Recombinant Gluconacetobacter diazotrophicus ATP synthase subunit c (atpE)

What is Gluconacetobacter diazotrophicus and why is it significant for agricultural research?

G. diazotrophicus is a plant growth-promoting (PGP) bacterium that can fix atmospheric nitrogen (diazotroph), allowing it to function as a biofertilizer and potentially minimize the need for industrially derived fertilizers in sustainable agricultural systems. This gram-negative bacterium was originally discovered as an endophyte in sugarcane plants but also forms associations with other agriculturally important crops . G. diazotrophicus requires microaerobic conditions for diazotrophic growth, and research has identified a streamlined set of essential genes involved in nitrogen fixation with less redundancy than other model diazotrophs .

The agricultural significance of G. diazotrophicus stems from its ability to:

• Fix atmospheric nitrogen while living inside plant tissues

• Function under the low oxygen conditions found within plant tissues

• Establish beneficial relationships with multiple crop species

• Constitutively produce enzymes like levansucrase that facilitate utilization of plant sucrose

What is the ATP synthase subunit c (atpE) and what role does it play in G. diazotrophicus metabolism?

ATP synthase subunit c, encoded by the atpE gene in G. diazotrophicus, is a critical component of the F₀ portion of the F₁F₀-ATP synthase complex. This enzyme complex is responsible for ATP production through oxidative phosphorylation. The c subunit forms the membrane-embedded c-ring structure that facilitates proton translocation across the bacterial membrane, which drives the synthesis of ATP.

In G. diazotrophicus, ATP production is particularly crucial for:

• Powering the energy-intensive process of biological nitrogen fixation

• Supporting endophytic colonization processes

• Enabling secretion of enzymes like levansucrase through the type II secretion pathway

• Maintaining cellular homeostasis under microaerobic conditions

While specific research on G. diazotrophicus atpE is limited in the current literature, its function can be inferred from the organism's metabolic requirements and the conserved nature of ATP synthase across bacterial species.

What techniques are currently used to express and purify recombinant G. diazotrophicus ATP synthase subunit c?

The expression and purification of recombinant G. diazotrophicus ATP synthase subunit c typically follows these methodological steps:

• Gene cloning and vector construction:

  • PCR amplification of the atpE gene from G. diazotrophicus genomic DNA

  • Insertion into an expression vector with appropriate promoter and affinity tags

  • Verification of the construct by sequencing

• Expression system optimization:

  • Selection of appropriate host (typically E. coli strains optimized for membrane protein expression)

  • Testing different induction conditions (temperature, inducer concentration, duration)

  • Small-scale expression trials to identify optimal conditions

• Scaled production and extraction:

  • Growth of bacteria under optimized conditions

  • Cell lysis (typically via French press or sonication)

  • Membrane fraction isolation through differential centrifugation

• Purification strategy:

  • Solubilization of membrane proteins using appropriate detergents

  • Affinity chromatography (typically Ni-NTA for His-tagged proteins)

  • Size exclusion chromatography for further purification

  • Protein concentration determination

As a small, hydrophobic membrane protein, special considerations must be taken during atpE purification to maintain its native structure and function, including careful detergent selection and buffer optimization.

How can researchers analyze the functional properties of recombinant G. diazotrophicus ATP synthase subunit c?

Functional analysis of recombinant ATP synthase subunit c should incorporate multiple complementary approaches:

• Structural characterization:

  • Circular dichroism spectroscopy to assess secondary structure

  • NMR spectroscopy for structural determination in membrane mimetics

  • Mass spectrometry for precise molecular weight determination

  • Protein-lipid interaction studies to evaluate membrane integration

• Functional reconstitution:

  • Incorporation into liposomes or nanodiscs

  • Proton translocation assays using pH-sensitive fluorescent dyes

  • Assembly with other ATP synthase subunits to assess c-ring formation

• Mutational analysis:

  • Site-directed mutagenesis of conserved residues

  • Functional complementation studies in ATP synthase-deficient strains

  • Assessment of effects on proton binding and translocation

• In situ studies:

  • Generation of fluorescently tagged variants to study localization

  • Crosslinking studies to identify protein-protein interactions

  • Proteolytic accessibility mapping to determine membrane topology

Integration of these methodologies provides a comprehensive understanding of both structural and functional properties of the recombinant protein.

How is the atpE gene organized and regulated in G. diazotrophicus?

