Recombinant Buchnera aphidicola subsp. Baizongia pistaciae Peptidoglycan synthase FtsI (ftsI), partial

What is Peptidoglycan synthase FtsI in Buchnera aphidicola and what is its function?

Peptidoglycan synthase FtsI in Buchnera aphidicola (also known as penicillin-binding protein 3 or PBP-3) is a crucial enzyme involved in bacterial cell division and cell wall biosynthesis. It functions by catalyzing the transpeptidation reaction that cross-links peptidoglycan strands in the bacterial cell wall . Despite Buchnera's extensive genome reduction through its evolutionary history, FtsI has been conserved, indicating its essential role in maintaining cellular integrity and division processes in this endosymbiont .

The enzyme belongs to the EC class 2.4.1.129 and plays a vital role in peptidoglycan glycosyltransferase activity. In the context of Buchnera's symbiotic relationship with aphids, which began 160-280 million years ago, the retention of this protein highlights its indispensable function even in highly reduced bacterial genomes .

How does FtsI in Buchnera aphidicola compare structurally and functionally to homologous proteins in free-living bacteria?

FtsI in Buchnera aphidicola retains core functional domains while potentially exhibiting streamlined structural features compared to its counterparts in free-living bacteria like Escherichia coli. The protein maintains essential catalytic residues required for peptidoglycan synthesis despite Buchnera's extensive genome reduction .

Due to its evolutionary history of gene loss and the resulting genetic stability, the FtsI protein likely represents a minimalist version that retains only essential functional domains required for bacterial cell division in the specialized intracellular environment of the aphid host .

What experimental challenges are associated with studying FtsI from an obligate endosymbiont?

Studying FtsI from Buchnera aphidicola presents several significant challenges inherent to research on obligate endosymbionts:

• Cultivation limitations: As Buchnera cannot be cultured outside its aphid host, obtaining native protein is extremely difficult, necessitating recombinant expression systems .

• Expression system optimization: Recombinant expression requires careful selection of host systems (E. coli, yeast, baculovirus, or mammalian cells) and optimization of conditions to ensure proper folding and activity .

• Genetic manipulation constraints: The obligate intracellular lifestyle of Buchnera makes direct genetic manipulation nearly impossible, limiting functional studies in the native environment .

• Codon usage bias: Buchnera's AT-rich genome creates potential codon optimization challenges when expressing its proteins in heterologous systems .

• Functional validation: Verifying the activity of recombinant FtsI requires sophisticated assays that can assess peptidoglycan synthesis in artificial systems since in vivo validation within Buchnera is technically challenging .

These challenges necessitate creative experimental approaches and often require integration of computational methods with biochemical techniques to study FtsI structure and function.

What are the optimal expression systems and conditions for producing functional recombinant FtsI from Buchnera aphidicola?

The optimal expression system for recombinant Buchnera aphidicola FtsI production depends on the specific research requirements, with several viable options available:

E. coli expression systems:
E. coli represents the most common and typically preferred expression host due to its phylogenetic similarity to Buchnera, relatively simple genetics, and rapid growth . For FtsI expression, BL21(DE3) or its derivatives are recommended, particularly with the following optimizations:

• Low-temperature induction (16-20°C) to improve protein folding

• Codon-optimization of the ftsI gene for E. coli expression

• Addition of rare tRNA-expressing plasmids to accommodate Buchnera's AT-rich codon bias

• Fusion tags (His6, MBP, or SUMO) to enhance solubility and facilitate purification

• Induction at mid-log phase (OD600 ~0.6) with reduced IPTG concentration (0.1-0.5 mM)

Alternative expression systems:
For cases where E. coli expression yields poorly folded protein, alternative systems include:

• Yeast systems (particularly Pichia pastoris) for improved folding and potential glycosylation

• Baculovirus-infected insect cells for enhanced post-translational modifications

• Cell-free expression systems for potentially toxic proteins

Typical purification yields from E. coli systems can reach ≥85% purity as determined by SDS-PAGE, with yields varying based on expression conditions and purification strategy .

What analytical methods are most effective for verifying the structure and function of recombinant FtsI?

Multiple complementary analytical approaches are recommended for comprehensive validation of recombinant FtsI:

Structural verification:

• Circular dichroism (CD) spectroscopy to assess secondary structure elements

• Thermal shift assays to evaluate protein stability and proper folding

• Limited proteolysis followed by mass spectrometry to verify domain organization

• Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) to determine oligomeric state

Functional validation:

• In vitro peptidoglycan synthesis assays measuring transpeptidase activity using fluorescently labeled substrates

• β-lactam binding assays to confirm the functionality of the penicillin-binding domain

• Cross-linking studies to identify interactions with other cell division proteins

Advanced structural characterization:
For high-resolution structural information, X-ray crystallography or cryo-electron microscopy can provide detailed insights into the three-dimensional structure of FtsI, though these approaches are technically challenging and resource-intensive.

