Recombinant Photobacterium profundum Leucine--tRNA ligase (leuS), partial

Basic Research Questions

• What is Photobacterium profundum and why is it significant for studying leuS?

  Photobacterium profundum is a deep-sea Gram-negative bacterium originally isolated from an amphipod homogenate collected from a depth of 2.5 km in the Sulu Sea . It belongs to the Photobacterium subgroup of the Vibrionaceae family and is closely related to other Vibrio species such as Vibrio cholerae . What makes P. profundum particularly valuable as a model organism is its ability to grow under a wide range of pressures, from atmospheric pressure (0.1 MPa) to 90 MPa, with optimal growth occurring at 28 MPa and 15°C . This classifies it as both a piezophile (thriving under high pressure) and a psychrophile (thriving under cold conditions) .

  The significance of P. profundum for leuS research stems from its adaptation to extreme environments, which provides a unique opportunity to study how essential enzymes like Leucine--tRNA ligase function under high-pressure conditions. Unlike many other piezophiles, P. profundum's ability to grow at atmospheric pressure enables easier genetic manipulation and culture, making it an ideal model organism for studying piezophily .

• What is the function of Leucine--tRNA ligase (leuS) in protein synthesis?

  Leucine--tRNA ligase (also known as Leucyl-tRNA synthetase or LeuRS) belongs to the family of aminoacyl-tRNA synthetases (aaRSs), which are ancient enzymes playing a fundamental role in protein synthesis . These enzymes catalyze the esterification of specific amino acids (in this case, leucine) to the 3'-end of their cognate tRNAs, a critical step in the translation process .

  The reaction catalyzed by LeuRS occurs in two steps:

  $\text{Leucine} + \text{ATP} \rightarrow \text{Leucyl-AMP} + \text{PP}_i$
  $\text{Leucyl-AMP} + \text{tRNA}^{\text{Leu}} \rightarrow \text{Leucyl-tRNA}^{\text{Leu}} + \text{AMP}$

  LeuRS contains both an aminoacylation domain for attaching leucine to tRNA and an editing domain for hydrolyzing mischarged tRNA, ensuring translational fidelity . In Photobacterium profundum, leuS is essential for protein synthesis under various pressure conditions, making it a key component for the organism's survival in deep-sea environments.

• What expression systems are effective for producing recombinant P. profundum leuS?

  Several expression systems have been successfully employed for producing recombinant Photobacterium profundum Leucine--tRNA ligase. According to available product information, the protein can be expressed in multiple host systems including E. coli, yeast, baculovirus, or mammalian cells .

  For research purposes, E. coli expression systems are most commonly used due to their efficiency and cost-effectiveness. The typical methodology involves:

  1. Cloning the leuS gene into an appropriate expression vector with a suitable tag (N-terminal and/or C-terminal)

  2. Transforming the construct into competent E. coli cells

  3. Growing cultures in liquid medium (such as LB) supplemented with appropriate antibiotics

  4. Inducing protein expression when the culture reaches optimal density

  5. Harvesting cells and purifying the recombinant protein

  For example, in similar protein expression studies, researchers have used the following conditions: growth in LB medium at pH 8.0 with appropriate antibiotics, incubation at 37°C with shaking at 325 rpm until A600 reached 0.4, followed by temperature reduction to 22°C for protein expression .

  The recombinant protein typically achieves ≥85% purity as determined by SDS-PAGE analysis .

• What are the optimal growth conditions for culturing P. profundum in laboratory settings?

  Culturing Photobacterium profundum requires specific conditions to mimic its native deep-sea environment. Based on established protocols, the following conditions are recommended:

  Temperature: 15-17°C is optimal for laboratory growth

  Pressure conditions:

  • For strain SS9: optimal growth at 28 MPa and 15°C

  • For strain 3TCK: optimal growth at 9°C and 0.1 MPa

  • For strain DSJ4: optimal growth at 10°C and 10 MPa

  Growth medium: Marine broth (28 g/liter 2216 medium) supplemented with:

  • 20 mM glucose

  • 100 mM HEPES buffer (pH 7.5)

  Method for high-pressure cultivation:

  1. Grow initial stock cultures in sterile plastic tubes at 17°C to an OD of 1.5 at 600 nm

  2. Inoculate 50 mL of marine broth with 100 μl of stock culture

  3. Aliquot into sterile plastic Pasteur pipettes (6 ml each), excluding air to ensure anaerobic conditions

  4. Seal pipettes with a Bunsen burner and bag sealer

  5. For atmospheric pressure growth (0.1 MPa): wrap pipettes in aluminum foil and incubate at 17°C

  6. For high pressure growth: incubate pipettes at desired pressure (e.g., 28 MPa) in a water-cooled pressure vessel at 17°C

  7. Culture to stationary phase (approximately 5 days)

  For genetic manipulation, conjugation with E. coli can be performed at room temperature for 12-16 hours on polycarbonate membrane filters (0.4-μm-pore-size) placed on dry 2216 plates .

