Recombinant Photobacterium profundum Chemotaxis response regulator protein-glutamate methylesterase 1 (cheB1)

What is Photobacterium profundum and why is it significant for chemotaxis studies?

Photobacterium profundum is a gram-negative marine bacterium belonging to the Vibrionaceae family and Photobacterium genus. It possesses significant scientific importance as it exhibits remarkable adaptability to extreme conditions, particularly high pressure (piezophilic) and low temperature (psychrophilic) environments. The bacterium contains two circular chromosomes and was originally isolated from the Sulu Sea in 1986 . P. profundum is especially valuable for chemotaxis studies because it demonstrates adaptive responses to varying environmental pressures (from 0.1 MPa to 70 MPa) and temperatures (0°C to 25°C), allowing researchers to investigate how chemotactic mechanisms function under extreme conditions that would be impossible to study in conventional model organisms . The strain SS9, with optimal growth at 15°C and 28 MPa, provides an excellent platform for understanding how chemotaxis regulation differs in deep-sea environments.

What is the general function of chemotaxis response regulator protein-glutamate methylesterase (cheB1) in bacteria?

The chemotaxis response regulator protein-glutamate methylesterase 1 (cheB1) plays a critical role in bacterial chemotaxis adaptation systems. This enzyme functions primarily by removing methyl groups from specific glutamate residues on methyl-accepting chemotaxis proteins (MCPs). In the bacterial chemotaxis signaling pathway, cheB1 acts as a negative regulator that counterbalances the methyltransferase activity, establishing a biochemical memory system that allows bacteria to compare current chemical conditions with previous environments. The activity of cheB1 is typically regulated through phosphorylation by the histidine kinase CheA, which increases its methylesterase activity. This phosphorylation occurs in response to changes in attractant or repellent concentrations, enabling the bacterium to reset its sensory system and respond appropriately to new chemical gradients. In Photobacterium profundum, this system likely includes adaptations for functioning under high pressure conditions, possibly through structural modifications that maintain enzymatic efficiency despite compression forces.

How does the structure of cheB1 relate to its function in P. profundum?

While the specific structure of P. profundum cheB1 has not been fully characterized in the provided sources, based on homologous proteins in related bacteria, we can infer that P. profundum cheB1 likely consists of two functional domains: an N-terminal regulatory domain that receives phosphoryl groups and a C-terminal catalytic domain with methylesterase activity. The regulatory domain undergoes conformational changes upon phosphorylation, activating the catalytic domain. In P. profundum, the protein structure likely incorporates adaptations for high-pressure environments, potentially including increased flexibility in certain regions or stabilizing hydrophobic interactions that prevent pressure-induced denaturation. These adaptations would be essential for maintaining proper protein folding and activity under deep-sea conditions where P. profundum naturally resides. The protein's structure-function relationship is particularly relevant as P. profundum navigates environments with significant pressure gradients, requiring precise chemotactic regulation.

What are the most effective methods for expressing and purifying recombinant P. profundum cheB1?

For expressing recombinant P. profundum cheB1, a methodology similar to that used for other bacterial methylesterases can be applied with specific modifications to accommodate this deep-sea bacterial protein. Begin by designing a codon-optimized synthetic gene construct based on the P. profundum cheB1 sequence, incorporating appropriate affinity tags (His6 or FLAG) and cloning into an expression vector with a strong, inducible promoter such as pET or pBAD systems. For expression, E. coli BL21(DE3) or Rosetta strains are recommended, with growth conducted at lower temperatures (15-18°C) following induction to mimic the natural cold environment of P. profundum and promote proper protein folding .

For purification, implement a multi-step approach:

• Initial capture using immobilized metal affinity chromatography (IMAC) with Ni-NTA resin

• Intermediate purification via ion exchange chromatography

• Final polishing step using size exclusion chromatography

During all purification steps, maintain buffers at slightly elevated pressures (5-10 MPa) using specialized high-pressure equipment to preserve the native conformation of this piezophilic protein. Additionally, include 0.5-0.7M NaCl in buffers to satisfy the halophilic requirements of this marine bacterial protein . Protein purity should be verified using SDS-PAGE and Western blotting with anti-His or custom anti-cheB1 antibodies, with expected yields of 2-5 mg per liter of bacterial culture.

How can one design experiments to assess the methylesterase activity of P. profundum cheB1 under varying pressure conditions?

