Recombinant Gloeobacter violaceus Chaperone protein ClpB (clpB), partial

What is Gloeobacter violaceus and why is it significant for evolutionary studies?

Gloeobacter violaceus PCC 7421 is a unique rod-shaped unicellular cyanobacterium isolated from calcareous rock in Switzerland. It is considered an evolutionary primordial cyanobacterium as molecular phylogenetic analyses have shown that it branched off from the main cyanobacterial tree at an early stage of evolution . Unlike other cyanobacteria, G. violaceus lacks thylakoid membranes, with photosynthesis taking place in the cytoplasmic membrane similar to anoxygenic photosynthetic bacteria . This distinctive characteristic makes it an excellent model organism for studying the evolution of photosynthetic systems. The genome of G. violaceus consists of a single circular chromosome 4,659,019 bp long with an average GC content of 62%, containing 4430 potential protein-encoding genes .

The absence of thylakoid membranes in G. violaceus represents a primitive trait that sets it apart from all other known oxygenic photosynthetic organisms. This characteristic offers researchers unique insights into the early evolutionary stages of photosynthetic apparatus and membrane organization in cyanobacteria.

What is the ClpB protein and what role does it play in bacterial systems?

ClpB is a member of the class 1 AAA+ (ATPases Associated with diverse cellular Activities) protein family that functions as a key molecular chaperone . Its primary function is disaggregation - the ability to resolubilize protein aggregates that form during stress conditions. This activity is crucial for bacterial survival under various forms of stress, particularly heat shock .

The primary mechanism of ClpB involves harnessing energy through ATP hydrolysis to unfold and remodel aggregated proteins, working cooperatively with the DnaK chaperone system to restore protein functionality. Beyond protein disaggregation, recent research has revealed that ClpB also regulates the secretion of bacterial effector molecules related to type VI secretion systems in some pathogenic bacteria . This versatility in function makes ClpB an essential component of bacterial stress response and potentially virulence.

How does the genomic context of clpB in G. violaceus differ from other cyanobacteria?

The genome of G. violaceus reveals several distinctive features compared to other cyanobacteria. While specific data about the clpB gene context isn't directly mentioned in the search results, the genome as a whole shows significant differences. G. violaceus possesses a large number of genes for sigma factors and transcription factors in the LuxR, LysR, PadR, TetR, and MarR families, while lacking genes for major elements of the circadian clock (kaiABC) .

These genomic differences likely reflect the phylogenetic distance between G. violaceus and other cyanobacteria. When studying clpB in this context, researchers should consider these evolutionary divergences. The G. violaceus genome contains many unique features that may influence gene expression and regulation patterns, potentially affecting how ClpB functions in stress response compared to other cyanobacteria.

What are the optimal conditions for expressing recombinant G. violaceus ClpB protein?

For expressing recombinant G. violaceus ClpB, researchers should consider the following methodological approach:

• Vector Selection: Use expression vectors with strong, inducible promoters such as pET series vectors with T7 promoter systems, which provide tight control over expression levels.

• Host Selection: E. coli BL21(DE3) or its derivatives are recommended as expression hosts due to their reduced protease activity and compatibility with T7 expression systems.

• Expression Conditions: Initial culture growth at 37°C until OD600 reaches 0.6-0.8, followed by temperature reduction to 18-25°C prior to induction often improves soluble ClpB yield. Induce with IPTG at lower concentrations (0.1-0.5 mM) to prevent excessive protein aggregation.

• Media Optimization: Rich media (such as Terrific Broth) supplemented with glucose (0.5-1%) can enhance biomass and protein yield. For structural studies requiring isotope labeling, minimal media formulations would be necessary.

• Induction Parameters: As ClpB is a large protein (approximately 95 kDa), extended expression times (16-20 hours) at lower temperatures often yield better results than short, high-temperature induction periods.

The high GC content (62%) of the G. violaceus genome may pose challenges for heterologous expression. Codon optimization of the clpB gene for the expression host may significantly improve protein yield.

What methodological approaches can be used to study the disaggregase activity of G. violaceus ClpB?

Studying the disaggregase activity of G. violaceus ClpB requires several specialized techniques:

• Aggregation Suppression Assays: Monitor the ability of ClpB to prevent aggregation of model substrates like luciferase or citrate synthase under heat stress (42-45°C) using light scattering at 320-360 nm.