The combination of these methods provides comprehensive validation of both structure and function of the recombinant protein before proceeding with more advanced experimental applications.

How can researchers design experiments to study the interaction between FtsI and other cell division proteins in an endosymbiotic context?

Studying protein-protein interactions in obligate endosymbionts requires innovative experimental approaches that overcome the limitations of traditional methods:

In vitro interaction studies:

• Pull-down assays: Using recombinantly expressed and tagged FtsI as bait to identify interaction partners from Buchnera protein extracts or with other recombinant division proteins

• Surface plasmon resonance (SPR): For quantitative measurement of binding affinities between FtsI and putative interaction partners

• Isothermal titration calorimetry (ITC): To determine thermodynamic parameters of protein-protein interactions

Cell-based approaches:

• Bacterial two-hybrid systems: Modified to accommodate the characteristics of Buchnera proteins in a surrogate host

• Split-protein complementation assays: Using fragments of reporter proteins fused to FtsI and potential interaction partners

• Fluorescence resonance energy transfer (FRET): For studying protein interactions in heterologous expression systems

Computational methods:

• Molecular docking simulations: To predict protein-protein interaction interfaces

• Coevolution analysis: Identifying coevolving residues that may indicate interaction surfaces

• Homology modeling: Building structural models based on known bacterial cell division complexes

A systematic experimental design would involve initial computational prediction of interaction partners, followed by in vitro validation and subsequent cellular studies in surrogate hosts. This multifaceted approach can provide insights into FtsI's role in the cell division machinery despite the experimental limitations associated with studying obligate endosymbionts.

What methods can be used to investigate the role of FtsI in maintaining the symbiotic relationship between Buchnera and aphids?

Investigating the role of FtsI in the Buchnera-aphid symbiosis requires interdisciplinary approaches that bridge molecular biology, cell biology, and symbiosis research:

Imaging and microscopy techniques:

• Transmission electron microscopy (TEM): To visualize Buchnera cell division within aphid bacteriocytes, focusing on septum formation where FtsI functions

• Fluorescence microscopy: Using specific antibodies against FtsI to monitor its localization during Buchnera cell division inside the host

• Super-resolution microscopy: For detailed visualization of FtsI distribution in the bacterial cell envelope within bacteriocytes

Molecular approaches:

• Quantitative proteomics: Comparing FtsI abundance under different physiological conditions of the aphid host

• RNA interference (RNAi): Targeting aphid genes that interact with the bacterial division machinery to indirectly assess FtsI function

• Metabolic labeling: Using D-amino acid-based probes to visualize peptidoglycan synthesis in vivo

Systems biology approaches:

• Transcriptome analysis: Examining coordination between host and symbiont gene expression related to bacterial division

• Metabolomic analysis: Investigating how disruptions in bacterial cell division affect metabolite exchange between Buchnera and the aphid host

• Mathematical modeling: Predicting how alterations in FtsI function might affect bacterial population dynamics within bacteriocytes

These methods collectively provide insights into how FtsI contributes to maintaining appropriate Buchnera population levels within the host, which is crucial for the nutritional symbiosis with aphids that has persisted for 160-280 million years .

How does the genomic reduction in Buchnera aphidicola impact the structure and function of FtsI compared to free-living relatives?

The extensive genomic reduction in Buchnera aphidicola has significant implications for FtsI structure and function. After 160-280 million years of symbiosis with aphids, Buchnera has lost numerous genes through reductive evolution while maintaining essential functions .

Comparative genomic analysis suggests that FtsI in Buchnera likely represents a streamlined version that retains only core functional domains essential for peptidoglycan synthesis and cell division. The evolutionary pressure to maintain FtsI despite massive gene loss (Buchnera has one of the smallest known genomes of any living organism) indicates its critical role in bacterial survival and symbiotic function .

Unlike most Gram-negative bacteria, Buchnera lacks genes for lipopolysaccharide synthesis for its outer membrane, potentially altering the cell envelope context in which FtsI operates . Additionally, the loss of regulatory factors in Buchnera's genome suggests that FtsI expression may lack sophisticated regulation found in free-living bacteria, potentially leading to constitutive expression .

These changes reflect adaptation to the stable, protected environment within aphid bacteriocytes, where the selection pressures differ substantially from those faced by free-living bacteria. Experimental approaches using recombinant FtsI from different Buchnera strains could provide direct evidence of functional adaptations resulting from this reductive evolution.

What are the evolutionary implications of FtsI conservation across different Buchnera aphidicola strains from diverse aphid hosts?

