Recombinant Photobacterium profundum tRNA (guanine-N (7)-)-methyltransferase (trmB)

What is Photobacterium profundum and why is it significant for tRNA modification studies?

Photobacterium profundum is a cosmopolitan marine bacterium capable of growth at low temperature and high hydrostatic pressure. Multiple strains have been isolated from different ocean depths, displaying remarkable differences in their physiological responses to pressure . The genome-sequenced strains include the deep-sea piezopsychrophilic (pressure and cold-loving) strain SS9 and the shallow-water non-piezophilic strain 3TCK, which provide excellent comparative models for studying environmental adaptations .

As an established model organism for studying high-pressure adaptation , P. profundum offers valuable insights into how fundamental cellular processes, including tRNA modifications, function under extreme conditions. The study of tRNA methyltransferases like TrmB in this organism can enhance our understanding of how essential enzymes adapt to environmental pressures, particularly in deep-sea environments where pressure and temperature differ significantly from standard laboratory conditions.

What is the biological function of tRNA (guanine-N(7)-)-methyltransferase (TrmB) in bacteria?

The tRNA (guanine-N(7)-)-methyltransferase, known as TrmB, catalyzes the formation of N7-methylguanosine (m7G) modifications in tRNA molecules. This post-transcriptional modification is highly conserved across prokaryotes, eukaryotes, and some archaea . In bacteria, TrmB typically modifies specific guanosine residues in the variable loop of tRNAs containing an "ABGWY" motif sequence .

While m7G tRNA modification is non-essential for yeast growth under normal conditions, it becomes critical for stress responses, such as heat shock . In bacteria like Acinetobacter baumannii, TrmB is crucial for responding to stressors encountered during infection, including oxidative stress, low pH, and iron deprivation . The enzyme plays a vital role in maintaining proper tRNA folding, stability, and function, which are essential for accurate and efficient protein translation under varying environmental conditions.

How does tRNA m7G modification affect cellular processes in bacteria?

The m7G modification catalyzed by TrmB affects several key cellular processes:

• tRNA stability: The modification helps maintain proper tRNA structural integrity, particularly under stress conditions .

• Translation efficiency: m7G modification impacts the decoding properties of tRNAs, affecting the efficiency and accuracy of protein synthesis .

• Stress response: TrmB-mediated modifications are critical for bacterial responses to environmental stressors. In A. baumannii, loss of TrmB dramatically attenuates bacterial survival under oxidative stress, low pH, and iron-limited conditions .

• Pathogenesis: In pathogenic bacteria, TrmB contributes to virulence by enabling adaptation to host environments. TrmB-deficient A. baumannii shows attenuated virulence in murine pulmonary infection models .

These findings suggest that TrmB may play similarly important roles in P. profundum, particularly in adaptation to the unique stressors of deep-sea environments, including high pressure and low temperature.

What are the standard methods for expressing recombinant bacterial tRNA methyltransferases?

Standard methods for expressing recombinant bacterial tRNA methyltransferases like TrmB include:

• Expression vector selection:

  • pET-based vectors with T7 promoter systems for high-level expression

  • pBAD vectors for arabinose-inducible, tunable expression

  • pGEX or pMAL vectors for fusion with solubility-enhancing tags (GST or MBP)

• Host strain considerations:

  • E. coli BL21(DE3) for standard high-yield expression

  • E. coli Arctic Express for cold-adapted proteins

  • E. coli Rosetta for rare codon optimization

• Expression conditions optimization:

  • Induction at lower temperatures (16-20°C) to enhance proper folding

  • Reduced IPTG concentrations (0.1-0.5 mM) to prevent inclusion body formation

  • Extended expression times (overnight) at reduced temperatures

• Protein purification approaches:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Affinity chromatography appropriate for fusion partners

  • Size exclusion and ion-exchange chromatography for further purification

For P. profundum TrmB specifically, considering its deep-sea origin, expression at lower temperatures with optimization for psychrophilic proteins may be particularly important for obtaining properly folded and functional enzyme.

How can researchers design experiments to evaluate TrmB activity under high hydrostatic pressure?

Designing experiments to evaluate TrmB activity under high hydrostatic pressure requires specialized equipment and methodological considerations:

Methodological approach:

• Prepare purified recombinant TrmB from both deep-sea (SS9) and shallow-water (3TCK) P. profundum strains.

• Set up parallel reaction mixtures containing:

  • Purified TrmB enzyme

  • Appropriate tRNA substrates

  • S-adenosylmethionine (methyl donor)

  • Buffer optimized for pressure stability

• Conduct reactions under precisely controlled pressure conditions using specialized high-pressure equipment.

