Recombinant Bdellovibrio bacteriovorus 60 kDa chaperonin (groL), partial

What is Bdellovibrio bacteriovorus and what role does its 60 kDa chaperonin (groL) play in its life cycle?

Bdellovibrio bacteriovorus is a predatory bacterium that invades and consumes Gram-negative bacteria to acquire nutrients necessary for growth and replication . Its life cycle consists of two main phases: the non-replicating attack phase (AP) where it swims at high speed searching for prey, and the intraperiplasmic phase (IP) which begins when the predator attaches to and invades a suitable prey .

The 60 kDa chaperonin (groL) in B. bacteriovorus likely functions similarly to other bacterial Group I chaperonins (Hsp60s), which are present in bacteria and endosymbiotic organelles of eukaryotes . Based on studies of similar chaperonins, groL would be involved in protein folding, particularly during stress conditions or when handling proteins prone to misfolding. The expression of chaperonin genes appears to be phase-specific, with certain genes predominantly expressed during either the attack phase or growth phase .

How does Bdellovibrio bacteriovorus' transcriptional program differ between attack and growth phases?

Research using RNA-seq has revealed a dramatic transcriptional switch between the attack phase (AP) and growth phase (GP) of B. bacteriovorus . Most genes in the Bdellovibrio genome are classified as either "AP only" or "GP only," demonstrating a largely mutually exclusive transcriptional program between phases .

The attack phase is associated with a specific set of promoters (140 AP promoters were experimentally mapped), with many containing a common sigma-like DNA binding site highly similar to the E. coli flagellar genes regulator sigma28 (FliA) . This suggests that FliA has evolved to become a global AP regulator in Bdellovibrio. Additionally, a non-coding RNA containing a c-di-GMP riboswitch is massively expressed during AP, potentially functioning as an intracellular reservoir for c-di-GMP and playing a role in the rapid switch from AP to GP .

What expression systems are most effective for producing recombinant Bdellovibrio bacteriovorus proteins?

Based on the available research, E. coli appears to be an effective heterologous expression system for B. bacteriovorus proteins. The recombinant Coxiella burnetii 60 kDa chaperonin described in the search results was expressed in E. coli , suggesting a similar approach would be suitable for B. bacteriovorus chaperonin.

Recent research has also developed the Golden Standard (GS) hierarchical assembly cloning technique, making it compatible with B. bacteriovorus HD100. The chromosomal integration of the Tn7 transposon's mobile element, combined with the GS technique, has allowed systematic characterization of constitutive and inducible promoters for controlling heterologous gene expression in this bacterium .

How can I optimize protein folding assays to assess the function of recombinant Bdellovibrio bacteriovorus chaperonin?

When designing folding assays for recombinant B. bacteriovorus chaperonin, consider the following methodological approaches based on studies with other chaperonins:

• Concentration Range: Conduct experiments at a range of chaperonin concentrations spanning those believed to occur in vivo. For reference, in E. coli, the GroEL concentration is estimated to be 35 μM of monomers. For experimental purposes, a range from 10 nM to 79.5 μM would be appropriate .

• Stability Assessment: Evaluate protein stability using techniques such as Circular Dichroism (CD) and Small X-Ray Angle Scattering (SAXS). Be aware that the different protein concentrations required by these methods (approximately 1 μM for CD and 20 μM for SAXS) may yield differing results, particularly if the chaperonin's stability is concentration-dependent .

• Denaturant Studies: Use chemical denaturants like guanidine hydrochloride to generate denaturation profiles and calculate free energy of unfolding .

• Functional Assays: Include ATP hydrolysis assays to measure chaperonin activity, as ATP binding and hydrolysis are essential for chaperonin function.

• Co-chaperonin Interaction: Test interaction with appropriate co-chaperonins, as these partnerships are crucial for chaperonin function. For bacterial systems, this would typically involve a GroES-like co-chaperonin .

What regulatory considerations should researchers be aware of when working with genetically modified Bdellovibrio bacteriovorus?

Research involving recombinant nucleic acids and engineered organisms is subject to regulatory oversight. Key considerations include:

• Institutional Biosafety Committee (IBC) Review: Projects involving recombinant DNA must be reviewed and approved by the IBC, which focuses on local oversight of biosafety concerns .

• NIH Guidelines Compliance: Research must comply with the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. The specific section applicable depends on the nature of the research .

• Human Subjects Research: If research involves human subjects, additional requirements apply. Human Gene Transfer (HGT) research involving the transfer of recombinant or synthetic nucleic acid molecules into human subjects requires IBC and IRB approval, and potentially FDA authorization .

• Laboratory Containment: Ensure appropriate biosafety level containment based on the characteristics of the modified organism and the nature of the experimental work.

• Predatory Nature Considerations: Given B. bacteriovorus's predatory behavior against other bacteria, special attention should be paid to containment and ecological impact assessments when modifying genes that might affect its predation capabilities or host range.