While the search results don't provide specific information about atpE gene organization in G. diazotrophicus, we can describe a methodological approach to characterizing its genetic context and regulation:

• Genomic context analysis:

  • Identification of the ATP synthase operon structure

  • Analysis of neighboring genes and potential co-regulation

  • Comparative genomics with related bacterial species

• Promoter characterization:

  • Similar to the approach used for the lsd genes, where a 548-bp region upstream from lsdX containing the putative promoter was analyzed

  • Identification of transcriptional start sites using 5′ RACE

  • Reporter gene fusion studies to determine promoter strength under different conditions

• Transcriptional analysis:

  • RT-qPCR to measure atpE expression under various growth conditions

  • RNA-seq to identify co-regulated genes and global expression patterns

  • Northern blot analysis to determine transcript size and operon structure

• Protein expression correlation:

  • Western blot analysis to measure AtpE protein levels

  • Correlation of expression with ATP synthase activity

  • Proteomic analysis under different growth conditions

From known G. diazotrophicus gene characteristics, researchers should expect high G+C content (64-74%) and codon usage with strong preference for C and G in the third position of triplets (78-91%), similar to other structural genes in this organism .

How does the expression of atpE correlate with nitrogen fixation activity in G. diazotrophicus?

To investigate the relationship between atpE expression and nitrogen fixation:

• Comparative expression analysis:

  • Measure atpE transcript and protein levels under:

    • Diazotrophic vs. non-diazotrophic growth conditions

    • Various oxygen concentrations (G. diazotrophicus requires microaerobic conditions for nitrogen fixation )

    • Different carbon sources that affect energy metabolism

• Correlation with nitrogenase activity:

  • Simultaneous measurement of:

    • ATP synthase activity (ATP production rate)

    • Nitrogenase activity (acetylene reduction assay)

    • Cellular ATP levels

    • Growth rates

• Genetic manipulation approaches:

  • Controlled modulation of atpE expression using:

    • Inducible promoter systems

    • Antisense RNA strategies

    • Partial knockdown using CRISPRi

  • Assessment of resulting effects on nitrogen fixation

• In planta studies:

  • Measurement of atpE expression during plant colonization

  • Correlation with in planta nitrogen fixation rates

  • Comparison between different plant hosts

This methodological framework would reveal whether atpE expression is constitutive or specifically regulated during nitrogen fixation, providing insights into the bioenergetic requirements of this process in G. diazotrophicus.

What genetic manipulation strategies are effective for studying atpE function in G. diazotrophicus?

Based on genetic approaches successfully applied to other G. diazotrophicus genes, the following methodologies can be adapted for atpE studies:

• Marker exchange mutagenesis:

  • Similar to the approach used for lsdG, lsdF, and lsdO genes

  • Construction of suicide plasmids containing:

    • Fragments of the target gene

    • Antibiotic resistance cassettes (e.g., kanamycin-bleomycin resistance)

    • Introduction into G. diazotrophicus by electroporation

  • Selection of recombinants on antibiotic-containing media

  • Confirmation of insertion by Southern hybridization

• Site-directed mutagenesis:

  • Similar to the approach used for Cys162 mutation in lsdG

  • PCR-based introduction of specific mutations

  • Cloning into appropriate vectors

  • Introduction into G. diazotrophicus

  • Phenotypic analysis of the mutants

• Complementation studies:

  • Construction of broad-host-range vectors carrying wild-type or mutated atpE

  • Introduction into ATP synthase-deficient strains

  • Assessment of functional restoration

• Conditional expression systems:

  • Development of inducible promoters for G. diazotrophicus

  • Construction of atpE expression cassettes under controlled regulation

  • Analysis of phenotypic effects upon induction/repression

Since atpE likely plays an essential role in cellular bioenergetics, conditional or partial disruption approaches may be necessary to avoid lethal phenotypes while studying gene function.

How can transposon insertion sequencing (Tn-seq) be applied to understand ATP synthase function in G. diazotrophicus?

