Recombinant Photobacterium profundum Isoleucine--tRNA ligase (ileS), partial

What is Photobacterium profundum and why is its isoleucine-tRNA ligase significant for research?

Photobacterium profundum SS9 is a Gram-negative bacterium originally isolated from the Sulu Sea at a depth of 2.5 km. It is classified as both a piezophile (pressure-loving) and psychrophile (cold-loving), with optimal growth conditions of 28 MPa and 15°C. The organism can grow across a remarkable pressure range from atmospheric pressure (0.1 MPa) to 90 MPa, making it an ideal model for studying pressure adaptation .

The genome of P. profundum consists of two chromosomes and an 80 kb plasmid, and the bacterium is closely related to other Vibrio species such as Vibrio cholerae . Its ability to grow at atmospheric pressure distinguishes it from many other piezophiles, allowing for easier genetic manipulation and cultivation in standard laboratory settings.

The isoleucine-tRNA ligase (ileS) from P. profundum is of particular interest because:

• It must function efficiently under high hydrostatic pressure conditions

• It likely contains structural and functional adaptations that maintain catalytic activity at elevated pressures

• Studying these adaptations provides fundamental insights into protein evolution in extreme environments

• It serves as a model system for investigating pressure effects on translation machinery

What expression systems are most effective for producing recombinant P. profundum ileS?

When expressing recombinant proteins from piezophilic organisms, several methodological considerations become essential:

• Temperature optimization: Expression at lower temperatures (15-17°C) often yields better results than standard expression temperatures, as this more closely mimics the native growth conditions of P. profundum .

• Culture conditions: Growth media containing marine broth supplemented with glucose and appropriate buffering systems (like HEPES pH 7.5) help maintain optimal conditions for protein expression .

• Anaerobic considerations: P. profundum is typically cultured under anaerobic conditions, which may influence protein folding and modification. Expression systems should account for this environmental factor .

• Pressure adaptation: While standard expression hosts like E. coli can be used, performing certain cultivation steps under pressure may improve proper folding and activity of the recombinant enzyme.

Based on methodologies used with other P. profundum proteins, the following expression protocol can be considered:

• Transform expression construct into an appropriate E. coli strain (e.g., BL21(DE3) or Arctic Express)

• Grow cultures at lower temperatures (15-17°C) in marine broth or LB with appropriate salt concentrations

• Induce expression with lower IPTG concentrations (0.1-0.5 mM) to promote slower, more accurate protein folding

• Harvest cells by centrifugation (800×g for 10 minutes) and process immediately or store at -80°C

How does hydrostatic pressure affect the catalytic activity of P. profundum ileS?

The catalytic function of aminoacyl-tRNA synthetases involves a two-step reaction: activation of the amino acid with ATP followed by transfer to the appropriate tRNA. For piezophilic enzymes like P. profundum ileS, pressure effects on catalysis are complex and multi-faceted:

• Conformational effects: Hydrostatic pressure can alter protein conformation, potentially affecting active site geometry and substrate binding. The P. profundum ileS likely possesses structural adaptations that maintain optimal conformation under pressure.

• Reaction volume changes: The reaction catalyzed by aminoacyl-tRNA synthetases involves changes in reaction volume, which are directly influenced by pressure according to thermodynamic principles.

• Substrate interactions: Pressure may alter the binding affinity for substrates (isoleucine, ATP, and tRNA) by affecting non-covalent interactions at the binding interface.

Experimental approaches to study these effects include:

• High-pressure stopped-flow spectroscopy

• Enzyme kinetic measurements in pressure vessels

• Comparative analysis of activity profiles across a pressure range (0.1-90 MPa)

What structural adaptations might enable P. profundum ileS to function optimally under high pressure?

Based on studies of other pressure-adapted proteins, several structural features may contribute to the function of P. profundum ileS at high hydrostatic pressure:

• Reduced void volumes: Piezophilic proteins often have fewer and smaller internal cavities to minimize pressure-induced conformational changes.

• Modified flexibility: Strategic placement of glycine residues and proline residues to provide necessary flexibility while maintaining structural integrity under pressure.

• Electrostatic interactions: Increased numbers of salt bridges and hydrogen bonds that become strengthened under pressure conditions.