To assess methylesterase activity of P. profundum cheB1 under varying pressure conditions, a specialized experimental setup is required that accommodates high-pressure biochemical assays. Begin by designing a pressure chamber system that allows for real-time monitoring of enzymatic reactions, similar to those used in studies of other piezophilic enzymes. The core methodology should include:

• Preparation of methylated substrates: Synthesize or isolate methylated peptides corresponding to the methylation sites on P. profundum MCPs. Alternatively, use pre-methylated MCP receptor fragments as substrates.

• Reaction setup: In pressure-resistant microplate wells or cuvettes, combine:

  • Purified recombinant cheB1 (50-200 nM)

  • Methylated substrate (5-20 μM)

  • Reaction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl₂)

• Pressure application: Place reactions in high-pressure vessels capable of generating pressures from atmospheric (0.1 MPa) to deep-sea conditions (up to 70 MPa) .

• Activity measurement: After reaction periods (5-60 minutes), quantify demethylation using:

  • HPLC analysis of released methyl groups

  • Mass spectrometry to detect changes in substrate mass

  • Colorimetric assays using methanol oxidase coupled reactions to detect released methanol

Analyze results by plotting enzymatic activity versus pressure to establish the pressure-activity profile. Compare activity at the native pressure environment of specific P. profundum strains (e.g., 28 MPa for strain SS9 versus 0.1 MPa for strain 3TCK) to determine pressure adaptation of the enzyme . This approach will reveal whether cheB1 exhibits pressure-optimized activity correlating with the strain's native deep-sea habitat.

What techniques are suitable for studying the interaction between cheB1 and other chemotaxis proteins in P. profundum?

For investigating protein-protein interactions between cheB1 and other chemotaxis components in P. profundum, a multi-faceted approach combining in vitro and in vivo techniques is recommended:

In vitro interaction studies:

• Pull-down assays: Immobilize purified His-tagged cheB1 on Ni-NTA resin and pass P. profundum lysate through the column. Analyze bound proteins by mass spectrometry to identify interacting partners.

• Surface Plasmon Resonance (SPR): Quantify binding kinetics between cheB1 and purified chemotaxis proteins (CheA, CheW, MCPs) by immobilizing one partner on a sensor chip and flowing the other partner at varying concentrations.

• Isothermal Titration Calorimetry (ITC): Measure thermodynamic parameters of binding between cheB1 and potential partners under varying pressure conditions using pressure-resistant ITC cells.

In vivo interaction studies:

• Bacterial Two-Hybrid system: Adapt conventional B2H systems for use in P. profundum by developing pressure-resistant reporter assays.

• Fluorescence microscopy: Express fluorescently-tagged cheB1 (similar to the GFP approach used for visualizing P. profundum in confocal microscopy ) and potential interacting partners with complementary fluorophores to observe co-localization patterns in living cells under various pressure conditions.

• Cross-linking coupled with mass spectrometry: Treat living P. profundum cells with membrane-permeable crosslinkers, then identify crosslinked peptides using tandem mass spectrometry to map the interaction interface.

For pressure-dependent interactions, conduct experiments at atmospheric pressure (0.1 MPa) and at the optimal growth pressure for the specific P. profundum strain (e.g., 28 MPa for strain SS9) . Compare interaction patterns to determine whether protein complex formation is pressure-modulated, which would indicate adaptive mechanisms for chemotaxis regulation in deep-sea environments.

How can mathematical modeling be applied to understand the role of cheB1 in P. profundum chemotaxis under varying pressure conditions?

Mathematical modeling of cheB1's role in P. profundum chemotaxis under varying pressure conditions requires integration of reaction kinetics, thermodynamics, and systems biology approaches. Begin by adapting established chemotaxis models (such as those developed for E. coli) with pressure-dependent parameters specific to P. profundum. The core modeling approach should include:

• Ordinary Differential Equation (ODE) system: Develop equations representing:

  • Receptor methylation/demethylation kinetics by cheB1 and methyltransferases

  • CheA autophosphorylation rates as functions of receptor state

  • Phosphotransfer to response regulators including cheB1

  • Signal amplification and adaptation timescales

• Pressure-dependent parameters: Incorporate pressure effects on:

  • Protein-protein interaction affinities (typically increasing with pressure)

  • Enzymatic reaction rates (often showing pressure optima corresponding to native environments)

  • Membrane fluidity and receptor clustering dynamics

Following the approach outlined in search result , implement model validation through:

• Creating multiple model variants with different hypothesized pressure-dependent connectivities

• Designing optimal experiments to differentiate between models using control theory concepts

• Applying MATLAB toolboxes like SOSTOOLS to determine experimental conditions that maximize observable differences between competing models

This methodology allows for systematic testing of hypotheses about how P. profundum cheB1 activity responds to pressure changes. For example, one could model whether cheB1 exhibits altered substrate specificity, modified phosphorylation kinetics, or changed interaction dynamics with the chemotaxis complex at different pressures. The modeling results can guide experimental design for validation studies, creating an iterative process between computational predictions and laboratory observations.

