Recombinant Prochlorococcus marinus subsp. pastoris tRNA (Ile)-lysidine synthase (tilS)

What methodologies can be used to express recombinant TilS for experimental studies?

Based on available research, recombinant TilS has been successfully expressed in Escherichia coli expression systems . When designing your expression system, consider using a vector with a strong inducible promoter and a purification tag that won't interfere with enzyme activity. After expression, purification typically involves affinity chromatography followed by size exclusion chromatography to ensure high purity for enzymatic assays.

For recombinant expression of Prochlorococcus proteins specifically, you may need to optimize codon usage for E. coli, as marine cyanobacteria often have different codon preferences. Additionally, consider expressing the protein at lower temperatures (15-18°C) to improve proper folding, especially when working with a complex enzyme like TilS.

How can I determine if my recombinant TilS is enzymatically active?

According to research findings, TilS activity can be assessed by its ability to modify tRNA Ile2 . A functional assay would involve incubating your purified recombinant TilS with in vitro transcribed tRNA Ile2 substrate, ATP, and lysine, then analyzing the modification status of the tRNA. Gel electrophoresis can be used to detect the modification, as demonstrated in previous studies .

A more definitive functional test would be to determine if the TilS-modified tRNA becomes a substrate for isoleucyl tRNA synthetase (IleRS). As confirmed in published research, "lysidine modification of tRNA Ile2 is both necessary and sufficient to convert this tRNA into a substrate for isoleucyl tRNA synthetase" . This aminoacylation assay provides a clear readout of successful TilS activity.

What are the key factors to consider when designing experiments to study TilS function?

When designing experiments to study TilS function, follow these established principles for effective experimental design :

• Clearly define your variables: Independent variables could include TilS concentration, substrate concentration, reaction time, temperature, or pH. The dependent variable would typically be the amount of modified tRNA produced.

• Write a specific, testable hypothesis: For example, "Recombinant P. marinus TilS will efficiently modify tRNA Ile2 at physiological temperatures relevant to marine environments."

• Design experimental treatments: Include appropriate controls such as reactions without ATP, without lysine, or with heat-inactivated enzyme.

• Plan how you will measure your dependent variable: Develop reliable assays to detect and quantify tRNA modification.

Additionally, consider potential extraneous variables that could influence your results, such as contaminating nucleases that could degrade your tRNA substrate, or oxidative stress that could affect enzyme activity (as suggested by discussions of ROS effects in Prochlorococcus) .

How might environmental factors like oxidative stress affect TilS activity in Prochlorococcus, and how can I design experiments to investigate this?

While research doesn't directly address TilS and oxidative stress, studies discuss how reactive oxygen species (ROS) and oxidative stress affect Prochlorococcus generally . To investigate potential effects on TilS activity:

Design an experiment with the following variables:

• Independent variable: Level of oxidative stress (e.g., different concentrations of hydrogen peroxide or exposure to different light intensities)

• Dependent variable: TilS activity (measured by tRNA modification efficiency)

• Control variable: Use antioxidants in some conditions to mitigate ROS effects

Research describes how investigators used cocultivation with the "helper" heterotrophic bacterium Alteromonas macleodii (which reduces ROS via catalase activity) as one approach to manipulate oxidative stress levels . A similar design could be adapted to study TilS:

The results would help determine if oxidative stress impacts TilS function in Prochlorococcus, which could have implications for understanding how environmental stressors affect translation fidelity in these ecologically important marine organisms.

What approaches can be used to characterize the substrate specificity of TilS from Prochlorococcus marinus compared to other bacterial species?

Based on research describing testing "a series of lysine analogs" with TilS, a systematic approach to characterize substrate specificity would involve :

• Substrate analog screening: Test a panel of lysine analogs and related compounds as alternative substrates for TilS. Research shows that "many of these analogs, including some simple alkyl amines, were alternative substrates" . Design your screening with compounds that vary in size, charge, and functional groups to probe the structural requirements of the TilS active site.

• Kinetic analysis: For each viable substrate, determine kinetic parameters (Km, kcat) to quantify substrate preference. This would allow you to create a substrate specificity profile for P. marinus TilS.

• Comparative analysis: Compare these kinetic parameters with those of TilS enzymes from other bacterial species to identify differences in substrate recognition that might reflect adaptations to different ecological niches.

• Structural analysis: If high-resolution structures are available (as mentioned in research: "Several high resolution protein structures of bacterial TilS are available"), use computational modeling to predict how substrate binding might differ between TilS from different species .

This methodological approach would provide insights into whether TilS from the marine cyanobacterium P. marinus has evolved unique substrate preferences compared to TilS from other bacteria, potentially reflecting adaptations to its specific environment.

How can I investigate the influence of horizontal gene transfer on TilS evolution in Prochlorococcus and related cyanobacteria?

Research discusses horizontal gene transfer (HGT) in Prochlorococcus, noting that "HGT events leading to homologous recombination between chromosomal segments result in cohesive 'gene-flow units'" . To investigate HGT's influence on TilS evolution:

• Phylogenetic analysis:

  • Collect TilS sequences from diverse Prochlorococcus strains and related cyanobacteria

  • Construct phylogenetic trees to identify potential incongruencies that might indicate HGT events

  • Compare the TilS gene tree with the species tree to detect discrepancies

• Synteny analysis:

  • Examine the genomic context of the TilS gene across different strains

  • Identify any mobile genetic elements or unusual GC content that might suggest recent HGT

• Recombination detection:

  • Use computational methods to detect potential recombination breakpoints in the TilS gene

  • Calculate recombination rates within different populations and ecotypes

• Experimental validation:

  • Design experiments to measure the frequency of TilS gene transfer under different environmental conditions

  • Test whether HGT events affecting TilS confer any selective advantage

This methodological approach would help determine whether TilS has been subject to HGT in Prochlorococcus populations and how this process might have contributed to the enzyme's evolution and the adaptation of different ecotypes to their specific niches .