• Analyze reaction products using techniques such as HPLC, mass spectrometry, or radiometric assays to quantify m7G formation.

• Calculate and compare enzymatic parameters (Km, Vmax, kcat) across different pressure conditions.

This experimental approach would reveal whether TrmB from piezophilic P. profundum strains maintains activity under high pressure more effectively than enzymes from shallow-water strains, providing insights into molecular adaptations to deep-sea environments.

What approaches can be used to identify tRNA substrates of P. profundum TrmB?

Several complementary approaches can be employed to identify and characterize tRNA substrates of P. profundum TrmB:

• TRAC-seq (tRNA reduction and cleavage sequencing):

  • This specialized method identifies m7G-modified tRNAs based on their cleavage patterns .

  • Approach: Extract total RNA from P. profundum, perform specific chemical treatments to cleave at modified positions, prepare sequencing libraries, and analyze patterns.

  • Expected outcome: Identification of tRNAs with the "ABGWY" motif sequence in the variable loop, which is associated with m7G modification .

• RNA mass spectrometry:

  • Provides direct chemical evidence of modifications.

  • Approach: Digest tRNAs into nucleosides, separate by liquid chromatography, and analyze by tandem mass spectrometry.

  • Expected outcome: Quantification of m7G levels in different tRNA species.

• Northwestern and northern blot assays:

  • Visualize specific modified tRNAs.

  • Approach: Use m7G-specific antibodies for northwestern blots and tRNA-specific probes for northern blots .

  • Expected outcome: Detection of m7G modifications and expression levels of modified tRNAs.

• In vitro modification assays:

  • Direct testing of substrate specificity.

  • Approach: Incubate purified recombinant TrmB with individual tRNA species and analyze modification.

  • Expected outcome: Determination of substrate preference hierarchy.

These approaches would reveal which tRNAs serve as substrates for P. profundum TrmB and how substrate specificity might differ between enzymes from deep-sea and shallow-water strains.

How might researchers investigate the relationship between TrmB activity and bacterial stress responses in P. profundum?

Investigating the relationship between TrmB activity and stress responses in P. profundum requires a multi-faceted approach:

• Gene deletion and complementation studies:

  • Generate a trmB deletion mutant in P. profundum using techniques similar to those described for other gene deletions in this organism .

  • Complement with wild-type trmB or catalytically inactive variants.

  • Assess phenotypes under various stress conditions (pressure, temperature, oxidative stress).

• Transcriptomic analysis:

  • Perform RNA-seq on wild-type and trmB mutant strains under normal and stress conditions.

  • Analyze the transcriptional landscape to identify differential expression patterns .

  • Focus on stress response pathways and translation-related genes.

• Translational efficiency assessment:

  • Conduct polysome profiling and ribosome footprinting to examine translation under stress.

  • Analyze codon usage in differentially translated mRNAs.

  • Correlate with codons decoded by m7G-modified tRNAs .

• Environmental stress tests:

  • Evaluate survival under conditions mimicking deep-sea environments:

    • High pressure (up to 60 MPa)

    • Low temperature (4°C)

    • Limited nutrients

    • Combinations of these stressors

• Photoactivation studies:

  • Given P. profundum's known responses to UV radiation and blue light recovery , investigate whether TrmB affects photoreactivation processes.

  • Methodology: Subject wild-type and trmB mutant strains to UV irradiation followed by recovery under different conditions.

This comprehensive approach would determine whether TrmB in P. profundum plays a role in stress responses similar to that observed in A. baumannii , particularly in relation to the unique stressors of the deep-sea environment.

What strategies can be used to compare the structural and functional differences between TrmB from deep-sea and shallow-water P. profundum strains?

Comparing TrmB from deep-sea (SS9) and shallow-water (3TCK) P. profundum strains requires integrated structural and functional approaches:

• Structural analysis:

  • X-ray crystallography or cryo-EM of both TrmB variants

  • Molecular dynamics simulations under different pressure conditions

  • Hydrogen-deuterium exchange mass spectrometry to identify regions with different flexibility

  • Circular dichroism spectroscopy to assess secondary structure stability under varying pressures

• Comparative biochemical characterization:

  • Enzyme kinetics under varying pressure and temperature conditions

  • Substrate specificity profiling

  • Thermal and pressure stability assays

  • Binding affinity measurements for tRNA substrates and cofactors

• Mutational analysis:

  • Site-directed mutagenesis targeting non-conserved residues

  • Creation of chimeric enzymes with domains from each strain

  • Activity and stability testing of mutant variants

• In vivo functional swapping:

  • Express deep-sea TrmB in shallow-water strain and vice versa

  • Assess growth and stress response under different conditions

  • Measure changes in tRNA modification profiles

This multi-faceted approach would identify specific adaptations in deep-sea TrmB that confer pressure tolerance or optimize activity under high-pressure conditions, providing insights into molecular mechanisms of deep-sea adaptation.