How can riboswitches be utilized to control gene expression in Bdellovibrio bacteriovorus?

Riboswitches represent a promising tool for controlling gene expression in B. bacteriovorus. Research has identified theophylline-activated riboswitches that function effectively in this organism . These can be strategically employed in several ways:

• Chromosomal Integration: Riboswitches can be inserted into the bacterial chromosome to regulate essential genes. For example, researchers successfully inserted a riboswitch to regulate expression of the flagellar sigma factor fliA, which is critical for predation .

• Predation Kinetics Modulation: The engineered strain with riboswitch-regulated fliA demonstrated faster predation kinetics in the presence of theophylline, indicating that riboswitch technology can effectively modulate predatory behavior .

• Phase-Specific Regulation: Given the distinct transcriptional programs of attack and growth phases, riboswitches could be strategically deployed to control phase-specific processes. The natural presence of a c-di-GMP riboswitch in a non-coding RNA highly expressed during the attack phase suggests this regulatory mechanism is already utilized by B. bacteriovorus .

• Experimental Design Considerations: When designing riboswitch-based regulatory systems:

  • Evaluate multiple riboswitch variants, as some constructs may function better than others in B. bacteriovorus

  • Consider the downstream sequence context, as including bases downstream of the transcription start site can affect the rate of transcription

  • Validate riboswitch function using reporter genes like mCherry before applying to genes of interest

What potential exists for engineering Bdellovibrio bacteriovorus chaperonin for enhanced predation or therapeutic applications?

Engineering B. bacteriovorus chaperonin offers several intriguing research avenues:

• Enhanced Predation Efficiency: Since chaperonins assist protein folding, engineering the chaperonin could potentially enhance the folding and activity of predation-related enzymes, particularly hydrolases secreted during prey consumption .

• Thermal Adaptation: Modified chaperonins might extend the temperature range in which B. bacteriovorus can effectively prey on pathogens, increasing its potential as a biocontrol agent in different environments.

• Substrate Specificity: Studies of chloroplast chaperonins have revealed that different chaperonin subunits demonstrate substrate specificity . Engineering B. bacteriovorus chaperonin to preferentially fold specific sets of proteins could potentially direct its predatory activity toward particular pathogenic targets.

• Biocontrol Applications: Due to its ability to prey on a wide range of Gram-negative pathogens, B. bacteriovorus has potential applications as a biocontrol agent . Engineered chaperonins could enhance its stability or effectiveness in various application environments.

• Living Antibiotic Development: B. bacteriovorus has been proposed as a "living antibiotic" in fields like agriculture or medicine . Engineering its chaperonin system could potentially enhance its predatory capabilities against specific bacterial pathogens of interest.

How do researchers analyze differences in chaperonin activity between recombinant forms and native proteins?

When comparing activity between recombinant and native chaperonins, researchers employ several methodological approaches:

• Stability Analysis: Studies comparing mitochondrial HSP60, naïve HSP60, and bacterial GroEL have revealed differences in protein stability that can be quantified through free energy of unfolding measurements . The following graph illustrates typical stability differences:

  Chaperonin Type	Free Energy of Unfolding (CD)	Free Energy of Unfolding (SAXS)
  Bacterial GroEL	Higher stability	Higher stability
  Naïve HSP60	Intermediate stability	Intermediate stability
  Mitochondrial HSP60	Lower stability	Lower stability

• Oligomerization Assessment: Native and recombinant chaperonins may differ in their oligomerization properties. Analytical ultracentrifugation, native PAGE, or size exclusion chromatography can be employed to analyze oligomeric states .

• Protein Folding Assays: Comparing the ability of native versus recombinant chaperonins to facilitate refolding of denatured substrate proteins provides functional insights. Typically, researchers monitor the recovery of enzymatic activity of model substrate proteins.

• ATP Hydrolysis: Measuring the ATPase activity of both native and recombinant chaperonins provides information about their functional state, as ATP hydrolysis is coupled to the conformational changes necessary for chaperonin function.

• Co-chaperonin Interaction: Evaluating interactions with co-chaperonins can reveal functional differences, particularly if the recombinant form shows altered binding affinity or cooperativity compared to the native form .

What are common challenges in expressing and purifying recombinant Bdellovibrio bacteriovorus chaperonin and how can they be addressed?

Researchers may encounter several challenges when working with recombinant B. bacteriovorus chaperonin:

• Low Expression Levels: The lac promoter shows weak expression in B. bacteriovorus . Solution: Use native B. bacteriovorus promoters active during the attack phase (P1753, P3184, PAPsRNA5, or PmerRNA) or the PJ ExD/EliR promoter/regulator system, which has proven exceptional for heterologous gene expression .

