommendations include maintaining the protein in 50% glycerol at -20°C to -80°C, with shelf life typically ranging from 6 months (liquid form) to 12 months (lyophilized) . Repeated freeze-thaw cycles should be avoided, with working aliquots stored at 4°C for no more than one week.

How can researchers investigate the interactions between ATP synthase subunit b and other components of the complex in B. bacteriovorus?

Investigating protein-protein interactions within the ATP synthase complex requires specialized techniques:

• Cross-linking studies: Use bifunctional chemical cross-linkers to capture interactions, although care must be taken in interpretation as previous studies showed that the N-terminus of two copies of subunit b may appear in close proximity despite being on opposite sides of subunit a .

• Bacterial two-hybrid systems: Useful for mapping interaction domains between subunit b and other components.

• Cryo-EM analysis: Has successfully revealed the structure of bacterial ATP synthases in multiple rotational states and can elucidate subunit b positioning .

• Site-directed mutagenesis: Target conserved residues in subunit b to assess functional impacts:

Table 2: Key Residues for Mutagenesis Studies in ATP Synthase Subunit b

How does ATP synthase activity in B. bacteriovorus correlate with its unique predatory lifestyle and energy requirements?

B. bacteriovorus has exceptional energy demands during predation, requiring ATP for both motility and prey cell degradation. Recent research suggests that ATP synthase plays a critical role in energy conversion during the intraperiplasmic stage.

Intracellular ATP levels can be measured using ATP bioluminescence assay kits, as described in study , where samples were centrifuged (13,000 × g, 15 min, 4°C) and resuspended in saline solution prior to measurement. This approach allows normalization to viable predator cell counts determined by double-layer method .

The predatory lifecycle requires careful energy management as B. bacteriovorus transitions between attack phase (seeking prey) and growth phase (consuming prey). ATP synthase activity appears to be differentially regulated during these phases, with evidence suggesting:

• Upregulation during the transition from attack to growth phase

• Peak activity coinciding with prey content digestion

• Possible coordination with nuclease activity (Bd1244, Bd1934) which peaks 30-45 minutes after prey invasion

Research methodologies should include:

• Comparisons of ATP synthesis rates between predatory wild-type and host-independent (HI) variants

• Measurement of proton motive force during different predatory stages

• Integration of transcriptomic and proteomic data to create a comprehensive model of energy utilization

What is the role of ATP synthase in host-independent (HI) growth of B. bacteriovorus, and how does it differ from its function during predation?

Host-independent (HI) variants of B. bacteriovorus can grow axenically (without prey) on rich media, representing a significant metabolic adaptation. Evidence suggests ATP synthase function differs between predatory and HI growth:

Table 3: Comparison of ATP Synthase Function in Different Growth Modes

HI variants show metabolic flexibility, with ATP synthase playing a crucial role in energy production from amino acid catabolism rather than prey-derived nutrients. Recent research indicates that proline and glutamate serve as primary carbon sources in amino acid-based rich media during HI growth .

How can researchers address the technical challenges in studying the function of ATP synthase subunit b mutations in B. bacteriovorus?

Studying ATP synthase mutations in B. bacteriovorus presents unique challenges due to its predatory lifestyle. Advanced researchers should consider:

• Genetic manipulation approaches:

  • Use suicide vectors like pK18mobsacB for targeted gene replacement

  • Consider conditional knockdowns if complete deletion affects viability

  • Implement CRISPR-Cas9 systems adapted for deltaproteobacteria

• Phenotypic analysis strategies:

  • Utilize luminescent prey assays to quantify predation efficiency

  • Measure swimming speeds using tracking microscopy to assess motility impact

  • Apply biofilm formation assays to identify changes in predatory behavior

• Energy metabolism assessment:

  • Measure membrane potential with fluorescent probes

  • Determine ATP/ADP ratios during different phases of predation

  • Conduct oxygen consumption measurements during predatory cycle

Critical considerations include the timing of measurements within the predatory cycle and distinguishing between effects on ATP synthesis versus prey cell invasion or digestion.

What is the relationship between B. bacteriovorus ATP synthase and its unusual motility patterns required for predation?