What are the optimal protocols for measuring TrmB enzymatic activity in vitro?

Optimal protocols for measuring TrmB enzymatic activity in vitro include several complementary approaches:

• Radiometric S-adenosylmethionine (SAM) incorporation assay:

  • Reaction components: Purified TrmB, tRNA substrate, [³H-methyl]-SAM, buffer system

  • Procedure: Incubate components at optimal temperature, precipitate tRNA, filter, wash, and measure radioactivity

  • Quantification: Calculate incorporation rates based on radioactive counts

  • Advantages: High sensitivity, direct measurement of methyl transfer

• HPLC-based nucleoside analysis:

  • Sample preparation: Enzymatic reaction followed by tRNA hydrolysis to nucleosides

  • Separation: Reverse-phase HPLC with appropriate column (e.g., C18)

  • Detection: UV absorbance at 254 nm with comparison to nucleoside standards

  • Quantification: Peak area integration for m7G relative to unmodified G

  • Advantages: Direct visualization of modification products

• Mass spectrometry approaches:

  • LC-MS/MS analysis: For precise quantification of modified nucleosides

  • MALDI-TOF analysis: For intact tRNA mass shift detection

  • Advantages: High specificity, ability to identify multiple modifications simultaneously

• Northwestern and northern blot assays:

  • Used to assess m7G modification levels and expression of m7G-modified tRNAs

  • Procedure: Use m7G-specific antibodies for detection of modifications

  • Advantages: Can be applied to complex mixtures of tRNAs

• TRAC-seq (tRNA reduction and cleavage sequencing):

  • Specialized method for identifying m7G-modified tRNAs

  • Advantages: Genome-wide analysis of modifications at specific positions

For optimal results, researchers should consider employing multiple complementary methods to cross-validate findings and obtain comprehensive insights into TrmB activity.

How can researchers establish a genetic manipulation system for P. profundum trmB?

Establishing a genetic manipulation system for P. profundum trmB requires consideration of the organism's specific characteristics:

• Vector selection and design:

  • Use broad-host-range vectors compatible with Vibrio-related species

  • Include appropriate antibiotic resistance markers (e.g., chloramphenicol, kanamycin)

  • Incorporate origin of replication functional in P. profundum

  • Design constructs for allelic exchange or integration

• Transformation methods:

  • Electroporation protocols optimized for marine bacteria

  • Conjugative transfer from E. coli donor strains

  • Natural transformation if applicable

  • Pressure-adaptation of protocols for deep-sea strains

• Selection strategies:

  • Antibiotic concentration optimization for marine media

  • Counter-selection systems (e.g., sacB for sucrose sensitivity)

  • Reporter gene integration for visualization (e.g., GFP, luciferase)

• Gene deletion approach:

  • Design homologous regions flanking trmB

  • Create deletion construct using overlap extension PCR

  • Transform using methods similar to those established for other P. profundum genes

  • Screen for successful deletion using PCR and phenotypic assays

• Complementation strategy:

  • Reintroduce wild-type trmB under native or inducible promoter

  • Create catalytically inactive variant (e.g., by point mutation in active site)

  • Express trmB from different P. profundum strains for comparative studies

This genetic system would enable the creation of defined trmB mutants, allowing for detailed analysis of TrmB function in its native host under various environmental conditions, including high pressure.

What approaches should be used to investigate the impact of trmB deletion on P. profundum phenotypes?

Investigating the impact of trmB deletion on P. profundum phenotypes requires a comprehensive phenotypic analysis:

• Growth and viability assessment:

  • Growth curves under standard conditions (1 atm, optimal temperature)

  • High-pressure growth analysis (10-60 MPa)

  • Temperature sensitivity (4-30°C range)

  • Viable cell counts under various conditions

• Stress response characterization:

  • Oxidative stress tolerance (H₂O₂, paraquat exposure)

  • pH tolerance range

  • Nutrient limitation responses

  • UV radiation sensitivity and photoreactivation

• Translation-related phenotypes:

  • Protein synthesis rates (radioactive amino acid incorporation)

  • Mistranslation frequency (reporter systems)

  • Ribosome profile analysis

  • Proteome composition (mass spectrometry)

• tRNA modification analysis:

  • Global tRNA modification profiling by LC-MS/MS

  • Specific analysis of m7G levels in tRNA

  • tRNA stability and abundance assessment

  • tRNA charging efficiency

• Comparative genomics and transcriptomics:

  • RNA-seq to identify differentially expressed genes

  • Analysis of the transcriptional landscape in wild-type vs. mutant

  • Focus on pressure-responsive gene expression

These analyses would reveal whether TrmB in P. profundum plays roles similar to those identified in other bacteria, such as stress response modulation , while also highlighting any unique functions related to deep-sea adaptation.