• Hydrophobic core modifications: Altered packing of hydrophobic residues to resist pressure-induced water penetration into the protein core.

• Surface charge distribution: Modified surface charge patterns that maintain protein-solvent interactions under pressure.

Similar to observations in P. profundum's proteome, the ileS enzyme likely shows differential expression and modification in response to pressure changes . Pressure-dependent proteomic studies have identified up-regulation of certain metabolic pathways (like glycolysis/gluconeogenesis) under high pressure, while other pathways (like oxidative phosphorylation) are up-regulated at atmospheric pressure .

What specialized equipment and techniques are required for investigating P. profundum ileS under high pressure?

Studying enzymes under high pressure requires specialized equipment and methodological approaches:

• Pressure vessels: High-pressure reactors capable of maintaining stable pressure (up to 90 MPa) while allowing for sample collection or spectroscopic measurement. These can be water-cooled pressure vessels maintained at appropriate temperatures (e.g., 17°C) .

• Pressure-resistant cultivation systems: For growing bacteria under pressure, specialized systems such as sealed Pasteur pipettes that can be pressurized in water-cooled pressure vessels have been used .

• Pressure-compatible spectroscopy: Modified spectrophotometers with high-pressure cells for real-time monitoring of enzyme activity.

• Sample handling under pressure: Techniques for introducing substrates or inhibitors while maintaining pressure conditions.

The culture methods described for P. profundum involve:

• Growing cultures in sealed, pressure-resistant containers excluding air to ensure even pressure distribution and anaerobic conditions

• Incubation at appropriate pressure (0.1 MPa for atmospheric or 28 MPa for high pressure) at controlled temperatures (17°C)

• Careful harvesting and processing to maintain protein integrity

How can the fidelity and editing function of P. profundum ileS be assessed in the context of pressure adaptation?

Aminoacyl-tRNA synthetases possess editing functions to ensure translational fidelity. For P. profundum ileS, evaluating this function under pressure conditions is critical:

• Misacylation assays: Techniques to measure the rate of incorrect amino acid incorporation (particularly the near-cognate amino acids valine and leucine) at different pressures.

• Pre-transfer vs. post-transfer editing: Methods to distinguish between these two editing mechanisms and their relative contributions under pressure.

• Structural analysis of the editing domain: Investigation of pressure-induced conformational changes in the CP1 editing domain through techniques such as hydrogen-deuterium exchange mass spectrometry.

Experimental design considerations should include:

• Comparative analysis with non-piezophilic ileS enzymes

• Assessment across the full range of pressures where P. profundum grows (0.1-90 MPa)

• Integration of kinetic, structural, and in vivo translation fidelity measurements

How can site-directed mutagenesis be used to identify pressure-adaptive residues in P. profundum ileS?

Site-directed mutagenesis provides a powerful approach for investigating specific residues hypothesized to contribute to pressure adaptation:

• Target selection strategies:

  • Comparison with non-piezophilic homologs to identify divergent residues

  • Focus on regions known to be pressure-sensitive in other proteins

  • Analysis of domains involved in substrate binding and catalysis

• Mutational approaches:

  • Conservative substitutions to assess the importance of specific chemical properties

  • Introduction of residues from non-piezophilic homologs to test pressure-sensitivity

  • Creation of chimeric enzymes by domain swapping with non-piezophilic homologs

• Functional analysis methods:

  • Pressure-dependent enzyme kinetics

  • Thermal stability assays at different pressures

  • In vivo complementation studies in ileS-deficient strains

The methodology for introducing and analyzing mutations would follow protocols similar to those used in other P. profundum genetic studies, which typically involve cultivation under anaerobic conditions and specialized pressure treatment .

What insights can comparative analysis between P. profundum ileS and other bacterial isoleucyl-tRNA synthetases provide?