• Disaggregation Assays: Pre-aggregate substrate proteins by heat treatment, then measure the resolubilization of these aggregates in the presence of ClpB, DnaK, DnaJ, and GrpE (the complete disaggregation system). Recovery can be monitored by:

  • Decrease in turbidity (light scattering)

  • Recovery of enzymatic activity for substrate enzymes

  • Differential centrifugation followed by SDS-PAGE analysis

• ATP Hydrolysis Assays: Measure ATPase activity using malachite green assays or coupled enzymatic assays to determine how substrate binding affects ATP consumption rates.

• Protein-Protein Interaction Studies: Investigate interactions between ClpB and substrate proteins or co-chaperones using:

  • Pull-down assays

  • Surface plasmon resonance

  • Isothermal titration calorimetry

• Structural Analysis: Employ cryo-electron microscopy or X-ray crystallography to determine the structural basis of ClpB function, particularly focusing on the middle domain (M-domain) which distinguishes ClpB from related AAA+ proteins.

When designing these experiments, it's important to consider that G. violaceus has adapted to unique ecological niches, potentially resulting in distinctive thermal stability properties of its ClpB compared to homologs from other species .

How does G. violaceus ClpB function in relation to the absence of thylakoid membranes?

The absence of thylakoid membranes in G. violaceus creates a unique cellular environment that likely influences ClpB function in several ways:

• Subcellular Localization: In typical cyanobacteria, photosynthetic apparatus is localized to thylakoid membranes, spatially organizing stress responses. In G. violaceus, photosynthesis occurs in the cytoplasmic membrane , potentially altering the distribution of protein aggregates during stress and subsequently affecting ClpB localization and substrate accessibility.

• Stress Response Integration: Without thylakoid membranes, G. violaceus may have evolved alternative stress signaling pathways that interact with ClpB differently than in other cyanobacteria. The photosynthetic apparatus directly integrated into the cytoplasmic membrane would expose these components to different microenvironments during stress conditions.

• Specialized Substrate Profile: G. violaceus ClpB may have evolved to recognize and disaggregate specific substrates related to its unique photosynthetic organization. This could include specialized membrane proteins or adaptations for disaggregating proteins at membrane interfaces.

• Evolutionary Adaptations: As a primordial cyanobacterium, G. violaceus ClpB may represent an ancestral form of the protein with potentially broader substrate specificity or alternative regulatory mechanisms compared to more evolved cyanobacterial species.

Research methodologies should include comparative studies between G. violaceus ClpB and homologs from thylakoid-containing cyanobacteria, focusing on substrate specificity and protein-protein interaction networks. Fluorescence microscopy with tagged ClpB could elucidate localization patterns during stress conditions.

What role might ClpB play in the type VI secretion system of G. violaceus?

While G. violaceus-specific information is limited in the search results, research on other bacterial species indicates that ClpB can serve as a functional homolog of ClpV in type VI secretion systems (T6SS) . Based on this information, we can hypothesize about its potential role in G. violaceus:

• Energy Provider: ClpB may harness energy through ATP hydrolysis for the assembly/disassembly of T6SS components, similar to its role in Francisella tularensis where it functions as a ClpV homolog .

• Sheath Recycling: In F. tularensis, ClpB is involved in depolymerization of the IglA-IglB sheath for recycling and reassembly . G. violaceus ClpB may perform similar functions if it possesses T6SS components.

• Virulence Regulation: If G. violaceus has T6SS or analogous secretion systems, ClpB might contribute to the secretion of effector molecules, potentially affecting interactions with other organisms in its ecological niche.

To investigate these possibilities, researchers should:

• Conduct genomic analysis to identify potential T6SS components in G. violaceus

• Generate ClpB knockout mutants and examine effects on protein secretion profiles

• Perform co-immunoprecipitation experiments to identify ClpB-interacting proteins

• Use bacterial two-hybrid systems to test direct interactions between ClpB and putative T6SS components

These approaches would help elucidate whether G. violaceus ClpB functions in secretion systems similar to other bacteria or has evolved distinct roles related to its unique cellular architecture.

How can structural studies of G. violaceus ClpB inform drug development targeting bacterial chaperones?