What are the evolutionary implications of studying cheB1 in piezophilic bacteria like P. profundum?

Studying cheB1 in piezophilic bacteria like P. profundum provides valuable insights into evolutionary adaptations to extreme environments. The evolutionary implications span multiple levels of biological organization:

• Molecular adaptation mechanisms:

  • Analysis of P. profundum cheB1 sequence compared to homologs from non-piezophilic bacteria can reveal specific amino acid substitutions that confer pressure resistance.

  • These adaptations may include increased proportions of small amino acids that reduce volume changes under pressure, or modified hydrophobic cores that maintain stability despite compression.

• Functional evolution:

  • Comparison of cheB1 activity profiles across P. profundum strains adapted to different depths (e.g., strain SS9 from deep sea versus strain 3TCK from shallow water) can reveal how enzymatic properties evolve in response to pressure gradients.

  • This functional comparison allows researchers to distinguish between adaptations specific to pressure versus other environmental factors like temperature.

• Systems-level evolution:

  • Examining the entire chemotaxis pathway across P. profundum strains can reveal whether cheB1 evolution occurs coordinately with changes in other pathway components.

  • This systems perspective addresses whether pathway modularity facilitates adaptation to extreme environments.

• Ecological implications:

  • Understanding how chemotaxis regulation via cheB1 functions under high pressure provides insights into microbial niche partitioning in the deep sea.

  • This knowledge contributes to our understanding of biogeochemical cycling in deep ocean environments, where P. profundum and related bacteria play significant roles.

By studying the evolutionary trajectory of cheB1 in organisms spanning pressure gradients, researchers can develop models of how essential cellular processes adapt to extreme environments, with potential applications to astrobiology and the search for life in high-pressure extraterrestrial environments.

How does the function of cheB1 in P. profundum compare with homologous proteins in non-piezophilic bacteria?

The function of cheB1 in piezophilic P. profundum likely exhibits significant adaptations compared to homologous proteins in non-piezophilic bacteria due to the unique challenges of deep-sea environments. A methodical comparative analysis reveals several key differences:

Table 1: Comparative Analysis of cheB1 Properties Between Piezophilic and Non-Piezophilic Bacteria

Functionally, P. profundum cheB1 must maintain methylesterase activity under conditions that would typically inhibit enzyme function in non-piezophilic bacteria. This adaptation may involve:

• Active site modifications that reduce the activation volume of the catalytic reaction

• Altered substrate binding pockets that maintain affinity despite pressure-induced conformational changes

• Modified regulatory domain interactions that preserve phosphorylation-dependent activation under pressure

When examining the broader chemotaxis pathway context, P. profundum likely exhibits co-evolution of multiple pathway components, including cheB1, to ensure robust chemotactic responses in the deep sea. This could involve compensatory mutations throughout the pathway that maintain system performance despite the extreme environment. Understanding these adaptations provides valuable insights into protein evolution under extreme conditions and potential applications in pressure-resistant enzyme engineering.

What are common challenges in expressing active recombinant P. profundum cheB1 and how can they be overcome?

Expressing active recombinant P. profundum cheB1 presents several challenges related to the piezophilic and psychrophilic nature of this protein. The following table outlines common issues and methodological solutions:

Table 2: Challenges and Solutions for P. profundum cheB1 Expression

Implementation of these solutions requires specialized equipment for high-pressure protein expression and purification. If such equipment is unavailable, alternative approaches include:

• Expressing P. profundum cheB1 from strain 3TCK, which grows optimally at atmospheric pressure rather than strain SS9 which requires high pressure

• Creating chimeric proteins combining the catalytic domain of P. profundum cheB1 with regulatory domains from non-piezophilic homologs

• Using computational modeling to predict pressure effects before attempting expression

By systematically addressing these challenges using the outlined methodological solutions, researchers can successfully obtain active recombinant P. profundum cheB1 suitable for structural and functional studies.