How should researchers analyze comparative enzymatic data from TrmB variants across pressure conditions?

Analyzing comparative enzymatic data from TrmB variants across pressure conditions requires specialized approaches:

• Enzyme kinetic parameter analysis:

  • Plot pressure-activity profiles (activity vs. pressure) for each variant

  • Calculate and compare pressure optima for different TrmB variants

  • Determine Km, Vmax, and kcat at each pressure point

  • Apply enzyme kinetic models modified to incorporate pressure effects

• Thermodynamic analysis:

  • Calculate activation volume (ΔV‡) from pressure-dependence of reaction rates

  • Determine volume changes associated with substrate binding

  • Analyze compressibility factors of enzyme-substrate complexes

  • Compare energetic profiles across TrmB variants

• Statistical approaches:

  • Apply non-linear regression models for pressure-dependent data

  • Use ANOVA to identify significant differences between variants

  • Perform principal component analysis to identify patterns in multidimensional data

  • Employ hierarchical clustering to group variants by pressure response profiles

• Visualization and interpretation:

  • Create 3D surface plots of activity as a function of pressure and temperature

  • Overlay structural information with functional data

  • Correlate kinetic parameters with specific amino acid differences between variants

  • Develop predictive models relating sequence features to pressure adaptation

This analytical framework would enable researchers to quantitatively characterize how TrmB variants from different P. profundum strains have adapted to their respective pressure environments and identify the molecular basis for these adaptations.

What insights can be gained from analyzing the codon usage in genes affected by trmB deletion?

Analysis of codon usage in genes affected by trmB deletion can provide significant insights into TrmB's functional role:

• Codon bias analysis in differentially expressed genes:

  • Compare codon usage patterns between up- and down-regulated genes

  • Focus on codons decoded by tRNAs that are targets for TrmB modification

  • Calculate codon adaptation index for each gene set

  • Identify enrichment of specific codons in stress response genes

• Translation efficiency correlation:

  • Analyze polysome profiles to identify mRNAs with altered translation efficiency

  • Examine codon usage in mRNAs with reduced translation efficiency (TE-down mRNAs)

  • Quantify the number of codons decoded by m7G-modified tRNAs in each mRNA

  • Correlate translation changes with codon composition

• Pathway and functional enrichment:

  • Perform gene ontology analysis on genes with high content of affected codons

  • Identify biological processes enriched in transcripts dependent on m7G-modified tRNAs

  • Similar to other systems, examine enrichment in processes like autophagy or mTOR signaling

  • Map affected pathways to known stress response mechanisms

• Compensatory mechanism investigation:

  • Analyze whether alternative tRNAs compensate for reduced modification

  • Examine changes in tRNA gene expression in response to trmB deletion

  • Investigate modifications by other enzymes that might become more prevalent

This codon-centric analysis would reveal how TrmB-mediated tRNA modifications influence translation of specific mRNAs and affect cellular processes, particularly those involved in environmental adaptation and stress response.

How can transcriptomic data be integrated with tRNA modification profiles to understand TrmB function?

Integrating transcriptomic data with tRNA modification profiles requires sophisticated multi-omic approaches:

• Data generation and preprocessing:

  • RNA-seq for gene expression profiling in wild-type and trmB mutant strains

  • tRNA-seq or TRAC-seq for tRNA abundance and modification analysis

  • Ribosome profiling to assess translation efficiency

  • Quality control and normalization of each data type

• Correlation analysis:

  • Map changes in m7G-modified tRNAs to codon usage in the transcriptome

  • Correlate tRNA modification levels with translation efficiency of corresponding codons

  • Identify genes whose expression correlates with modification status of specific tRNAs

  • Analyze temporal dynamics if time-course data is available

• Pathway-level integration:

  • Perform gene set enrichment analysis on genes affected by trmB deletion

  • Map effects to specific cellular pathways and processes

  • Similar to studies in other systems, examine enrichment in processes like autophagy or mTOR signaling

  • Construct regulatory networks linking tRNA modifications to gene expression changes

• Visualization and interpretation:

  • Develop integrative visualizations showing relationships between tRNA modifications and gene expression

  • Create modification-centric pathway maps highlighting affected cellular processes

  • Compare patterns with known stress response mechanisms

  • Identify feedback loops between transcription and translation

This integrated analysis would provide a systems-level understanding of how TrmB-mediated tRNA modifications influence the transcriptome and translatome, particularly under environmental stress conditions relevant to P. profundum's deep-sea habitat.