Structural studies of G. violaceus ClpB could significantly contribute to antimicrobial drug development strategies:

• Exploiting Structural Differences: The search results indicate that human ClpB (Skd3) differs significantly from bacterial ClpB proteins in domain structure. While bacterial ClpB contains a characteristic coiled-coil domain, human Skd3 lacks this domain and instead contains a unique ankyrin-repeat domain . These structural differences provide potential targets for selective inhibition.

• Nucleotide Binding Pocket Analysis: Detailed structural characterization of the ATP-binding sites in G. violaceus ClpB could reveal bacterial-specific features for the design of selective ATP-competitive inhibitors that would not affect human Skd3.

• Allosteric Site Identification: Crystal structures or cryo-EM models of G. violaceus ClpB could reveal unique allosteric sites for small molecule binding that could modulate protein function without competing with ATP.

• Interaction Interface Mapping: Structural studies could identify critical protein-protein interaction surfaces between ClpB and its co-chaperones or substrates, providing targets for disrupting the disaggregation machinery.

The methodological approach should include:

• Obtaining high-resolution structures using X-ray crystallography or cryo-EM

• Performing molecular dynamics simulations to identify druggable pockets

• Using structure-based virtual screening to identify potential inhibitors

• Validating hits with biochemical assays measuring disaggregase activity

This approach leverages the evolutionary distance between bacterial and human chaperones to develop antimicrobials with reduced potential for off-target effects .

What protein purification strategies are most effective for recombinant G. violaceus ClpB?

A comprehensive purification strategy for recombinant G. violaceus ClpB should include:

• Affinity Chromatography:

  • Incorporate a His6 or His10 tag at the N-terminus of the recombinant ClpB

  • Use immobilized metal affinity chromatography (IMAC) with Ni-NTA or Co-NTA resins

  • Include 5-10 mM ATP in the buffers to prevent non-specific binding of other ATPases

• Buffer Optimization:

  • Maintain buffer pH between 7.5-8.0 (typically HEPES or Tris-based)

  • Include 5-10% glycerol to enhance protein stability

  • Add 100-150 mM NaCl and 5 mM MgCl₂ to maintain native conformation

  • Consider including 1 mM DTT or 2-5 mM β-mercaptoethanol to prevent oxidation

• Secondary Purification:

  • Ion exchange chromatography (typically Q-Sepharose) to remove contaminants

  • Size exclusion chromatography to separate oligomeric states and ensure homogeneity

• Oligomerization Considerations:

  • ClpB exists in equilibrium between monomers, dimers, and hexamers depending on protein concentration and nucleotide binding

  • The addition of ATP or ATP analogs (ATPγS) can stabilize the hexameric form

• Quality Control:

  • Verify ATPase activity using malachite green phosphate detection assay

  • Confirm disaggregation activity using model substrates

For structural studies, an additional ultracentrifugation step may be necessary to ensure sample homogeneity. The purification process should be performed at 4°C throughout to minimize protein degradation and aggregation.

How can researchers design mutation studies to investigate critical functional domains of G. violaceus ClpB?

Designing effective mutation studies for G. violaceus ClpB requires systematic targeting of functional domains:

• Nucleotide Binding Domains (NBDs):

  • Create Walker A motif mutations (K→A) in both NBD1 and NBD2 to disrupt ATP binding

  • Modify Walker B motif (E→Q) to allow ATP binding but prevent hydrolysis

  • These mutations help distinguish between ATP binding and hydrolysis requirements for different ClpB functions

• Middle Domain (M-domain):

  • The coiled-coil M-domain is critical for interaction with the DnaK system

  • Create point mutations in conserved residues at the tip of the coiled-coil

  • Design truncation variants with partial or complete M-domain deletions

  • These mutations help elucidate co-chaperone interactions

• N-terminal Domain:

  • Create N-terminal deletion mutants to investigate substrate recognition

  • Identify and mutate conserved hydrophobic residues potentially involved in substrate binding

  • These mutations clarify the role of the N-terminal domain in disaggregation specificity

• Oligomerization Interface:

  • Target residues at subunit interfaces that contribute to hexamer formation

  • These mutations help determine the relationship between oligomerization and function

• Substrate Threading Pore:

  • Modify conserved tyrosine residues in the central channel that engage substrates

  • These mutations affect the protein unfolding and translocation capabilities

For each mutation, analyze:

• Oligomeric state (by size exclusion chromatography or analytical ultracentrifugation)

• ATPase activity (basal and substrate-stimulated)

• Disaggregation activity with model substrates

• Ability to complement ClpB-deficient bacterial strains under stress conditions

A systematic alanine-scanning approach of conserved residues can also identify previously unknown functional regions of the protein.