Advanced Research Questions

• How does pressure affect the expression and activity of leuS in P. profundum?

  Pressure significantly influences protein expression patterns in Photobacterium profundum, including the expression of tRNA synthetases. A shotgun proteomic analysis using label-free quantitation and mass spectrometry revealed differential expression of proteins when P. profundum was grown at atmospheric pressure versus high pressure (28 MPa) .

  While specific data for leuS expression isn't explicitly detailed in the available sources, related research on P. profundum shows that several stress response genes are up-regulated in response to atmospheric pressure, including heat shock proteins like htpG, dnaK, dnaJ, and groEL . These findings suggest a complex regulatory response to pressure changes.

  In terms of metabolic pathways, proteins involved in glycolysis/gluconeogenesis were up-regulated at high pressure, while several proteins involved in oxidative phosphorylation were up-regulated at atmospheric pressure . This metabolic shift likely affects the demand for protein synthesis and, consequently, the activity of leuS.

  Research has shown that the types and abundance of fatty acid chains in the cell membrane respond to changes in pressure and temperature. At low temperature and high pressure, P. profundum strain SS9 increases the proportion of unsaturated fatty acids in its membrane , which suggests potential adaptation mechanisms that might involve altered protein synthesis pathways.

  For researchers studying leuS activity under different pressure conditions, the HP/LP (high pressure/low pressure) ratio methodology can be valuable. This involves:

  1. Growing cultures at both high pressure (30 MPa) and atmospheric pressure (0.1 MPa)

  2. Measuring the optical density (OD600) when the low-pressure culture enters early logarithmic phase

  3. Calculating the ratio of growth between HP and LP conditions

• What evidence exists for recombination in P. profundum genes and how might this affect leuS evolution?

  While specific data on recombination in Photobacterium profundum leuS is limited in the provided sources, research on other bacterial systems provides insights into how recombination might influence leuS evolution in this species.

  Studies on recombination in bacterial genomes, such as those conducted on Lactobacillus casei, have shown clear evidence for homologous recombination during the diversification of bacterial clones . In L. casei, several genes including leuS showed evidence of recombination, as indicated by statistical tests such as Sawyer's test and chi-square intragenic recombination test .

  The frequency of recombination can vary significantly between genes. In L. casei, leuS had 12 alleles, making it one of the more variable genes analyzed in multilocus sequence typing (MLST) schemes . This suggests that leuS might be subject to relatively high recombination rates in some bacterial species.

  In the context of P. profundum, which inhabits extreme and fluctuating environmental conditions, recombination could provide an important mechanism for adaptive evolution. The selective pressures of the deep-sea environment might favor genetic variations in essential genes like leuS that optimize protein synthesis under high pressure.

  Research on aminoacyl-tRNA synthetases in other extremophiles, such as the archaeal family Sulfolobaceae, has revealed gene duplication events resulting in two distinct copies of LeuRS with different functions . One copy (LeuRS-F) maintained the canonical editing function, while the other (LeuRS-I) had key amino acid substitutions in the editing domain that would disrupt hydrolytic editing of mischarged tRNA . Similar evolutionary processes could potentially occur in P. profundum through recombination events.

  For researchers investigating recombination in P. profundum leuS, methodological approaches could include:

  1. Sequencing leuS from multiple P. profundum strains

  2. Performing phylogenetic analyses to detect incongruencies

  3. Using statistical tests (e.g., Sawyer's test, chi-square intragenic recombination test) to identify potential recombination events

  4. Analyzing synonymous vs. non-synonymous substitution patterns to detect selective pressures

• How can researchers distinguish between pressure-specific and general stress responses in leuS regulation?

