Recombinant Polynucleobacter necessarius ATP synthase subunit beta (atpD)

What is Polynucleobacter necessarius and why is it significant in research?

Polynucleobacter necessarius is a betaproteobacterium that represents an excellent model system for studying genome reduction in bacteria. Its significance stems from several unique features:

• It exists in both symbiotic and free-living forms within the same species, allowing for comparative genomic studies

• The symbiotic forms live within ciliated protists (specifically Euplotes)

• Free-living strains have unusually small genomes and reduced metabolic flexibility despite being highly abundant in freshwater systems worldwide

• It serves as a valuable model for understanding bacterial adaptation and symbiotic relationships

The study of P. necessarius provides insights into fundamental evolutionary processes including genome erosion, metabolic adaptation, and the transition from free-living to symbiotic lifestyles. This makes it particularly valuable for research into microbial ecology, evolution, and host-microbe interactions.

What is the structure and function of ATP synthase subunit beta (atpD) in P. necessarius?

ATP synthase subunit beta (atpD) in P. necessarius is a critical component of the F1F0-ATP synthase complex, which is responsible for ATP production via oxidative phosphorylation. The protein:

• Consists of 466 amino acids with a complete sequence available in protein databases (UniProt: B1XSD4)

• Contains multiple functional domains for ATP binding and catalysis

• Participates in the catalytic conversion of ADP to ATP using the proton gradient across the membrane

The protein sequence includes essential regions for nucleotide binding and interaction with other ATP synthase subunits. According to the product datasheet, the recombinant protein has a purity of >85% as determined by SDS-PAGE and is expressed as a full-length protein (amino acids 1-466) .

How do the metabolic capabilities of symbiotic and free-living P. necessarius strains differ?

Comparative analysis of symbiotic and free-living P. necessarius strains reveals significant metabolic differences:

The symbiotic strain shows clear evidence of genome reduction, having lost the glyoxylate cycle and several amino acid biosynthetic pathways. This metabolic streamlining reflects its adaptation to the host environment where it can obtain certain metabolites directly from its ciliate host. Neither strain can utilize sugars as carbon sources, highlighting their unusual metabolism even among free-living bacteria .

What are the optimal storage and handling conditions for recombinant P. necessarius ATP synthase subunit beta?

For optimal preservation of recombinant P. necessarius ATP synthase subunit beta (atpD) activity and stability, researchers should follow these evidence-based protocols:

• Short-term storage: Store at -20°C

• Extended storage: Conserve at -20°C or -80°C

• Reconstitution procedure:

  • Briefly centrifuge the vial before opening to bring contents to the bottom

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (50% is recommended)

  • Aliquot for long-term storage at -20°C/-80°C

• Avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

The shelf life of the liquid form is approximately 6 months at -20°C/-80°C, while the lyophilized form remains stable for 12 months at -20°C/-80°C. These conditions help maintain protein structure and functionality for experimental applications.

How can recombinant P. necessarius ATP synthase subunit beta be used to study bacterial adaptation mechanisms?

Recombinant P. necessarius ATP synthase subunit beta serves as a valuable tool for investigating bacterial adaptation mechanisms through several methodological approaches:

• Comparative functional studies: Researchers can compare the enzymatic properties of ATP synthase from symbiotic versus free-living strains to understand adaptations in energy metabolism. This involves:

  • Purified protein activity assays under varying pH, temperature, and ion concentrations

  • Measuring ATP synthesis/hydrolysis rates to assess functional differences

• Structural biology approaches: Utilizing the recombinant protein for:

  • X-ray crystallography to determine high-resolution structures

  • Cryo-EM analysis to visualize the complete ATP synthase complex

  • Binding studies with inhibitors or activators

• Interaction studies: Investigating how atpD interacts with other components of energy metabolism in the context of genome reduction, particularly given the streamlined central metabolism of P. necessarius .

These approaches provide insights into how essential proteins maintain functionality despite evolutionary pressures in bacteria undergoing genome reduction.

What methodological approaches are recommended for studying differential expression of atpD in P. necessarius?