What experimental design would be most appropriate for testing the effects of temperature on recombinant P. marinus TilS activity, considering its marine origin?

Following established experimental design principles, a robust approach would be :

• Define variables:

  • Independent variable: Temperature (range should include temperatures relevant to Prochlorococcus' natural environment, typically 15-30°C)

  • Dependent variable: TilS activity (measured by rate of tRNA modification)

  • Control variables: pH, salt concentration, ATP and lysine concentrations

• Create a temperature profile:

  • Test TilS activity at multiple temperatures (e.g., 10, 15, 20, 25, 30, 35°C)

  • Include temperatures outside the natural range as controls

• Experimental setup:

  • Use a thermal cycler or water bath to maintain precise temperatures

  • Include appropriate controls at each temperature (e.g., no-enzyme controls)

  • Run reactions for standardized time periods

• Data analysis:

  • Calculate enzyme activity at each temperature

  • Plot temperature vs. activity to identify the optimal temperature

  • Use Arrhenius plots to determine activation energy

• Correlation with environmental data:

  • Compare the temperature profile with the known distribution of P. marinus in the ocean

  • Correlate optimal TilS activity temperature with the temperature of the water column where this subspecies is most abundant

This methodological approach would provide insights into the temperature adaptation of P. marinus TilS and how it might be optimized for the organism's specific marine niche.

How can I design experiments to test whether TilS activity is affected by specific mutations in the tRNA substrate?

Based on principles of experimental design and information about TilS function, a systematic approach would be :

This methodological approach would systematically map the structural requirements of the tRNA substrate for TilS recognition and modification, providing insights into the enzyme's specificity.

What are the most effective controls to include when assessing the impact of potential inhibitors on TilS activity?

Research mentions testing "a series of lysine analogs were tested as potential inhibitors during the mechanistic characterization of tRNA Ile-lysidine synthetase" . When designing inhibitor studies, include these essential controls:

• Positive control:

  • Standard reaction with TilS, tRNA substrate, ATP, and lysine

  • Establishes baseline activity level

• Negative control:

  • Reaction mixture without TilS

  • Confirms that observed modification is enzyme-dependent

• Substrate competition control:

  • Vary lysine concentration in the presence of fixed inhibitor concentration

  • Helps determine if inhibition is competitive with respect to lysine

• ATP competition control:

  • Vary ATP concentration in the presence of fixed inhibitor concentration

  • Determines if inhibition affects ATP binding

• Time-dependent controls:

  • Pre-incubate TilS with inhibitor before adding substrates

  • Tests for slow-binding or irreversible inhibition

• Specificity controls:

  • Test inhibitors against related enzymes

  • Ensures observed effects are specific to TilS

• Dose-response analysis:

  • Test multiple inhibitor concentrations to generate IC50 values

  • Allows quantitative comparison between different inhibitors

This comprehensive set of controls would provide robust data on the mechanism of inhibition and help avoid misinterpretation of results when characterizing potential TilS inhibitors.

What steps should be taken to troubleshoot inactive recombinant TilS enzyme preparations?

If your recombinant P. marinus TilS shows low or no activity, a systematic troubleshooting approach would include:

• Protein quality assessment:

  • Check protein purity by SDS-PAGE

  • Verify protein concentration using multiple methods (Bradford, BCA, A280)

  • Analyze protein folding using circular dichroism or fluorescence spectroscopy

• Expression system optimization:

  • Try different E. coli strains (BL21, Rosetta for rare codons)

  • Vary induction conditions (temperature, IPTG concentration, duration)

  • Test different fusion tags (His, GST, MBP) that might improve solubility

• Buffer optimization:

  • Test different pH values around the physiological range

  • Vary salt concentration to mimic marine conditions

  • Include stabilizing agents (glycerol, reducing agents)

• Substrate quality verification:

  • Confirm integrity of in vitro transcribed tRNA substrate

  • Verify ATP quality and prepare fresh solutions

  • Use high-purity lysine

• Potential solutions for common issues:

This methodological troubleshooting approach addresses the most common issues encountered when working with recombinant enzymes from marine organisms.

How can I adapt experimental protocols for studying TilS to better reflect the natural marine environment of Prochlorococcus?

Standard laboratory conditions often differ significantly from the natural environment of marine organisms like Prochlorococcus. To design more ecologically relevant experiments:

• Buffer composition:

  • Use artificial seawater-based buffers rather than standard laboratory buffers

  • Adjust salt concentration to match oceanic values (~35 g/L)

  • Include trace elements found in seawater

• Temperature conditions:

  • Conduct experiments at temperatures relevant to Prochlorococcus habitats (15-25°C)

  • Consider testing temperature ranges rather than single points

• Light conditions:

  • If studying enzyme expression, include appropriate light cycles

  • Test the effect of different light intensities, as research suggests light affects Prochlorococcus physiology

• pH considerations:

  • Account for ocean acidification by testing TilS activity across relevant pH ranges

  • Include controls that mimic projected future ocean conditions

• Oxidative stress:

  • As mentioned in research, ROS levels affect Prochlorococcus

  • Include oxidative stress variables that mimic conditions encountered in the photic zone

• Experimental design recommendations:

This methodological approach would provide insights into TilS function under conditions that better reflect the actual marine environment where Prochlorococcus lives, potentially revealing adaptations not apparent under standard laboratory conditions .