• Protein Solubility Issues: Chaperonins are large, complex proteins that may form inclusion bodies when overexpressed. Solutions:

  • Express at lower temperatures (16-18°C)

  • Add solubility tags (e.g., MBP, SUMO)

  • Optimize induction conditions (lower IPTG concentrations)

  • Include appropriate folding additives in lysis buffer (e.g., ATP, low concentrations of denaturants)

• Oligomerization Challenges: Chaperonins function as large oligomeric complexes, and recombinant versions may not assemble correctly. Solutions:

  • Include appropriate co-factors during purification (Mg²⁺, ATP)

  • Use gentle purification methods that preserve native structure

  • Consider co-expression with co-chaperonins or other assembly factors

• Purification Optimization: Based on available data , the following purification protocol can be suggested:

  • Use His-tag or other affinity tags for initial purification

  • Include 5-50% glycerol in storage buffer to maintain stability

  • Consider buffer composition similar to Tris/PBS-based buffer, pH 8.0

  • Aliquot and store at -20°C for long-term storage

How should researchers interpret contradictory results when comparing Bdellovibrio bacteriovorus chaperonin with other bacterial chaperonins?

When faced with contradictory results, consider these methodological approaches:

• Concentration Effects: Different experimental techniques require different protein concentrations, which can significantly affect results, particularly for human chaperonins. For example, CD typically uses concentrations around 1 μM while SAXS requires approximately 20 μM .

• Analysis Model Limitations: Be aware that analytical models may introduce bias. For instance, CD data analysis often employs a two-state unfolding model that works well for GroEL but may not accurately represent the unfolding behavior of all chaperonins .

• Oligomeric State Variations: Chaperonins can exist in different oligomeric states, and the distribution of these states may vary between experimental conditions. Consider whether discrepancies might result from analyzing different populations of oligomers.

• Phase-Specific Variations: B. bacteriovorus undergoes dramatic transcriptional changes between attack and growth phases . Ensure that samples being compared represent the same life cycle phase to avoid misinterpreting phase-specific variations as intrinsic differences between chaperonins.

• Co-Chaperonin Interactions: Consider the symmetry challenges observed in chloroplast chaperonin systems, where even-numbered co-chaperonin complexes must interact with odd-numbered chaperonin rings . Such symmetry mismatches might lead to apparently contradictory results depending on the specific experimental conditions.

• Statistical Analysis Framework: When analyzing chaperonin activity data:

  • Apply appropriate statistical tests based on data distribution

  • Consider biological replicates from independent protein preparations

  • Use positive controls (known functional chaperonins) to benchmark activity levels

  • Account for background activity in ATP hydrolysis assays

What emerging technologies could advance research on Bdellovibrio bacteriovorus chaperonin?

Several cutting-edge approaches show promise for advancing B. bacteriovorus chaperonin research:

• Synthetic Biology Tools: The recent adaptation of the Golden Standard hierarchical assembly cloning technique for B. bacteriovorus HD100, combined with Tn7 transposon-based chromosomal integration, provides powerful new tools for genetic manipulation .

• Cryo-Electron Microscopy: High-resolution structural analysis of B. bacteriovorus chaperonin in different conformational states could provide insights into its function during different life cycle phases.

• Single-Molecule Techniques: Applying single-molecule FRET or optical tweezers to study chaperonin dynamics could reveal mechanistic details of protein folding assistance specific to B. bacteriovorus.

• In vivo Imaging: Developing fluorescent reporters linked to chaperonin activity could allow visualization of when and where chaperonin activity is highest during predation events.

• Systems Biology Approaches: Integration of transcriptomics, proteomics, and metabolomics could provide a comprehensive understanding of how chaperonin function relates to the broader cellular processes during the attack and growth phases.

How might Bdellovibrio bacteriovorus chaperonin research contribute to our understanding of bacterial predation mechanisms?

Chaperonin research offers several insights into predation mechanisms:

• Phase-Specific Protein Folding: Chaperonins may play critical roles in ensuring proper folding of phase-specific proteins essential for predation. The dramatic transcriptional switch between attack and growth phases likely requires corresponding protein folding assistance for newly synthesized proteins.

• Stress Response During Predation: Predation likely induces stress in both predator and prey. Understanding how chaperonins mitigate stress effects could reveal adaptations specific to the predatory lifestyle.

• Evolutionary Insights: Comparative analysis of chaperonin sequences and structures across predatory and non-predatory bacteria could illuminate evolutionary adaptations associated with the predatory lifestyle.

• Environmental Adaptation: Chaperonins assist protein folding under various environmental conditions. Research could reveal how B. bacteriovorus chaperonin has adapted to function optimally in the diverse environments encountered during predation.

• Therapeutic Applications: Understanding the molecular mechanisms of B. bacteriovorus predation, including the role of chaperonins, could inform the development of novel antimicrobial strategies or the optimization of B. bacteriovorus as a "living antibiotic" .