For researchers investigating atpD expression patterns in P. necessarius, the following methodological approaches are recommended:

• RNA-Seq analysis: Following the approach used in related Polynucleobacter studies, researchers can:

  • Culture P. necessarius under various conditions (different carbon sources, stress factors, or with/without host factors)

  • Extract total RNA using established protocols for bacterial samples

  • Perform rlog transformation of expression data for normalization

  • Conduct principal component analysis to identify major factors affecting gene expression

  • Identify differentially expressed genes using statistical methods (similar to the approach that identified 150 differentially expressed genes in P. asymbioticus under different conditions)

• qRT-PCR verification: For targeted analysis of atpD expression:

  • Design primers specific to atpD gene regions

  • Use appropriate reference genes for normalization

  • Validate expression patterns observed in RNA-Seq data

• Operon structure analysis: Since many bacterial genes are co-expressed in operons, researchers should:

  • Analyze the genomic context of atpD to identify potential co-regulated genes

  • Study the expression of entire operons rather than isolated genes

  • Identify potential regulatory elements in the promoter region

These approaches enable comprehensive analysis of atpD expression dynamics in response to various experimental conditions.

How does P. necessarius ATP synthase contribute to our understanding of bacterial adaptation to symbiosis?

The ATP synthase complex in P. necessarius provides critical insights into bacterial adaptation to symbiotic lifestyles through several research perspectives:

As a core component of energy metabolism, ATP synthase must be maintained for cellular viability even as genomes erode during symbiotic adaptation. Research shows that despite significant genome reduction in symbiotic P. necessarius strains, the ATP synthase complex remains intact and functional. This conservation highlights its essential role even as other metabolic pathways are lost .

The retention of ATP synthase in the symbiont suggests that:

• Energy production remains under bacterial control rather than being outsourced to the host

• The protein structure maintains sufficient functionality despite potential sequence changes

• Selection pressure preserves energy production even as biosynthetic pathways are lost

By studying the structural and functional characteristics of ATP synthase in both free-living and symbiotic strains, researchers can trace evolutionary adaptations that occur during the transition to symbiosis. This comparison provides a rare opportunity to examine how essential complexes evolve during genome reduction processes.

What insights can structural studies of P. necessarius ATP synthase provide regarding energy metabolism in bacteria with reduced genomes?

Structural studies of P. necessarius ATP synthase offer valuable insights into energy metabolism adaptations in bacteria with reduced genomes:

These structural insights contribute to our understanding of how bacteria maintain energy homeostasis despite genome reduction, providing principles that may apply across diverse host-associated bacteria.

How can recombinant P. necessarius ATP synthase be used in comparative studies with other bacterial ATP synthases?

Recombinant P. necessarius ATP synthase subunit beta enables sophisticated comparative studies with other bacterial ATP synthases through several methodological approaches:

• Biochemical characterization:

  • Enzymatic assays comparing ATP synthesis/hydrolysis rates

  • Analysis of temperature and pH optima across species

  • Determination of kinetic parameters (Km, Vmax) for substrate binding and catalysis

  • Inhibitor sensitivity profiles to identify structural and functional differences

• Structural biology approaches:

  • Comparative crystallography or cryo-EM studies to identify structural adaptations

  • Analysis of protein-protein interactions within the ATP synthase complex

  • Investigation of subunit interface conservation across species

• Evolutionary analysis:

  • Sequence-structure-function relationships across bacterial lineages

  • Identification of conserved versus variable regions in the context of different ecological niches

  • Correlation of ATP synthase adaptations with genome size and metabolic capabilities

These comparative studies are particularly valuable given P. necessarius' unusual position as a bacterium with naturally streamlined metabolism and distinct ecological forms (free-living versus symbiotic) . The insights gained contribute to understanding both fundamental principles of ATP synthase function and specific adaptations in diverse bacterial lineages.

What are the key considerations for experimental design when studying recombinant P. necessarius ATP synthase subunit beta?

When designing experiments involving recombinant P. necessarius ATP synthase subunit beta, researchers should consider these methodological factors:

• Protein preparation:

  • Expression system: The recombinant protein is produced in mammalian cells, which may influence post-translational modifications

  • Purity requirements: The standard preparation achieves >85% purity by SDS-PAGE; higher purity may be required for certain applications

  • Tag considerations: The type of tag is determined during manufacturing and may affect protein function or detection methods

• Functional assays:

  • Buffer composition: ATP synthase activity is sensitive to ionic conditions, particularly Mg²⁺ and Na⁺/K⁺ concentrations

  • pH optimization: Activity assays should account for the natural environmental pH of P. necessarius

  • Temperature range: Consider testing at both standard laboratory temperatures and temperatures relevant to the natural habitat

• Comparative framework:

  • Include appropriate controls when comparing with other bacterial ATP synthases

  • Consider the unusual metabolic context of P. necessarius, which lacks glycolytic pathways and has a restricted carbon metabolism

  • Account for differences between free-living and symbiotic strains in comparative studies

• Storage and stability:

  • Implement proper storage protocols (-20°C/-80°C) to maintain protein integrity

  • Avoid repeated freeze-thaw cycles

  • Consider adding glycerol (5-50%) to prevent activity loss during storage

These considerations ensure robust experimental results and reliable interpretations when working with this unique bacterial ATP synthase.