Transposon insertion sequencing offers a powerful approach to study ATP synthase components in G. diazotrophicus, as demonstrated by previous Tn-seq applications in this organism . A comprehensive methodology would include:

• Transposon library generation:

  • Construction of a comprehensive transposon mutant library in G. diazotrophicus

  • Ensuring sufficient coverage (>3,200 genes were probed in previous work )

  • Verification of library diversity by preliminary sequencing

• Condition-specific selection:

  • Culture of the transposon library under various conditions relevant to ATP synthase function:

    • Diazotrophic vs. non-diazotrophic growth

    • Different oxygen tensions

    • Varying carbon sources

    • pH variations affecting proton motive force

• Sequencing and analysis:

  • Next-generation sequencing of transposon insertion sites

  • Quantification of mutant abundance before and after selection

  • Calculation of fitness scores for each gene under each condition

  • Statistical analysis to identify significant fitness effects

• ATP synthase-focused analysis:

  • Detailed examination of fitness defects associated with:

    • Insertions in ATP synthase genes

    • Insertions in genes affecting proton motive force

    • Insertions in genes involved in energy metabolism pathways

  • Identification of synthetic interactions through combinatorial analysis

• Validation experiments:

  • Confirmation of key findings using:

    • Targeted gene deletions

    • Complementation studies

    • Physiological measurements

This approach would reveal not only the importance of ATP synthase components under different conditions but also identify unexpected genetic interactions affecting ATP synthase function in G. diazotrophicus.

What are the predicted structural features of G. diazotrophicus ATP synthase subunit c?

While specific structural data for G. diazotrophicus ATP synthase subunit c is not provided in the search results, a methodological approach to predicting its structure would include:

• Sequence analysis:

  • Multiple sequence alignment with known ATP synthase c subunits

  • Identification of conserved residues involved in:

    • Proton binding (typically a conserved carboxyl group)

    • Subunit-subunit interactions within the c-ring

    • Interactions with other ATP synthase components

• Secondary structure prediction:

  • Computational prediction of alpha-helical content

  • Transmembrane helix prediction using hydropathy analysis

  • Identification of the characteristic hairpin structure (two transmembrane helices connected by a polar loop)

• Tertiary structure modeling:

  • Homology modeling based on resolved structures from other bacteria

  • Molecular dynamics simulations in membrane environment

  • Validation of models using experimental constraints

• Quaternary structure prediction:

  • Prediction of c-ring stoichiometry based on sequence features

  • Modeling of the c-ring assembly

  • Analysis of potential interfaces with other ATP synthase components

• Functional site identification:

  • Prediction of the proton binding site

  • Identification of residues involved in the proton translocation pathway

  • Mapping of potential sites for post-translational modifications

This structural information would guide experimental approaches to study the function of specific residues and domains within the G. diazotrophicus ATP synthase subunit c.

How does G. diazotrophicus ATP synthase adapt to microaerobic conditions required for nitrogen fixation?

To investigate the adaptations of G. diazotrophicus ATP synthase to microaerobic conditions, researchers should consider:

• Comparative structural analysis:

  • Sequence comparison of ATP synthase components between:

    • Aerobic bacteria

    • Microaerobic bacteria

    • Anaerobic bacteria

  • Identification of unique features in G. diazotrophicus ATP synthase

• Functional characterization under varying oxygen tensions:

  • Measurement of:

    • ATP synthesis rates

    • Proton translocation efficiency

    • ATP synthase assembly

  • Under precisely controlled oxygen concentrations

• Regulatory analysis:

  • Investigation of:

    • Oxygen-dependent expression of ATP synthase components

    • Post-translational modifications affecting enzyme activity

    • Protein-protein interactions modulating ATP synthase function

• Energy balance assessment:

  • Analysis of:

    • ATP/ADP ratios under different oxygen tensions

    • Proton motive force maintenance strategies

    • Alternative electron transport pathways supporting ATP synthesis

Since G. diazotrophicus requires microaerobic conditions for diazotrophic growth , its ATP synthase may exhibit specific adaptations to function efficiently under low oxygen tensions while supporting the high energy demands of nitrogen fixation.

How can understanding ATP synthase function contribute to enhancing G. diazotrophicus as a biofertilizer?

Knowledge of ATP synthase function in G. diazotrophicus can contribute to biofertilizer development through several methodological approaches:

• Strain optimization strategies:

  • Enhancement of ATP production efficiency through:

    • Targeted genetic modifications of ATP synthase components

    • Selection of natural variants with improved energy metabolism

    • Adaptation to specific plant host environments

• Plant colonization improvement:

  • Investigation of energy requirements during:

    • Initial plant colonization

    • Establishment of endophytic relationship

    • Long-term persistence within plant tissues

  • Development of strains with optimized energy allocation

• Nitrogen fixation enhancement:

  • Balancing energy allocation between:

    • Growth and reproduction

    • Nitrogen fixation

    • Metabolite exchange with plant host

  • Creating strains with improved ATP availability for nitrogenase

• Stress tolerance engineering:

  • Improving ATP synthase function under:

    • Variable oxygen conditions in different plant tissues

    • pH fluctuations in the plant environment

    • Temperature variations in field conditions

The unique characteristics of G. diazotrophicus as a nitrogen-fixing endophyte with applications as a biofertilizer make ATP synthase an important target for optimization to enhance its beneficial effects in sustainable agriculture.