  Distinguishing between pressure-specific and general stress responses in leuS regulation requires a multi-faceted experimental approach. Based on methodologies used in P. profundum research, the following strategies can be employed:

  Comparative transcriptomics/proteomics approach:

  1. Expose P. profundum cultures to multiple stress conditions:

     • High pressure (28 MPa)

     • Low pressure (0.1 MPa)

     • Low temperature (4°C at 0.1 MPa)

     • Nutrient limitation

     • Osmotic stress

  2. Perform RNA isolation and quantitative reverse transcriptase PCR (qRT-PCR) to measure leuS expression levels under each condition:

     • Use the gyrB gene (PBPRA0011) as an internal reference

     • Calculate differences using the threshold cycle (ΔΔC) method

  3. Conduct shotgun proteomic analysis using label-free quantitation and mass spectrometry:

     • Extract proteins from cells grown under different conditions

     • Perform LC-MS/MS analysis

     • Use software like Progenesis (Nonlinear Dynamics, UK) for label-free quantitation

     • Consider proteins differentially expressed with an absolute ratio of at least 1.5 and p<0.05

  Genetic manipulation approach:

  1. Create leuS mutants with modifications in potential pressure-sensing domains

  2. Evaluate growth of these mutants under various stress conditions

  3. Complement mutants with wild-type leuS to confirm phenotype specificity

  Suppressor analysis:

  Isolation of suppressor strains can reveal genetic interactions that distinguish between pressure-specific and general stress pathways:

  1. Culture P. profundum under high pressure conditions

  2. Monitor for suppressor mutants showing altered growth characteristics

  3. Isolate and sequence these strains to identify genetic changes

  4. Test suppressors under various stress conditions to determine specificity

  A key consideration is that pressure affects multiple cellular processes simultaneously. For instance, pressure increases membrane rigidity (similar to cold temperatures) but can also affect protein folding and oligomeric protein assembly in ways distinct from other stressors .

• What approaches can be used to investigate potential moonlighting functions of P. profundum leuS?

  Aminoacyl-tRNA synthetases, including Leucyl-tRNA synthetase (LeuRS), are known to perform functions beyond their canonical roles in protein synthesis. Human cytosolic LeuRS, for example, functions as a leucine sensor in the mTORC1 pathway . Similar moonlighting functions might exist in P. profundum leuS, particularly in relation to pressure adaptation.

  The following methodological approaches can be used to investigate potential moonlighting functions:

  Protein interaction studies:

  1. Immunoprecipitation (IP) followed by mass spectrometry:

     • Express tagged versions of P. profundum leuS

     • Perform pull-down experiments under different pressure conditions

     • Identify interaction partners by mass spectrometry

  2. Yeast two-hybrid or bacterial two-hybrid screening:

     • Use leuS as bait to screen for interacting proteins

     • Validate interactions using co-immunoprecipitation

  Structural analysis:

  1. X-ray crystallography or cryo-EM to determine the structure of P. profundum leuS:

     • Compare with structures of LeuRS from other organisms (e.g., human cytosolic LeuRS)

     • Identify unique structural features that might relate to moonlighting functions

  2. Domain-specific functional analysis:

     • Generate constructs expressing specific domains of leuS

     • Test these domains for novel functions independent of aminoacylation activity

  Phenotypic analysis of mutants:

  1. Create deletion or point mutants that maintain canonical function but might disrupt moonlighting functions

  2. Characterize phenotypes under various conditions, particularly focusing on pressure adaptation

  3. Perform high-throughput phenotypic microarrays to identify conditions where mutants show unexpected phenotypes

  Metabolomic studies:

  1. Compare metabolite profiles of wild-type and leuS mutant strains under different pressure conditions

  2. Look for changes in metabolites not directly related to protein synthesis pathways

  The key to this investigation is designing experiments that can distinguish between the canonical aminoacylation function and potential moonlighting functions. One approach is to create mutations that specifically affect one function while preserving the other, similar to the LeuRS-I and LeuRS-F paralogs identified in Sulfolobaceae .

• What are the implications of leuS duplication and divergence for evolutionary adaptation in deep-sea bacteria?

  Gene duplication provides raw material for evolutionary innovation, allowing one copy to maintain the original function while the other is free to evolve new functions. The case of LeuRS duplication in the archaeal family Sulfolobaceae provides valuable insights that may apply to deep-sea bacteria like P. profundum.

  In Sulfolobaceae, bioinformatics analyses identified two distinct leucyl-tRNA synthetase (LeuRS) genes within all genomes of this family . One copy (LeuRS-F) maintained the canonical function of accurately charging leucine to tRNA for protein translation, while the other copy (LeuRS-I) had key amino acid substitutions in its editing domain that would disrupt hydrolytic editing of mischarged tRNA .