What are the main technical challenges in studying G. violaceus ClpB and how can they be addressed?

Researchers investigating G. violaceus ClpB face several technical challenges:

• Protein Solubility and Stability Issues:

  • Challenge: ClpB is prone to aggregation during overexpression and purification

  • Solution: Express at lower temperatures (16-18°C); include stabilizing agents like arginine (50-100 mM) and glycerol (10%) in purification buffers; consider fusion tags like MBP that enhance solubility

• Functional Reconstitution:

  • Challenge: Full disaggregase activity requires the complete chaperone system (ClpB + DnaK/DnaJ/GrpE)

  • Solution: Co-express or separately purify compatible DnaK system components; test compatibility with DnaK systems from different species if G. violaceus components are unavailable

• ATP Consumption:

  • Challenge: High ATP consumption during functional assays increases cost and complicates analysis

  • Solution: Implement ATP regeneration systems (phosphoenolpyruvate + pyruvate kinase); use ATP analogs like ATPγS for binding studies

• Substrate Availability:

  • Challenge: Identifying physiologically relevant substrates specific to G. violaceus

  • Solution: Use proteomic approaches to identify aggregation-prone proteins in G. violaceus under stress; develop fluorescently labeled model substrates for high-throughput assays

• Heterologous Expression:

  • Challenge: G. violaceus high GC content (62%) may cause expression problems in E. coli

  • Solution: Optimize codon usage for the expression host; consider specialized expression strains designed for high-GC genes

• Structural Analysis:

  • Challenge: ClpB conformational flexibility complicates structural determination

  • Solution: Use cryo-EM rather than crystallography; employ FRET-based approaches to capture dynamic states; consider ClpB variants with restricted conformational freedom for initial structural studies

• Measuring Disaggregation Activity:

  • Challenge: Quantitative assessment of disaggregation can be difficult and variable

  • Solution: Standardize aggregate preparation protocols; use multiple complementary assays (turbidity, enzyme reactivation, fluorescence); include positive controls with well-characterized ClpB homologs

By addressing these technical challenges methodically, researchers can more effectively study the unique properties of G. violaceus ClpB and its role in this primordial cyanobacterium.

How does G. violaceus ClpB compare functionally and structurally to ClpB proteins from other cyanobacteria?

A comparative analysis of G. violaceus ClpB with homologs from other cyanobacteria reveals important evolutionary insights:

• Structural Conservation and Divergence:

  • The core AAA+ domains are likely highly conserved due to the fundamental importance of ATP binding and hydrolysis

  • The middle domain (M-domain) may show greater sequence divergence reflecting adaptations to different cellular environments and stress conditions

  • As a primordial cyanobacterium, G. violaceus ClpB might represent an ancestral form with potentially different domain arrangements or regulatory elements

• Functional Adaptations:

  • G. violaceus lacks thylakoid membranes , suggesting its ClpB may have adapted to function in a cellular environment fundamentally different from thylakoid-containing cyanobacteria

  • The absence of certain photosystem components in G. violaceus may correlate with specialized ClpB substrate specificity tailored to its unique photosynthetic apparatus

  • Temperature adaptations may differ based on the ecological niche of G. violaceus compared to other cyanobacteria

• Co-evolution with Partner Chaperones:

  • The DnaK system, which collaborates with ClpB in protein disaggregation, may have co-evolved specifically in G. violaceus

  • Interactions between ClpB and the DnaK/DnaJ/GrpE system might show species-specific optimizations

• Regulatory Mechanisms:

  • The distinctive transcription factor profile of G. violaceus suggests potentially unique regulation of clpB expression

  • The absence of circadian clock genes (kaiABC) in G. violaceus may result in different temporal regulation of ClpB expression compared to other cyanobacteria

Methodological approaches for comparative studies should include:

• Phylogenetic analysis of ClpB sequences across cyanobacterial species

• Cross-species complementation assays with ClpB from different cyanobacteria

• Comparative biochemical characterization of purified ClpB proteins

• Heterologous expression studies examining regulation in different genetic backgrounds

What insights can G. violaceus ClpB provide about the evolution of protein quality control systems?