The conservation of FtsI across different Buchnera aphidicola strains from diverse aphid hosts provides valuable insights into bacterial evolution under symbiotic constraints:

• Essential function maintenance: The preservation of FtsI across Buchnera strains from aphids such as Acyrthosiphon pisum, Schizaphis graminum, Baizongia pistacea, and Cinara cedri demonstrates its indispensable role in bacterial cell division despite different symbiotic contexts .

• Selective pressure patterns: Comparative sequence analysis of FtsI from different Buchnera strains can reveal patterns of positive selection (adaptation) versus purifying selection (conservation), providing insights into which protein regions are most critical for function.

• Co-evolutionary dynamics: As Buchnera and aphids have co-evolved for 160-280 million years, strain-specific adaptations in FtsI may reflect particular requirements of different aphid hosts .

• Minimal functional requirements: By identifying conserved domains across all strains, researchers can determine the minimal structural and functional elements required for cell division in highly reduced bacterial genomes.

• Differential genomic reduction: The varying degree of genome reduction across Buchnera strains (from ~640kb in B. aphidicola BAp to ~420kb in B. aphidicola BCc) provides a natural experiment to observe which components of cell division machinery are retained even in the most extremely reduced genomes .

This evolutionary perspective on FtsI conservation contributes to our understanding of the minimal genetic requirements for bacterial life and the molecular basis of long-term symbiotic relationships.

How does metabolic complementation between Buchnera and secondary symbionts affect cell division processes involving FtsI?

In some aphid species, particularly Cinara cedri, Buchnera aphidicola has established obligate metabolic complementation with secondary symbionts like "Candidatus Serratia symbiotica," creating a complex symbiotic consortium . This metabolic interdependence has significant implications for cell division processes involving FtsI:

• Resource allocation: The metabolic complementation between symbionts affects the availability of metabolic resources required for peptidoglycan synthesis and cell division. For example, in C. cedri, B. aphidicola has lost many metabolic genes, potentially affecting precursor availability for cell wall components .

• Coordinated regulation: The presence of multiple bacterial symbionts necessitates coordinated regulation of cell division to maintain appropriate proportions of each symbiont within the host.

• Evolutionary trajectory: In aphids with co-primary symbionts, FtsI function may be under different selective pressures compared to aphids with only Buchnera as the primary symbiont.

• Complementary pathways: Secondary symbionts may provide metabolites or cellular components that complement Buchnera's limited metabolic capabilities, potentially affecting peptidoglycan precursor synthesis pathways upstream of FtsI function .

• Bacterial interactions: The physical proximity of different bacterial symbionts within aphid bacteriocytes may facilitate direct interactions that influence cell division processes.

The study of FtsI in these complex symbiotic systems requires consideration of the entire holobiont rather than isolated components, as the metabolic interdependence likely influences fundamental cellular processes including cell division .

What approaches can be used to study potential adaptations in FtsI function under different environmental stresses affecting the aphid host?

Studying how environmental stresses affecting aphid hosts influence FtsI function in Buchnera requires integrated approaches spanning multiple biological scales:

Experimental approaches:

• Controlled stress experiments: Exposing aphids to controlled environmental stresses (temperature, nutrition, predation, etc.) and examining:

  • Changes in Buchnera population dynamics within bacteriocytes

  • Alterations in FtsI expression levels or localization patterns

  • Modifications to cell division rates or morphology

• Comparative proteomics: Quantifying changes in FtsI abundance, post-translational modifications, or interaction partners under different stress conditions.

• Functional assays with recombinant proteins: Testing whether recombinant FtsI proteins from different Buchnera strains exhibit different sensitivities to stress conditions in vitro.

• Metabolic analysis: Measuring changes in peptidoglycan synthesis pathways under stress conditions to identify potential bottlenecks or adaptations.

Analytical frameworks:

• Phenotypic plasticity assessment: Determining whether observed changes in FtsI function represent transient responses or adaptive traits.

• Systems biology models: Developing mathematical models that predict how stress-induced changes propagate through the symbiotic system.

• Ecological context integration: Relating molecular-level changes to ecological consequences for both symbiont and host.

This research is particularly relevant given that environmental stresses can destabilize symbiotic relationships, and understanding how fundamental processes like cell division adapt to stress provides insights into symbiosis resilience mechanisms that have evolved over the 160-280 million year history of the Buchnera-aphid relationship .

How do the structural and functional characteristics of FtsI compare across different Buchnera aphidicola subspecies?

Comparison of FtsI across Buchnera aphidicola subspecies from different aphid hosts reveals important insights into evolutionary constraints and adaptations:

These comparative analyses provide insights into the minimal functional requirements for bacterial cell division and reveal how selective pressures in different symbiotic contexts shape protein evolution while maintaining essential functions.

What insights can be gained from comparing the expression and purification methods for FtsI across different bacterial expression systems?