What potential applications might emerge from studying pressure-adapted TrmB enzymes?

Studying pressure-adapted TrmB enzymes from P. profundum could lead to several innovative applications:

• Biotechnological applications:

  • Development of pressure-stable enzymes for industrial biocatalysis

  • Creation of expression systems for high-pressure protein production

  • Design of pressure-resistant translation systems for synthetic biology

  • Engineering of pressure-adapted microorganisms for deep-sea bioremediation

• Biomedical applications:

  • Similar to proposals for TrmD , TrmB could serve as a novel antimicrobial target

  • Insights into translational control mechanisms under stress conditions

  • Development of therapeutic approaches targeting tRNA modifications

  • Understanding fundamental principles of enzyme adaptation to extreme conditions

• Astrobiology and extremophile research:

  • Models for potential life in high-pressure extraterrestrial environments

  • Understanding fundamental limits of biological systems under extreme conditions

  • Development of biomarkers for detecting life in extreme environments

  • Insights into evolutionary adaptation mechanisms

• Biophysical tools and methodologies:

  • Novel pressure-based methods for studying protein-RNA interactions

  • Development of pressure-stable reagents for molecular biology

  • Improved high-pressure experimental systems for biochemical research

  • Computational models for predicting pressure effects on macromolecular interactions

These applications would leverage the unique adaptations of P. profundum TrmB to expand our technological capabilities in high-pressure environments and deepen our understanding of life's adaptability to extreme conditions.

How might research on P. profundum TrmB contribute to understanding tRNA modification systems across diverse bacterial species?

Research on P. profundum TrmB can significantly advance our understanding of tRNA modification systems across diverse bacterial species:

• Evolutionary insights:

  • Comparative analysis of TrmB across bacteria from different environments

  • Identification of conserved and variable features related to environmental adaptation

  • Reconstruction of evolutionary trajectories of tRNA modification systems

  • Understanding of selection pressures on tRNA modification enzymes

• Structure-function relationships:

  • Correlation of structural adaptations with environmental parameters

  • Identification of critical residues for catalysis vs. environmental adaptation

  • Development of predictive models for enzyme function based on sequence

  • Understanding of how environmental pressures shape enzyme evolution

• Regulatory networks:

  • Comparison of trmB regulation across diverse bacteria

  • Identification of common and species-specific regulatory mechanisms

  • Integration of tRNA modification into broader stress response networks

  • Understanding how modification systems are coordinated across different species

• Methodological advances:

  • Development of improved techniques for studying tRNA modifications

  • Standardization of approaches for comparing modification systems

  • Creation of databases linking tRNA modifications to bacterial adaptations

  • Establishment of model systems representing different environmental niches

By studying TrmB in an extremophilic organism like P. profundum, researchers can identify fundamental principles governing tRNA modification across bacteria while highlighting specific adaptations that enable survival in extreme environments like the deep sea.

What interdisciplinary approaches might enhance our understanding of TrmB's role in bacterial adaptation to extreme environments?

Advancing our understanding of TrmB's role in bacterial adaptation to extreme environments requires interdisciplinary approaches:

• Integration of biophysics and biochemistry:

  • High-pressure structural biology techniques

  • Measurement of thermodynamic parameters under extreme conditions

  • Single-molecule studies of enzyme function under pressure

  • Computational modeling of pressure effects on enzyme-substrate interactions

• Combination of systems biology with ecological perspectives:

  • Metatranscriptomic analysis of deep-sea microbial communities

  • Correlation of tRNA modification patterns with environmental parameters

  • Network analysis of stress response systems across pressure gradients

  • Comparison of adaptation strategies across diverse piezophilic bacteria

• Application of synthetic biology and genetic engineering:

  • Creation of minimally modified organisms with engineered trmB variants

  • Development of biosensors for monitoring translation under pressure

  • Design of pressure-responsive genetic circuits

  • Directed evolution of TrmB under pressure selection

• Integration of oceanography and molecular biology:

  • In situ sampling and preservation methods for deep-sea tRNA analysis

  • Correlation of ocean depth profiles with modification patterns

  • Development of pressure-maintaining sampling technologies

  • Real-time monitoring of translation processes in deep-sea environments

These interdisciplinary approaches would provide a comprehensive understanding of how TrmB contributes to bacterial adaptation to extreme environments, particularly the deep sea, and would establish broadly applicable principles for studying biological adaptation to environmental stressors.