G. violaceus, as a primordial cyanobacterium, offers unique evolutionary insights into protein quality control systems:

• Ancestral Protein Quality Control:

  • G. violaceus represents an early-branching cyanobacterial lineage , suggesting its ClpB may retain ancestral features lost in more derived lineages

  • Studying G. violaceus ClpB can provide insights into the minimal required elements for effective disaggregation machinery in early photosynthetic organisms

  • The protein could represent an evolutionary intermediate between bacterial and more complex cyanobacterial systems

• Adaptation to Membrane Organization:

  • The absence of thylakoid membranes creates a fundamentally different cellular architecture for protein quality control

  • This unique cellular organization may have driven specific adaptations in ClpB function to manage aggregation in a simpler membrane system

  • Comparative analysis could reveal how protein quality control systems adapted during the evolution of complex membrane organizations

• Co-evolution with Photosynthetic Systems:

  • G. violaceus lacks several photosystem components present in other cyanobacteria

  • ClpB may have co-evolved with these simplified photosynthetic systems, potentially revealing the ancestral state of chaperone-photosystem interactions

  • The high GC content (62%) of the G. violaceus genome might reflect selective pressures that also shaped the evolution of its protein quality control systems

• Functional Conservation:

  • Despite its evolutionary distance from other cyanobacteria, core ClpB functions are likely conserved

  • Identifying which aspects of ClpB function and regulation are conserved versus divergent can highlight the evolutionary pressures acting on protein quality control systems

Research approaches should include:

• Reconstruction of ancestral ClpB sequences using phylogenetic methods

• Functional characterization of chimeric ClpB proteins combining domains from G. violaceus and other species

• Comparative genomic analysis of stress response pathways across cyanobacterial lineages

• Experimental evolution studies exposing G. violaceus to novel stress conditions

These studies would contribute to understanding how protein quality control systems evolved alongside increasing cellular complexity in photosynthetic organisms.

What novel applications might emerge from further characterization of G. violaceus ClpB?

Further characterization of G. violaceus ClpB could lead to several innovative applications:

• Biotechnological Applications:

  • Development of ClpB-based reagents for protein aggregation prevention in recombinant protein production

  • Creation of engineered ClpB variants with enhanced activity or altered substrate specificity for industrial enzyme stabilization

  • Utilization in cell-free protein synthesis systems to increase yield of difficult-to-express proteins

• Therapeutic Potential:

  • As bacterial ClpB proteins differ structurally from human homologs , G. violaceus ClpB could serve as a template for selective antimicrobial development

  • Exploration of ClpB inhibitors as novel antibacterials targeting stress response mechanisms

  • Potential applications in protein misfolding diseases by adapting ClpB's disaggregation mechanisms for therapeutic purposes

• Synthetic Biology Tools:

  • Development of ClpB-based protein degradation systems for targeted removal of misfolded proteins

  • Creation of synthetic stress response circuits incorporating ClpB for enhanced cellular resilience

  • Use as a molecular tool for controlled protein activity through reversible aggregation/disaggregation

• Structural Biology Platforms:

  • G. violaceus ClpB could serve as a model system for studying AAA+ protein dynamics

  • Development of fluorescent biosensors based on conformational changes in ClpB domains

  • Utilization as a platform for studying protein-protein interactions in disaggregation networks

• Environmental Applications:

  • Engineering of stress-resistant cyanobacteria for bioremediation using optimized ClpB systems

  • Development of biosensors for environmental stress using ClpB-based detection systems

Research methodologies should incorporate interdisciplinary approaches combining structural biology, protein engineering, synthetic biology, and applied biotechnology to fully explore these potential applications.

How might CRISPR-Cas9 gene editing be applied to study ClpB function in G. violaceus?

CRISPR-Cas9 technology offers powerful approaches for investigating ClpB function in G. violaceus:

Methodological considerations should include:

• Optimization of transformation protocols for G. violaceus

• Design of guide RNAs accounting for the high GC content (62%)

• Development of appropriate selection markers

• Screening strategies to verify successful genomic modifications

• Phenotypic characterization under various stress conditions

These approaches would significantly advance our understanding of ClpB function in this primordial cyanobacterium and potentially reveal unique adaptations not present in more evolutionarily recent species.