Comparative analysis of expression and purification strategies for Buchnera FtsI across different expression systems reveals important methodological considerations:

• For membrane-associated domains of FtsI, detergent screening or membrane-mimetic systems may be necessary regardless of the expression host

• When high-throughput screening is needed, cell-free systems offer rapid production despite lower yields

• For structural studies requiring highly purified and properly folded protein, insect cell systems may provide superior results despite higher complexity

This systematic comparison guides researchers in selecting appropriate expression strategies based on their specific experimental requirements for Buchnera FtsI studies.

How can research on FtsI in Buchnera aphidicola contribute to broader understanding of bacterial cell division in minimal genomes?

Research on FtsI in Buchnera aphidicola provides valuable insights into fundamental aspects of bacterial cell division in minimal genomes:

• Minimal divisome requirements: By studying which components of the cell division machinery are retained in Buchnera's highly reduced genome, researchers can identify the core essential components of bacterial cell division . This contributes to understanding the minimal genetic requirements for bacterial replication.

• Evolutionary plasticity vs. conservation: The analysis of FtsI across different Buchnera strains with varying genome sizes (from ~640kb to ~420kb) reveals which protein domains and functions have remained under strict purifying selection despite extensive genome reduction . This helps differentiate between essential and accessory aspects of FtsI function.

• Regulatory simplification: Buchnera has lost many regulatory genes during its genome reduction, providing insights into how cell division can function with minimal regulatory control . This contrasts with the complex regulatory networks controlling cell division in free-living bacteria.

• Host-microbe coordination: The study of FtsI in Buchnera reveals mechanisms by which bacterial cell division becomes integrated with host physiology in long-term symbiotic relationships .

• Synthetic biology applications: Understanding the minimal requirements for bacterial cell division from systems like Buchnera informs efforts to design minimal synthetic cells with reduced genomes.

These insights extend beyond symbiosis research to fundamental questions in bacterial cell biology and evolution, contributing to our understanding of the minimal genetic and molecular requirements for bacterial life.

What research techniques can be applied to study the three-dimensional structure of FtsI and its interaction with peptidoglycan substrates?

Elucidating the three-dimensional structure of FtsI and its interactions with peptidoglycan substrates requires a combination of advanced structural biology techniques:

Experimental structural determination approaches:

• X-ray crystallography: The gold standard for high-resolution protein structure determination, requiring:

  • Large-scale purification of highly pure, homogeneous recombinant FtsI

  • Crystallization screening under various conditions

  • Diffraction data collection and structure solution

• Cryo-electron microscopy (cryo-EM): Particularly valuable for membrane-associated proteins like FtsI:

  • Sample preparation in vitreous ice

  • Single-particle analysis or tomography approaches

  • Potential for visualizing FtsI in complex with other divisome components

• Nuclear Magnetic Resonance (NMR) spectroscopy: For studying dynamics and ligand interactions:

  • Isotopic labeling of recombinant FtsI

  • Solution-state NMR for smaller domains or protein fragments

  • Solid-state NMR for membrane-associated regions

Substrate interaction studies:

• Enzyme kinetics: Determining catalytic parameters using synthetic peptidoglycan analogs

• Surface Plasmon Resonance (SPR): Measuring binding kinetics to immobilized substrates

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Identifying regions involved in substrate binding

• Molecular docking and molecular dynamics simulations: Predicting binding modes and conformational changes

Integrated structural biology approach:

An optimal research strategy would combine low-resolution techniques (small-angle X-ray scattering, negative-stain EM) with high-resolution methods (X-ray crystallography, cryo-EM) and dynamic analyses (NMR, HDX-MS) to build a comprehensive model of FtsI structure and function in the context of peptidoglycan synthesis and cell division.

The structural insights gained would enhance our understanding of how this essential protein functions in the minimal genomic context of Buchnera aphidicola.

What are the most significant research gaps in our understanding of FtsI function in Buchnera aphidicola?

Despite progress in understanding Buchnera aphidicola genomics and evolution, significant research gaps remain regarding FtsI function in this obligate endosymbiont:

• Structure-function relationship: No high-resolution structural data exists for Buchnera FtsI, limiting our understanding of potential adaptations to endosymbiotic life.

• Regulatory mechanisms: How FtsI expression and activity are regulated in the absence of many traditional regulatory systems remains poorly understood.

• Host-symbiont coordination: The mechanisms coordinating Buchnera cell division with host cell cycles and developmental stages are largely unknown.

• Evolutionary adaptation: While genome reduction is well-documented, the functional consequences of these reductions on FtsI activity and the cell division process require further investigation.

• Metabolic integration: How peptidoglycan synthesis and cell division are integrated with the specialized metabolic functions Buchnera performs for its aphid host remains to be elucidated.

Addressing these gaps will require innovative experimental approaches that overcome the challenges of studying obligate endosymbionts while leveraging comparative genomics and advanced structural biology techniques.