Recombinant Bdellovibrio bacteriovorus ATP synthase gamma chain (atpG)

What is the role of ATP synthase gamma chain in Bdellovibrio bacteriovorus?

The ATP synthase gamma chain (atpG) serves as a critical central component of the F1F0 ATP synthase complex in B. bacteriovorus. This complex generates ATP through oxidative phosphorylation, which appears particularly important for B. bacteriovorus given its reliance on amino acid metabolism rather than glycolysis for energy production. The gamma chain functions as the central rotor shaft connecting the F1 catalytic domain to the membrane-embedded F0 domain.

In B. bacteriovorus, ATP synthesis is particularly crucial during its predatory lifecycle transitions, which demand significant energy for both attack-phase motility and intraperiplasmic replication. Unlike many bacteria that primarily utilize glycolysis, B. bacteriovorus demonstrates high activity of citric acid cycle enzymes, suggesting it generates ATP primarily through oxidative phosphorylation . This makes the ATP synthase complex, including the gamma chain, essential for harvesting energy from amino acids that serve as its primary carbon sources .

ATP generation must be tightly regulated during the complex predatory lifecycle of B. bacteriovorus, which involves transitions between extracellular hunting and intraperiplasmic replication phases. These transitions likely involve shifts in ATP synthase activity to accommodate changing energy demands throughout predation.

How does B. bacteriovorus energy metabolism differ from other bacteria?

B. bacteriovorus exhibits a distinctive metabolic profile that differentiates it from most bacteria. Research has demonstrated that B. bacteriovorus is unlikely to utilize polysaccharides as primary energy substrates, instead preferentially respiring amino acids to fulfill energy requirements during intraperiplasmic growth . This metabolic preference is supported by observations of high activities of citric acid cycle enzymes and comparatively low activities of glycolytic enzymes in cell extracts .

Interestingly, despite reduced reliance on glycolysis, B. bacteriovorus maintains high activity of specific glycolytic enzymes like phosphoglucose isomerase (PGI) and contains a complete complement of enzymes required for glycolytic ATP production . This suggests a complex metabolic regulatory system that may adapt based on life cycle phase and environmental conditions.

B. bacteriovorus can grow in amino acid-rich media without prey while maintaining its predatory capability, with glutamate, serine, aspartate, isoleucine, and threonine serving as its main carbon sources . This metabolic flexibility likely enables B. bacteriovorus to survive in diverse environments while maintaining its predatory capacity. The ATP synthase complex would therefore need to function efficiently across these varied metabolic states, potentially requiring specializations in components like the gamma chain.

What are the predicted structural features of B. bacteriovorus atpG protein?

While the search results contain limited specific structural information about B. bacteriovorus atpG, we can make informed predictions based on conserved features of ATP synthase gamma chains across species and the unique metabolic characteristics of this predatory bacterium.

The gamma chain typically consists of a long alpha-helical coiled-coil structure that extends from the membrane-embedded F0 domain into the center of the F1 catalytic domain. It contains conserved regions that interact with the c-ring of F0 and the alpha/beta subunits of F1, facilitating the mechanical rotation that drives ATP synthesis.

Given B. bacteriovorus' unique predatory lifestyle and metabolic adaptations, we might predict potential specializations in its atpG structure. These could include modifications to binding interfaces or regulatory regions that optimize ATP synthesis under the distinctive conditions encountered during predation and intraperiplasmic growth. The structure might also include features that enable rapid adaptation to changing energy demands during transitions between attack phase and growth phase.

Structural analysis approaches similar to those used for the B. bacteriovorus phosphoglucose isomerase, which has been solved to high resolution (1.74 Å and 1.67 Å for different forms) , would be valuable for characterizing atpG structure and identifying potential adaptations for predatory metabolism.

What are the optimal expression systems for producing recombinant B. bacteriovorus atpG?

Producing recombinant B. bacteriovorus atpG requires careful consideration of expression systems to maximize yield and functionality. Based on B. bacteriovorus' growth characteristics, the following expression strategies can be recommended:

E. coli BL21(DE3) remains a standard choice for bacterial protein expression, but researchers should consider several B. bacteriovorus-specific factors:

• Temperature optimization: Since B. bacteriovorus grows optimally at 30-35°C , expression at similar temperatures may better preserve native folding compared to standard 37°C expression. A temperature series testing expression at 28°C, 30°C, and 32°C could identify optimal conditions.