B. bacteriovorus relies on flagellar motility for locating prey, with motility ceasing during invasion and resuming inside the bdelloplast prior to lysis . The relationship between ATP synthase and motility is complex:

• Energy coupling: ATP synthase provides the energy for the three different MotAB flagellar motor protein pairs encoded in the B. bacteriovorus genome .

• Proton gradient utilization: Both ATP synthase and flagellar motors utilize the proton gradient, suggesting potential competition for this resource. Experiments with protonophores like CCCP have shown that all three Mot pairs are proton-driven .

• Regulatory coordination: ATP production must be coordinated with motility requirements during different predatory phases.

Table 4: Relationship Between ATP Synthase Activity and Motility Phases

Research approaches should include:

• ATP synthesis measurements during motility transitions

• Correlation of proton motive force with swimming speed

• Analysis of ATP synthase mutants for motility defects

How might comparative genomics of ATP synthase components across predatory bacteria inform our understanding of energy requirements for predation?

Comparative genomic analysis of ATP synthase subunits across predatory bacteria offers valuable insights into energy adaptation strategies. B. bacteriovorus is a model for bacterial predation , but other predatory bacteria may have evolved different ATP synthase adaptations.

Research approach recommendations:

• Compare ATP synthase gene organization and sequence conservation across predatory species

• Analyze selection pressure on different subunits, particularly peripheral stalk components

• Investigate horizontal gene transfer patterns in ATP synthase components

• Correlate ATP synthase variations with predatory efficiency and host range

This approach can identify conserved features essential for predatory lifestyle versus species-specific adaptations.

What role does ATP synthase play in the formation and regulation of B. bacteriovorus biofilms?

Recent research indicates that B. bacteriovorus can form biofilms, with nucleases like Bd1244 influencing this process . The relationship between ATP synthase activity and biofilm formation represents an unexplored frontier.

Key research questions include:

• How does ATP synthase expression change during biofilm formation?

• Is the proton motive force maintained differently in biofilm versus planktonic cells?

• Do ATP synthase inhibitors affect biofilm formation or stability?

Methodological approaches should include:

• Biofilm formation assays in PVC microtitre plates

• Confocal microscopy with ATP indicators in biofilm structures

• Transcriptomic analysis comparing planktonic and biofilm cells

How can structural information about B. bacteriovorus ATP synthase inform the development of selective inhibitors for biocontrol applications?

As B. bacteriovorus has potential as a "living antibiotic" , understanding its ATP synthase structure could guide the development of selective inhibitors that target pathogenic bacteria while sparing beneficial predatory species.

Research priorities include:

• High-resolution structural determination of B. bacteriovorus ATP synthase

• Identification of unique structural features distinguishing it from pathogenic species

• In silico screening of compounds that selectively bind to pathogen ATP synthases

• Development of assays to measure differential inhibition across bacterial species

This research direction has significant implications for addressing antibiotic resistance by harnessing rather than hindering predatory bacteria's natural abilities.

What are the optimal conditions for measuring ATP synthase activity in B. bacteriovorus samples?

Measuring ATP synthase activity in B. bacteriovorus requires careful consideration of its unique lifecycle and energy metabolism:

Recommended Experimental Parameters:

• Sample preparation:

  • For predatory cells: Synchronize predation by adding predator cells to prey at high MOI (>3)

  • For HI cells: Harvest during exponential growth phase

  • Membrane preparation should use gentle lysis methods to preserve enzyme integrity

• Activity assay options:

  • ATP synthesis: Measure formation of ATP from ADP and Pi in the presence of a proton gradient

  • ATP hydrolysis: Monitor release of Pi from ATP using colorimetric assays

  • Proton pumping: Measure pH changes with pH-sensitive dyes

• Buffer conditions:

  • pH: Test range from 6.5-8.0 (optimal likely ~7.4)

  • Salt: Consider the potential adaptation to different environments

  • Divalent cations: Mg²⁺ concentration critical (typically 2-5 mM)

• Controls and validations:

  • Include specific ATP synthase inhibitors (oligomycin, DCCD)

  • Prepare comparative measurements from model organisms (E. coli)

  • Include uncoupled controls (CCCP-treated samples)

By accounting for these specialized considerations, researchers can obtain reliable measurements of ATP synthase activity in this unique predatory bacterium.