  For P. profundum and other deep-sea bacteria, similar duplication events could potentially provide adaptive advantages:

  Potential adaptive benefits of leuS duplication:

  1. Enhanced proteome diversity: A LeuRS with reduced editing fidelity would introduce amino acid substitutions, generating proteome diversity that might be beneficial under fluctuating environmental conditions .

  2. Pressure-specific adaptations: One copy could evolve specialization for optimal function under high pressure, while the other maintains activity at lower pressures.

  3. Functional divergence: Beyond aminoacylation, duplicated copies might evolve new functions related to stress response or sensing environmental changes.

  Methodological approaches to investigate leuS duplication events:

  1. Comparative genomic analysis:

     • Search for paralogous leuS genes in P. profundum and related deep-sea bacteria

     • Analyze sequence divergence patterns and selection pressures

     • Compare with known cases like Sulfolobaceae

  2. Functional characterization:

     • Express and purify recombinant proteins from any identified leuS paralogs

     • Measure aminoacylation and editing activities under different pressure conditions

     • Assess growth phenotypes of strains with deletions of individual paralogs

  3. Experimental evolution:

     • Subject P. profundum to long-term cultivation under fluctuating pressure conditions

     • Monitor for duplication events in leuS or other tRNA synthetase genes

     • Characterize the functional consequences of any observed duplications

  Table 1: Comparative Analysis of LeuRS Paralogs in Sulfolobaceae and Potential Implications for Deep-Sea Bacteria

  Feature	LeuRS-F (Sulfolobaceae)	LeuRS-I (Sulfolobaceae)	Potential Implications for Deep-Sea Bacteria
  Essential for growth	Yes	No	Duplication could allow maintenance of essential function while permitting divergence
  Editing activity	Present	Disrupted	Reduced fidelity could generate proteome diversity beneficial under pressure stress
  Growth optimization	Normal growth	Optimal growth support	Paralogs could specialize for different pressure ranges
  Evolutionary origin	Ancestral	Duplicated and diverged	Similar duplication events may occur in response to deep-sea selective pressures

  The study of leuS duplication in deep-sea bacteria could provide insights into how these organisms adapt to extreme environments and the role of genetic redundancy in facilitating evolutionary innovation.

• How can deep-sea bacterial tRNA synthetases inform the development of pressure-stable enzymes for biotechnology?

  The study of tRNA synthetases from piezophilic organisms like Photobacterium profundum offers valuable insights for engineering pressure-stable enzymes with biotechnological applications. These naturally evolved enzymes provide models for understanding protein stability and activity under high-pressure conditions.

  Research approaches for studying pressure stability of P. profundum leuS:

  1. Structural analysis:

     • Determine crystal structure of P. profundum leuS and compare with homologs from non-piezophilic organisms

     • Identify specific amino acid substitutions or structural features associated with pressure adaptation

     • Use molecular dynamics simulations to analyze protein behavior under pressure

  2. Domain swapping experiments:

     • Create chimeric proteins combining domains from P. profundum leuS and homologs from non-piezophilic organisms

     • Test activity and stability under different pressure conditions

     • Identify domains critical for pressure adaptation

  3. Site-directed mutagenesis:

     • Introduce specific amino acid substitutions based on comparative sequence analysis

     • Measure effects on enzyme activity and stability under pressure

     • Identify key residues that confer pressure resistance

  Biochemical characterization methods:

  1. Aminoacylation assays under pressure:

     • Measure leucylation activity at various pressures (0.1-90 MPa)

     • Determine kinetic parameters (Km, kcat) as a function of pressure

     • Compare wild-type enzyme with engineered variants

  2. Thermal stability analysis:

     • Use differential scanning calorimetry to measure melting temperatures under pressure

     • Determine if pressure adaptation correlates with thermal stability

  3. Limited proteolysis under pressure:

     • Assess conformational changes induced by pressure through susceptibility to proteases

     • Compare digestion patterns at atmospheric vs. high pressure

  Biotechnological applications:

  Pressure-stable enzymes derived from P. profundum leuS research could find applications in:

  1. High-pressure biocatalysis processes, which can offer advantages in terms of reaction rates, substrate solubility, and reduced microbial contamination

  2. Development of biosensors functional in deep-sea environments for environmental monitoring

  3. Creation of pressure-resistant cell-free protein synthesis systems for specialized applications

  4. Engineering microorganisms with enhanced tolerance to industrial processes involving high pressure

  Understanding the molecular basis of pressure adaptation in P. profundum leuS not only advances our knowledge of extremophile biology but also provides valuable tools for biotechnological innovation in high-pressure applications.