• Codon optimization: B. bacteriovorus has distinct codon usage patterns, making expression strains like Rosetta or CodonPlus that supply rare tRNAs potentially beneficial for atpG expression.

• Expression construct design:

  • Include affinity tags (His6 or GST) with cleavable linkers

  • Test both N-terminal and C-terminal tag placements, as the gamma chain has functional interfaces at both termini

  • Consider fusion proteins with solubility enhancers like SUMO or MBP if initial expression yields insoluble protein

• Induction parameters:

  • IPTG concentration: Test range from 0.1 mM to 1.0 mM

  • Expression duration: 4-6 hours versus overnight induction

  • Cell density at induction: mid-log (OD600 0.6-0.8) versus late-log phase

Given the challenges of expressing membrane-associated proteins, co-expression with molecular chaperones like GroEL/ES might improve atpG folding and solubility. Additionally, researchers might consider cell-free expression systems if cellular expression proves challenging.

What are the challenges in purifying functional recombinant atpG from B. bacteriovorus?

Purifying functional recombinant B. bacteriovorus atpG presents several challenges that researchers should anticipate and address through methodological optimization:

• Solubility challenges: The gamma chain typically interacts extensively with other ATP synthase subunits and may expose hydrophobic regions when expressed in isolation. Researchers can address this through:

  • Addition of solubility enhancers (0.5-1.0 M sorbitol, 5-10% glycerol) to lysis buffers

  • Inclusion of mild detergents at concentrations below CMC to stabilize hydrophobic regions

  • Optimization of salt concentration (300-500 mM NaCl) to reduce aggregation

  • Testing extraction under various pH conditions within B. bacteriovorus' optimal range (pH 7.0-8.0)

• Conformational heterogeneity: The gamma chain undergoes substantial rotational movement during ATP synthesis, potentially leading to structural heterogeneity. Consider:

  • Addition of nucleotides (ATP/ADP) at low concentrations to stabilize specific conformations

  • Size exclusion chromatography as a final purification step to isolate conformationally homogeneous populations

  • Analysis of oligomeric state using dynamic light scattering or analytical ultracentrifugation

• Stability concerns: Isolated ATP synthase components often show reduced stability compared to the assembled complex. Implement:

  • Immediate analysis or appropriate storage conditions (liquid nitrogen or -80°C with cryoprotectants)

  • Addition of reducing agents (1-5 mM DTT or TCEP) to prevent oxidation of cysteine residues

  • Protease inhibitors throughout purification to prevent degradation

• Functional verification: Unlike enzymes with simple catalytic assays, verifying isolated atpG functionality is challenging. Consider developing:

  • Binding assays with other ATP synthase components

  • Structural assessment via circular dichroism to confirm proper folding

  • Limited proteolysis to assess structural integrity compared to predictions

How can researchers assess the interaction between recombinant atpG and other ATP synthase components?

Investigating interactions between recombinant atpG and other ATP synthase components requires specialized approaches that account for the complex nature of these interactions:

• In vitro reconstitution studies:

  • Express and purify multiple ATP synthase components separately

  • Perform controlled assembly experiments with varying component ratios

  • Analyze complex formation via native PAGE or size exclusion chromatography

  • Use analytical ultracentrifugation to determine binding stoichiometry and affinity

• Surface Plasmon Resonance (SPR) analysis:

  • Immobilize purified atpG on a sensor chip using a site-specific approach that maintains accessibility of interaction surfaces

  • Flow solutions containing potential binding partners at physiologically relevant concentrations

  • Monitor real-time association and dissociation kinetics

  • Calculate binding constants (Ka, Kd) for each interaction

  • Test interactions under varying conditions (pH, ionic strength) that mimic different stages of the B. bacteriovorus lifecycle

• Microscale Thermophoresis (MST):

  • Label atpG with a fluorescent dye at sites that won't interfere with interactions