What methodologies are available for monitoring ATP synthase activity during plant-microbe interactions?

To study ATP synthase function during plant-microbe interactions, researchers can employ:

• In planta imaging techniques:

  • Construction of fluorescent reporter systems for:

    • ATP synthase component localization

    • ATP concentration measurement using ATP-sensitive fluorescent proteins

    • Membrane potential visualization with voltage-sensitive dyes

  • Confocal microscopy of plant tissues colonized by labeled bacteria

• Transcriptomic and proteomic approaches:

  • RNA-seq analysis of bacterial genes during plant colonization

  • Proteomics to quantify ATP synthase components in planta

  • Phosphoproteomics to detect regulatory modifications

• Metabolic profiling:

  • Measurement of:

    • ATP/ADP ratios in bacteria isolated from plants

    • Metabolic intermediates indicating energy status

    • Isotope labeling to track carbon flow through energy-generating pathways

• Genetic reporter systems:

  • Construction of:

    • Promoter-reporter fusions to monitor ATP synthase gene expression

    • Biosensors responsive to ATP concentration or proton motive force

    • Conditional expression systems activated by energy limitation

These methodologies would provide insights into how G. diazotrophicus regulates its energy metabolism during endophytic growth and nitrogen fixation within plant tissues, information that could be leveraged to develop improved biofertilizer strains.

How does the type II secretion system of G. diazotrophicus interact with energy metabolism during levansucrase secretion?

The relationship between the type II secretion system and energy metabolism in G. diazotrophicus can be investigated through:

• Energetic requirements analysis:

  • Measurement of:

    • ATP consumption during protein secretion

    • Proton motive force dependence of the secretion machinery

    • Effects of ATP synthase inhibitors on levansucrase secretion

• Genetic interaction studies:

  • Construction of:

    • Conditional ATP synthase mutants

    • Type II secretion system component mutants

    • Double mutants to detect synthetic phenotypes

• Co-regulation investigation:

  • Analysis of:

    • Transcriptional coordination between secretion and energy metabolism genes

    • Shared regulatory elements

    • Metabolic signals affecting both systems

• Structural and functional coupling:

  • Investigation of:

    • Physical proximity of ATP synthase and secretion machinery in membranes

    • Local ATP concentration effects on secretion efficiency

    • Membrane organization and potential microdomains

The search results indicate that G. diazotrophicus secretes levansucrase (LsdA) through a type II secretory pathway , which requires energy input. Understanding the interplay between ATP production and protein secretion would provide insights into how this bacterium allocates energy resources during plant colonization.

What are the most promising emerging technologies for studying ATP synthase dynamics in living G. diazotrophicus cells?

Cutting-edge approaches for studying ATP synthase dynamics include:

• Advanced microscopy techniques:

  • Single-molecule fluorescence microscopy to visualize:

    • ATP synthase rotational dynamics

    • Assembly/disassembly processes

    • Subcellular localization patterns

  • Super-resolution microscopy to overcome diffraction limits

  • Cryo-electron tomography of frozen-hydrated cells

• Genetically encoded biosensors:

  • Development of:

    • FRET-based sensors for ATP concentration

    • Conformational reporters integrated into ATP synthase components

    • pH sensors for proton gradient measurement

  • Real-time monitoring in living cells

• Microfluidic approaches:

  • Design of:

    • Devices for precise control of microaerobic conditions

    • Single-cell analysis platforms

    • Gradient generators to mimic plant microenvironments

  • Integration with live-cell imaging

• Genome editing technologies:

  • Application of:

    • CRISPR-Cas9 for precise genetic manipulation

    • Base editing for specific nucleotide changes

    • Prime editing for targeted insertions or deletions

  • Creation of conditional or tissue-specific expression systems

These technologies would enable researchers to observe ATP synthase function with unprecedented spatial and temporal resolution, particularly during the microaerobic diazotrophic growth that characterizes G. diazotrophicus in its endophytic lifestyle .