How can researchers establish appropriate control experiments when studying the effects of pressure on P. profundum cheB1 activity?

Establishing appropriate controls for pressure experiments with P. profundum cheB1 is crucial for distinguishing specific pressure effects from experimental artifacts. A methodological framework for control experiments should include:

• Strain-specific controls:

  • Compare cheB1 from P. profundum strain SS9 (piezophilic, optimal growth at 28 MPa) with cheB1 from strain 3TCK (non-piezophilic, optimal growth at atmospheric pressure)

  • This comparison provides a natural control for distinguishing adaptation-specific effects from general pressure effects

• Protein structural integrity controls:

  • Monitor secondary structure stability under pressure using:
    a. Circular dichroism spectroscopy in pressure-resistant cells
    b. Intrinsic tryptophan fluorescence to detect unfolding
    c. Differential scanning calorimetry at varying pressures

  • These measurements ensure that observed activity changes reflect functional adaptations rather than protein denaturation

• Enzymatic parameter controls:

  • Determine complete enzyme kinetics (Km, Vmax, kcat) at each pressure point

  • Plot Lineweaver-Burk or Eadie-Hofstee transformations to identify whether pressure affects substrate binding or catalytic steps

• Time-course controls:

  • Monitor activity over extended time periods at each pressure to distinguish between:
    a. Immediate pressure effects on enzyme conformation
    b. Slower effects due to pressure-induced structural reorganization
    c. Potential enzyme degradation during high-pressure incubation

• Technical controls for high-pressure equipment:

  • Include pressure-insensitive enzymatic reactions (e.g., alkaline phosphatase activity) in parallel experiments

  • These serve as internal standards to verify pressure application and release are functioning correctly

• Reversibility controls:

  • After high-pressure exposure, return samples to atmospheric pressure and re-measure activity

  • This distinguishes reversible functional adaptations from irreversible structural changes

By implementing this comprehensive control framework, researchers can confidently attribute observed changes in cheB1 activity to specific pressure adaptations rather than experimental artifacts or general protein responses to pressure.

What approaches can be used to study the in vivo function of cheB1 in P. profundum under deep-sea conditions?

Studying the in vivo function of cheB1 in P. profundum under deep-sea conditions requires specialized approaches that combine molecular genetics with high-pressure cultivation technologies. A methodological framework includes:

• Genetic manipulation strategies:

  • Develop pressure-resistant transposon mutagenesis systems for P. profundum

  • Create targeted cheB1 deletion mutants using allelic exchange vectors

  • Construct complementation strains expressing fluorescently-tagged cheB1 variants

  • Design CRISPR-Cas9 systems optimized for function in piezophilic bacteria

• High-pressure cultivation systems:

  • Utilize pressure vessels equipped with:
    a. Observation windows for microscopy
    b. Sampling ports for time-course experiments
    c. Gradient-generating capabilities for chemotaxis assays

  • Maintain cultures at strain-specific optimal pressures (e.g., 28 MPa for strain SS9)

• In vivo cheB1 activity assessment:

  • Develop fluorescence-based reporters for receptor methylation state

  • Implement FRET-based biosensors to monitor cheB1-substrate interactions in living cells

  • Establish chemotaxis assays using microfluidic devices capable of withstanding high pressure

• Behavioral phenotype analysis:

  • Quantify chemotaxis efficiency using:
    a. Capillary assays adapted for high-pressure environments
    b. Tracking algorithms for bacterial movement under pressure
    c. Population distribution analysis in chemical gradients

  • Compare wild-type, cheB1 deletion, and complemented strains

• Adaptation to environmental stressors:

  • Test cheB1 function during combined stresses (pressure, temperature, UV radiation)

  • Implement the photoreactivation methodology described in search result to assess how cheB1 function affects bacterial survival after UV damage under pressure

  • Quantify survival rates using protocols similar to those described for P. profundum photoreactivation experiments

• Systems biology approaches:

  • Perform transcriptomics and proteomics analyses under various pressures

  • Correlate cheB1 expression levels with expression of other chemotaxis genes

  • Use the modeling approaches described in search result to design experiments that maximize information about network connectivity

Through this comprehensive methodological framework, researchers can gain insights into how cheB1 functions within the complex cellular environment of P. profundum under native deep-sea conditions, revealing adaptations that may not be apparent in isolated protein studies.

How should researchers interpret differential expression of cheB1 in P. profundum under varying pressure conditions?