  • Mix with varying concentrations of binding partners

  • Measure changes in thermophoretic mobility upon binding

  • This technique requires minimal protein amounts (nM range) and can work in complex buffers

• Cross-linking coupled with mass spectrometry:

  • Use bifunctional cross-linkers with varied spacer lengths to capture transient interactions

  • Digest cross-linked complexes for mass spectrometry analysis

  • Identify specific residues involved in interactions

  • Map identified interactions onto structural models to create interaction maps

• Co-expression approaches:

  • Design constructs for co-expression of atpG with other ATP synthase components

  • Test for improved solubility and stability compared to individual expression

  • Purify complexes via tandem affinity purification if components have different tags

  • Analyze composition and stoichiometry of purified complexes

How does atpG function differ during host-dependent versus host-independent growth of B. bacteriovorus?

B. bacteriovorus exhibits a complex lifecycle with distinct host-dependent predatory phases and host-independent growth modes. The function of ATP synthase and specifically its gamma chain likely varies significantly between these phases to accommodate different energy demands and metabolic states.

During the host-dependent predatory phase, B. bacteriovorus invades prey bacteria and utilizes their cellular contents for nutrients. This phase involves several energy-intensive processes:

• Initial high-energy flagellar motility during prey hunting

• Invasion into the prey periplasm

• Rapid replication inside the prey periplasm

• Growth and septation into multiple progeny cells

• Coordinated exit from prey remnants via gliding motility

In contrast, during host-independent growth, B. bacteriovorus must generate ATP from environmental nutrients, primarily amino acids . The ATP synthase complex may be regulated differently in this phase to maximize energy efficiency from free-living metabolism.

To investigate these differences, researchers should consider:

• Comparative transcriptomics/proteomics analysis:

  • Compare atpG expression levels between growth phases

  • Identify post-translational modifications specific to each phase

  • Map regulatory networks controlling ATP synthase expression in different growth modes

• Bioenergetic measurements:

  • Measure ATP/ADP ratios in cells from different phases

  • Compare membrane potential and proton motive force between growth modes

  • Assess oxygen consumption rates as indicators of respiratory activity

• Structure-function analysis:

  • Examine if atpG undergoes conformational changes between growth phases

  • Test for interaction with different regulatory partners in each phase

  • Investigate potential modifications that might alter rotational efficiency

The ability of B. bacteriovorus to exit prey remnants through gliding motility, which requires signaling via second messengers like cGAMP produced by Bd0367 , suggests potential links between ATP production and the regulation of lifecycle transitions that should be explored.

What role might the ATP synthase gamma chain play in the unique predatory lifecycle of B. bacteriovorus?

The ATP synthase gamma chain may serve specialized functions in B. bacteriovorus beyond its canonical role in ATP synthesis, particularly in relation to the organism's predatory lifecycle:

• Energy sensing and lifecycle coordination:

  • The rotational state of the gamma chain reflects cellular energy status

  • This mechanical information could potentially couple to signaling pathways

  • The deletion strain of Bd0367, which produces the second messenger cGAMP, shows defects in completing the predatory cycle, specifically in exiting prey remnants via gliding motility

  • This suggests potential links between energy metabolism and predatory cycle signaling

• Adaptation to fluctuating nutrient environments:

  • The transition from extracellular hunting to intraperiplasmic growth requires significant metabolic shifts

  • ATP synthase activity may need to respond rapidly to changing nutrient availability

  • The gamma chain might contain regulatory features that enable quick adaptation

• Coordination with motility mechanisms:

  • B. bacteriovorus uses both flagellar and gliding motility at different lifecycle stages

  • ATP supply for these processes must be precisely regulated

  • The gamma chain might interact with motility components directly or indirectly

  • Research shows that cGAMP signaling is required for gliding motility needed for progeny escape from the bdelloplast on solid surfaces

• Response to prey-derived signals:

  • Predation requires sensing prey status and coordinating appropriate responses

  • ATP synthase activity might respond to prey-derived metabolites

  • The gamma chain could serve as a regulatory interface for these adaptations

Research approaches to investigate these possibilities include:

• Site-directed mutagenesis of potential regulatory sites in atpG

• Interaction studies with components of signaling pathways, particularly those involving cGAMP

• Creation of fluorescently tagged atpG to track its behavior during predation

• Correlation of ATP synthase activity with predatory cycle transitions

How do the unique metabolic pathways of B. bacteriovorus influence atpG function?