What techniques are most effective for analyzing the interaction between P. necessarius ATP synthase and other components of energy metabolism?

For researchers investigating interactions between P. necessarius ATP synthase and other components of energy metabolism, these advanced methodological approaches are recommended:

• Protein-protein interaction studies:

  • Co-immunoprecipitation with antibodies against atpD or interacting partners

  • Pull-down assays using tagged recombinant atpD

  • Crosslinking mass spectrometry to identify interaction interfaces

  • Bacterial two-hybrid systems for in vivo interaction validation

• Membrane complex analysis:

  • Blue native PAGE to isolate intact ATP synthase complexes

  • Clear native PAGE followed by in-gel activity assays

  • Gradient ultracentrifugation for membrane complex separation

  • Cryo-electron tomography for visualization of membrane-embedded complexes

• Functional coupling experiments:

  • Reconstitution of ATP synthase with respiratory chain components in liposomes

  • Measurement of proton pumping coupled to ATP synthesis

  • Analysis of the impact of electron transport chain inhibitors on ATP synthesis

• Metabolic context analysis:

  • Metabolic flux analysis using isotope-labeled substrates

  • Integration of ATP synthase activity with TCA cycle function

  • Comparative analysis with other bacterial systems that lack glycolytic pathways

These techniques are particularly valuable given the unusual metabolic configuration of P. necessarius, which relies primarily on TCA cycle and related pathways for energy generation rather than glycolysis .

What are the emerging research questions regarding P. necessarius ATP synthase in the context of bacterial adaptation?

Several cutting-edge research questions are emerging regarding P. necessarius ATP synthase and bacterial adaptation:

• Structural adaptation mechanisms:

  • How does ATP synthase maintain functionality despite genome reduction pressures?

  • Are there unidentified compensatory mechanisms that preserve ATP synthase efficiency in the absence of certain metabolic pathways?

  • Do symbiotic strains exhibit structural modifications that optimize function in the host environment?

• Evolutionary dynamics:

  • What selection pressures maintain the integrity of ATP synthase genes during genome erosion?

  • How does the rate of sequence evolution in ATP synthase compare to other essential proteins during the transition to symbiosis?

  • Can comparative genomics across the Polynucleobacter genus reveal adaptive signatures in ATP synthase components?

• Functional interactions in a streamlined metabolism:

  • How does ATP synthase function coordinate with the unusual carbon metabolism of P. necessarius?

  • What compensatory mechanisms exist for energy production given the absence of glycolytic pathways?

  • How does the loss of translesion DNA polymerases in symbiotic strains affect the genetic stability of ATP synthase genes?

• Host-symbiont energy exchange:

  • Does the host ciliate (Euplotes) influence ATP synthase activity in symbiotic strains?

  • Is there evidence for ATP export from the symbiont to the host?

  • How do energy production requirements differ between free-living and symbiotic lifestyles?

These questions represent fertile ground for researchers seeking to understand the fundamental principles of bacterial adaptation and energy metabolism in the context of symbiosis and genome reduction.

How might studies of P. necessarius ATP synthase contribute to broader research on minimal bacterial genomes?

Research on P. necessarius ATP synthase offers significant contributions to the broader field of minimal bacterial genome studies through several key perspectives:

• Natural model of genome reduction:

  • P. necessarius provides a rare natural experiment in genome reduction, with both free-living and symbiotic strains available for comparison

  • The maintenance of ATP synthase despite genome streamlining highlights essential energy production requirements in minimal genomes

  • Comparison between strains reveals which aspects of energy metabolism are dispensable versus essential

• Metabolic network simplification:

  • The absence of glycolytic pathways in both strains demonstrates alternative energy production strategies in streamlined genomes

  • The loss of the glyoxylate cycle in symbiotic strains provides insights into carbon utilization requirements in host-associated bacteria

  • These patterns inform synthetic biology approaches to designing minimal bacterial genomes

• Methodological approaches for minimal systems:

  • Techniques developed to study ATP synthase in P. necessarius can inform approaches to other minimal bacterial systems

  • The recombinant expression and characterization of proteins from bacteria with reduced genomes provides valuable protocols for similar studies

  • Comparative analyses between free-living and symbiotic strains offer templates for studying other bacteria undergoing genome reduction

These contributions extend beyond P. necessarius to inform fundamental principles of bacterial genome reduction, minimal metabolic requirements, and the evolution of host-microbe interactions across diverse biological systems.