Comparative analysis can reveal evolutionary patterns and convergent adaptations to pressure:

• Sequence-based analysis:

  • Multiple sequence alignment of ileS from piezophilic and non-piezophilic organisms

  • Identification of conserved motifs specific to piezophilic lineages

  • Analysis of amino acid composition biases associated with pressure adaptation

• Structural comparisons:

  • Homology modeling based on crystal structures of related isoleucyl-tRNA synthetases

  • Analysis of predicted flexibility, void volumes, and electrostatic interactions

  • Identification of potential pressure-adaptive structural elements

• Cross-species complementation studies:

  • Similar to findings with M. tuberculosis ileS which could complement E. coli ileS-deficient mutants

  • Testing whether P. profundum ileS can function in non-piezophilic hosts

  • Assessment of pressure tolerance conferred by heterologous expression

Studies of tRNA synthetases from other organisms have shown that these enzymes can exhibit species-specific tRNA recognition and varying sensitivities to inhibitors . Similar principles may apply to the pressure adaptations in P. profundum ileS.

How is the expression of P. profundum ileS regulated in response to changing pressure conditions?

The regulation of gene expression in response to pressure is a critical aspect of piezophilic adaptation:

• Transcriptional regulation:

  • P. profundum shows differential gene expression patterns under varying pressure conditions

  • Specific transcription factors or pressure-responsive promoter elements may control ileS expression

  • RNA polymerase adaptation to pressure may involve sigma factors like RpoX, which has been identified in related P. profundum strains

• Post-transcriptional regulation:

  • Pressure effects on mRNA stability and structure

  • Potential role of small RNAs in pressure-responsive regulation

  • Alterations in translation efficiency under different pressure conditions

Proteomics studies have shown that P. profundum differentially expresses proteins involved in key metabolic pathways in response to pressure changes . The regulation of translational machinery components, including aminoacyl-tRNA synthetases, would be expected to participate in this adaptive response.

What techniques are available for investigating pressure-dependent changes in P. profundum ileS expression and activity in vivo?

Several methodological approaches can be employed to study pressure effects on ileS expression and function:

• Transcriptomics:

  • RNA-seq analysis of cultures grown at different pressures

  • qRT-PCR quantification of ileS transcript levels

  • Promoter-reporter fusion constructs to monitor transcriptional activity

• Proteomics:

  • Shotgun proteomic analysis comparing protein expression at different pressures

  • Targeted mass spectrometry to quantify ileS protein levels

  • Pulse-chase experiments to determine protein turnover rates

• In vivo activity assessment:

  • Translation rate measurements at different pressures

  • tRNA charging level analysis

  • Polysome profiling to assess global translation activity

The methodology for culturing P. profundum under pressure conditions has been established using sealed Pasteur pipettes in pressure vessels . These techniques can be adapted for specific gene expression and protein function studies focused on ileS regulation.

What role might P. profundum ileS play in understanding broader principles of protein adaptation to extreme environments?

The study of P. profundum ileS has implications that extend beyond this specific enzyme:

• Fundamental biophysical principles:

  • Pressure effects on protein structure and function

  • Volume changes in enzyme-catalyzed reactions

  • Solvent interactions under extreme conditions

• Evolutionary biology insights:

  • Convergent vs. divergent adaptations to pressure

  • Evolutionary trajectories in extreme environment adaptation

  • Molecular basis of habitat expansion into the deep sea

• Biotechnological applications:

  • Design principles for pressure-stable enzymes

  • Potential applications in high-pressure biocatalysis

  • Insights for protein engineering in other extreme environments

How might the study of P. profundum ileS contribute to understanding translational fidelity under pressure stress?

Protein synthesis accuracy under extreme conditions is a critical aspect of cellular adaptation:

• Pressure effects on translation accuracy:

  • Potential shifts in error rates at different pressures

  • Mechanisms for maintaining translational fidelity under stress

  • Trade-offs between translation speed and accuracy

• Methodological approaches:

  • Reporter systems to measure mistranslation rates in vivo

  • In vitro translation systems incorporating purified P. profundum components

  • Comparative analysis with non-piezophilic translation systems

• Evolutionary implications:

  • Selection pressure on translational accuracy in piezophilic organisms

  • Co-evolution of tRNA and aminoacyl-tRNA synthetases

  • Adaptation of the entire translation apparatus to pressure

The insights gained from studying P. profundum ileS can provide a foundation for understanding how fundamental cellular processes adapt to extreme environmental conditions, with potential applications in both basic science and biotechnology.