B. bacteriovorus displays unusual metabolic patterns, including high reliance on amino acids rather than carbohydrates for energy generation . This distinctive metabolism likely influences ATP synthase function and potentially requires specialized adaptations in the gamma chain.

The primary carbon sources identified for B. bacteriovorus include glutamate, serine, aspartate, isoleucine, and threonine . Metabolism of these amino acids feeds into the TCA cycle at various points, potentially creating unique patterns of electron transport chain substrate availability that would affect the proton motive force driving ATP synthase rotation.

Furthermore, B. bacteriovorus shows high activities of citric acid cycle enzymes but relatively low activities of glycolytic enzymes (with exceptions like phosphoglucose isomerase) . This suggests a metabolic network optimized for amino acid catabolism rather than carbohydrate utilization.

Potential research areas to explore this relationship include:

• Metabolic flux analysis:

  • Trace carbon flow from labeled amino acids to respiratory substrates

  • Measure ATP production rates with different amino acid combinations

  • Correlate respiratory chain activity with ATP synthase performance

• Structural and functional adaptations:

  • Compare B. bacteriovorus atpG sequence with organisms using different metabolic strategies

  • Identify unique residues that might reflect adaptation to amino acid-based metabolism

  • Test how these specific features affect coupling efficiency or regulatory properties through site-directed mutagenesis

• Regulatory network integration:

  • Map relationships between amino acid metabolism and ATP synthase regulation

  • Identify metabolic intermediates that might allosterically regulate ATP synthase

  • Investigate whether atpG responds differently to metabolites from amino acid versus carbohydrate metabolism

• Comparative bioenergetics:

  • Measure ATP synthase activity using inverted membrane vesicles from cells grown on different nutrient sources

  • Compare P/O ratios (ATP produced per oxygen consumed) between predatory and non-predatory growth

  • Assess how metabolic inhibitors differentially affect ATP production in B. bacteriovorus versus non-predatory bacteria

What analytical techniques are most effective for studying recombinant B. bacteriovorus atpG structure and function?

Studying the structure and function of recombinant B. bacteriovorus atpG requires a multi-faceted approach combining various analytical techniques:

• Structural analysis methods:

  • X-ray crystallography: Can provide high-resolution structural data if high-quality crystals can be obtained, similar to the approach used for B. bacteriovorus phosphoglucose isomerase which was solved to 1.74 Å

  • Cryo-electron microscopy: Particularly valuable for visualizing atpG in the context of reconstituted ATP synthase complexes

  • Small-angle X-ray scattering (SAXS): Provides lower-resolution information about protein shape and conformational changes in solution

  • Hydrogen-deuterium exchange mass spectrometry: Maps protein dynamics and solvent accessibility

• Functional characterization assays:

  • ATP hydrolysis assays: Measure inorganic phosphate release using colorimetric methods (Malachite Green, EnzChek)

  • Proton pumping assays: Monitor pH changes using pH-sensitive fluorescent dyes in reconstituted systems

  • ATP synthesis measurements: Quantify ATP production in reconstituted liposomes under established proton gradients

  • Rotation assays: Attach fluorescent beads or gold nanoparticles to the gamma subunit and track rotation using single-molecule microscopy

• Interaction analysis techniques:

  • Surface plasmon resonance: Measure real-time binding kinetics with other ATP synthase components

  • Isothermal titration calorimetry: Determine thermodynamic parameters of binding interactions

  • Native mass spectrometry: Analyze intact complexes and their composition

  • Cross-linking mass spectrometry: Identify interaction interfaces at amino acid resolution

• Computational approaches:

  • Molecular dynamics simulations: Model rotational motion and energy transduction

  • Homology modeling: Generate structural models based on related ATP synthases

  • Molecular docking: Predict interactions with other ATP synthase components or regulatory molecules

For initial characterization, circular dichroism spectroscopy combined with ATP hydrolysis assays provides accessible starting points. For detailed mechanistic studies, reconstitution with other ATP synthase components followed by functional assays would yield the most complete picture of atpG function in the context of B. bacteriovorus' unique metabolism.