Interpreting differential expression of cheB1 in P. profundum under varying pressure conditions requires a multifaceted analytical approach that considers both direct pressure effects and integrated cellular responses. Researchers should employ the following methodological framework:

• Expression analysis contextualization:

  • Compare cheB1 expression changes with known pressure-responsive genes in P. profundum (htpG, dnaK, dnaJ, groEL)

  • Determine whether cheB1 follows similar expression patterns to these stress response genes

  • Assess whether expression changes correlate with the optimal pressure for the specific strain (e.g., 28 MPa for SS9 vs. 0.1 MPa for 3TCK)

• Regulatory network mapping:

  • Identify potential pressure-responsive promoter elements upstream of cheB1

  • Perform ChIP-seq to identify transcription factors binding to the cheB1 promoter region under different pressures

  • Conduct reporter gene assays with truncated promoter constructs to isolate pressure-responsive elements

• Functional correlation analysis:

  • Correlate cheB1 expression levels with:
    a. Receptor methylation states as measured by mass spectrometry
    b. Chemotactic efficiency in pressure gradient environments
    c. Growth rate and cell morphology changes

  • This reveals whether expression changes have functional significance

• Comparative strain analysis:

  • Create an expression ratio table comparing cheB1 levels across strains:

Table 3: Hypothetical cheB1 Expression Ratios Under Different Pressure Conditions

• Patterns in this table would reveal whether cheB1 expression is tuned to each strain's ecological niche

• Evolutionary interpretation:

  • Analyze whether cheB1 expression differences represent:
    a. Adaptive responses that optimize chemotaxis under native pressure conditions
    b. General stress responses to non-optimal pressures
    c. Compensatory changes balancing other pressure-affected cellular processes

By applying this analytical framework, researchers can distinguish between direct pressure regulation of cheB1 and indirect effects mediated through broader cellular stress responses. This approach allows for meaningful interpretation of expression data within the context of P. profundum's remarkable adaptation to deep-sea environments.

What are promising future research directions for understanding the role of cheB1 in P. profundum adaptation to deep-sea environments?

Future research on P. profundum cheB1 should explore several promising directions that leverage emerging technologies and interdisciplinary approaches:

• Structural biology under pressure:

  • Determine the high-resolution structure of P. profundum cheB1 using:
    a. Cryo-electron microscopy with samples vitrified under pressure
    b. NMR spectroscopy in high-pressure cells
    c. X-ray crystallography of crystals grown under pressure

  • Compare structures obtained at atmospheric versus deep-sea pressures to identify conformational adaptations

• Single-molecule approaches:

  • Develop high-pressure single-molecule FRET systems to observe:
    a. Real-time conformational changes in cheB1 upon phosphorylation
    b. Interactions between cheB1 and receptors at the molecular level
    c. Pressure effects on protein dynamics with nanometer resolution

• Synthetic biology applications:

  • Engineer pressure-sensing bacterial biosensors using modified P. profundum cheB1

  • Create chimeric chemotaxis systems combining elements from piezophilic and non-piezophilic bacteria

  • Develop pressure-resistant industrial enzymes inspired by P. profundum adaptations

• Ecological and evolutionary studies:

  • Conduct metagenomics surveys of cheB1 variants across marine depth gradients

  • Perform experimental evolution of P. profundum under varying pressure regimes

  • Reconstruct ancestral cheB1 sequences to trace the evolutionary path to piezophilicity

• Systems biology integration:

  • Apply the chemotaxis modeling approaches outlined in search result to:
    a. Create predictive models of P. profundum chemotaxis under pressure
    b. Design optimal experiments for validating model predictions
    c. Identify network motifs responsible for pressure adaptation

• Medical and biotechnological applications:

  • Investigate pressure-adapted enzymes like cheB1 as catalysts in high-pressure industrial processes

  • Explore applications in food preservation using pressure-resistant enzymatic systems

  • Develop deep-sea biomimetic materials inspired by pressure adaptations

• Astrobiology connections:

  • Examine P. profundum cheB1 as a model for potential chemotaxis systems in high-pressure extraterrestrial environments

  • Develop protocols for detecting similar systems in samples from icy moons with subsurface oceans

These research directions offer complementary approaches to understanding not only how cheB1 functions under pressure but also how deep-sea adaptations evolve and how they might be harnessed for practical applications. By pursuing these avenues, researchers can develop a comprehensive understanding of chemotaxis in extreme environments while potentially discovering novel applications for pressure-adapted biological systems.