How can researchers design effective mutational studies of B. bacteriovorus atpG?

Designing effective mutational studies of B. bacteriovorus atpG requires thoughtful consideration of structure-function relationships and the unique aspects of this predatory bacterium's energy metabolism:

• Strategic target selection:

  • Conserved catalytic residues: Identify amino acids conserved across bacterial species that likely have critical functions

  • B. bacteriovorus-specific residues: Focus on unique residues that may reflect adaptation to predatory lifestyle

  • Interface residues: Target amino acids predicted to interact with other ATP synthase subunits

  • Regulatory sites: Identify residues that might participate in allosteric regulation or respond to second messengers like cGAMP

• Mutation types to implement:

  • Conservative substitutions: Replace with similar amino acids to subtly affect function

  • Charge inversions: Change positive to negative residues (or vice versa) to disrupt electrostatic interactions

  • Alanine scanning: Systematically replace regions with alanine to identify functional segments

  • Domain swapping: Exchange regions with gamma chains from non-predatory bacteria to identify specialized domains

• Expression and purification strategy:

  • Use a standardized expression system for all variants to ensure comparable results

  • Include wild-type controls in all experiments

  • Verify protein folding for each mutant using circular dichroism or fluorescence spectroscopy

  • Assess oligomeric state and stability before functional testing

• Functional analysis approach:

  • ATP hydrolysis assays: Measure effects on catalytic activity

  • Binding assays: Test interactions with other ATP synthase components

  • Thermal stability analysis: Determine if mutations affect protein stability

  • In vivo complementation: Test if mutants can restore function in atpG-deficient strains

• Structure-function correlation:

  • Map mutations onto structural models to interpret effects

  • Use molecular dynamics simulations to predict conformational changes

  • Correlate functional defects with structural alterations

A particularly informative approach would be to create mutations analogous to the S214D substitution in Bd0367, which converted this enzyme from primarily producing cGAMP to producing c-di-GMP . This type of targeted mutation that switches protein specificity could reveal key functional determinants in atpG.

What approaches can researchers use to study potential links between ATP synthase and cyclic nucleotide signaling in B. bacteriovorus?

Given B. bacteriovorus' use of cyclic nucleotide signaling molecules like cGAMP for lifecycle regulation , potential interactions with ATP synthase components merit investigation through multiple complementary approaches:

• Direct interaction studies:

  • Pull-down assays: Use immobilized cyclic nucleotides to capture potential binding partners

  • Surface plasmon resonance: Assess real-time binding kinetics between purified atpG and cyclic nucleotides

  • Isothermal titration calorimetry: Directly measure binding thermodynamics

  • Fluorescence-based assays: Use fluorescently labeled cyclic nucleotides to detect binding

• Structural approaches:

  • Co-crystallization attempts with atpG and cyclic nucleotides

  • Hydrogen-deuterium exchange mass spectrometry to identify regions protected upon nucleotide binding

  • NMR chemical shift analysis to map binding interfaces at atomic resolution

  • In silico docking to predict potential binding sites

• Functional correlation analyses:

  • Measure ATP synthase activity in the presence of various cyclic nucleotides

  • Compare ATP synthase function in wild-type vs. Bd0367 deletion strains that lack cGAMP production

  • Test if cyclic nucleotides affect proton translocation or rotational coupling

  • Investigate if ATP synthase subunits are modified in response to changing cyclic nucleotide levels

• Genetic and cellular approaches:

  • Create reporter strains with fluorescently tagged atpG and cyclic nucleotide-related proteins

  • Perform co-localization studies during different lifecycle stages

  • Generate strains with modified cyclic nucleotide production and assess effects on ATP synthase

  • Use chemical biology approaches with modified cyclic nucleotides to identify potential binding partners

Research should particularly focus on potential connections between ATP synthase function and the cGAMP signaling pathway involving Bd0367, which has been implicated in controlling gliding motility required for progeny escape from prey remnants . This represents a clear link between energy metabolism and a critical stage of the predatory lifecycle.