Recombinant Photobacterium profundum tRNA threonylcarbamoyladenosine biosynthesis protein RimN (rimN)

What is the primary function of RimN in P. profundum and how does it differ from homologs in other bacteria?

RimN in P. profundum functions as a tRNA threonylcarbamoyladenosine biosynthesis protein involved in the t6A modification pathway. This universal tRNA modification is found at position 37 of tRNAs decoding ANN codons across all three domains of life . Unlike similar proteins in non-piezophilic bacteria, the P. profundum RimN likely exhibits structural adaptations that maintain functionality under high hydrostatic pressure conditions. The protein participates in a complex enzymatic pathway requiring multiple components including TsaC/TsaC2 (or their orthologs Sua5/Tcs1/Tcs2) and TsaD (or its orthologs Kae1/Tcs3/Qri7) . Experimental approaches to characterize these differences should include comparative structural analysis using X-ray crystallography or cryo-EM under varying pressure conditions, coupled with site-directed mutagenesis to identify pressure-adaptive residues.

How is RimN expression regulated in response to environmental conditions in P. profundum?

RimN expression in P. profundum is likely regulated in response to pressure changes, similar to other proteins involved in adaptation to deep-sea environments. Transcriptomic analysis of P. profundum has revealed complex expression patterns with differential regulation of genes in response to hydrostatic pressure . The transcriptional regulator ToxR, a transmembrane DNA-binding protein whose abundance and activity are influenced by hydrostatic pressure, may be involved in regulating rimN expression . Methodologically, researchers should utilize RNA-seq under varying pressure conditions (0.1 MPa vs. 28 MPa) to quantify rimN transcript levels, complemented by proteomic analysis to correlate transcript abundance with protein expression .

What are the optimal conditions for expressing recombinant P. profundum RimN in heterologous systems?

When expressing recombinant P. profundum RimN, researchers should consider the piezophilic nature of the source organism. Optimal expression conditions should include:

For pressure adaptation studies, expressed protein should be purified and subjected to activity assays under varying pressure conditions using high-pressure bioreactors to determine pressure optima .

What assays can be used to measure RimN enzymatic activity in vitro?

RimN activity can be assessed by monitoring the formation of threonylcarbamoyladenosine (t6A) modified tRNA. A comprehensive methodological approach includes:

• In vitro reconstitution of t6A synthesis using purified recombinant RimN together with other necessary components (TsaC/TsaD or homologs)

• Substrate preparation of unmodified tRNA transcripts via in vitro transcription

• Reaction monitoring via:

  • Reversed-phase nano liquid chromatography mass spectrometry of ribonucleosides after tRNA hydrolysis, similar to methods used for detecting t6A modifications in Candida albicans

  • Thin-layer chromatography of 32P-labeled tRNA fragments

  • HPLC analysis of nucleosides after complete digestion of tRNA substrates

Activity assays should be performed under varying pressure conditions (0.1-70 MPa) to characterize pressure-dependent enzyme kinetics, providing insights into adaptive mechanisms for deep-sea function .

How does the absence of t6A modification affect translation efficiency in bacteria under different environmental conditions?

The absence of t6A modification has significant effects on translation efficiency, particularly under stress conditions. Research indicates t6A serves as a strong positive determinant for aminoacylation of tRNA by bacterial-type isoleucyl-tRNA synthetases . Without t6A, bacteria experience:

• Decreased translation fidelity, particularly at ANN codons

• Impaired recognition of start codons, affecting translation initiation

• Potential activation of proteotoxic stress responses, as observed in t6A-deficient D. radiodurans strains

Under high-pressure conditions characteristic of P. profundum's native environment, these effects may be magnified. Ribosome profiling of t6A-deficient P. profundum strains would reveal codon-specific translational pauses and can be correlated with pressure conditions. Quantitative proteomics comparing wild-type and t6A-deficient strains grown at different pressures would identify proteins most affected by this modification .

What is the relationship between RimN function and bacterial stress responses, particularly in deep-sea environments?

RimN function and t6A modification likely play critical roles in bacterial adaptation to deep-sea environmental stresses. In P. profundum, several stress response genes including htpG, dnaK, dnaJ, and groEL are upregulated in response to atmospheric pressure , suggesting a connection between pressure stress and translational regulation.

A methodological approach to investigate this relationship would include:

• Construction of conditional rimN mutants in P. profundum using techniques similar to those employed for recD mutations

• Transcriptomic and proteomic profiling of these mutants under varying pressure conditions

• Analysis of growth, survival, and morphological characteristics under combined stresses (pressure, temperature, UV exposure)

• Assessment of translational fidelity using reporter constructs with programmed frameshifting or stop codon readthrough elements

Research indicates that t6A modification might be particularly important for accurate translation under stress conditions, potentially explaining the essentiality of t6A synthetic genes in most prokaryotes despite being dispensable in some species .

How do strains of P. profundum differ in their pressure adaptation, and what role might RimN play in these differences?

Different P. profundum strains exhibit distinct pressure adaptation profiles:

These differences in pressure adaptation may be partially attributed to variations in tRNA modification systems, including RimN function. Research methodology to investigate RimN's role should include:

• Comparative genomics and sequence analysis of rimN genes across P. profundum strains

• Heterologous expression of rimN variants from different strains and characterization of their enzymatic properties under varying pressures

• Construction of strain hybrids through allelic exchange of rimN genes between strains with different pressure optima

• Transcriptome and proteome analysis of these strains at different pressures to identify pressure-responsive pathways

The piezophilic phenotype of P. profundum is growth condition dependent, with more pronounced piezophily in minimal medium , suggesting that nutrient availability interacts with translation efficiency mediated by tRNA modifications.

What experimental approaches are most effective for studying the function of RimN under high hydrostatic pressure conditions?

Studying RimN function under high pressure requires specialized approaches:

• High-pressure bioreactors for bacterial cultivation under controlled pressure conditions (0.1-70 MPa)

• High-pressure spectroscopy systems for real-time monitoring of enzymatic reactions

• Pressure-resistant reaction chambers for in vitro biochemical assays

Methodology should include:

• Pressure-shift experiments to analyze rapid transcriptomic and proteomic responses to pressure changes

• In vivo reporter systems to monitor translation efficiency and accuracy under pressure

• High-pressure structural biology techniques (HP-NMR, HP-XRD) to characterize conformational changes

• Complementation assays using recombinant RimN variants in P. profundum pressure-sensitive mutants

These approaches will help determine if RimN from P. profundum possesses unique structural or kinetic properties compared to homologs from non-piezophilic organisms, potentially explaining its adaptation to high-pressure environments .

How conserved is RimN across piezophilic bacteria, and what can this tell us about the evolution of deep-sea adaptation?

RimN conservation across piezophilic bacteria provides insights into the evolution of deep-sea adaptation mechanisms. A comprehensive analysis would involve:

• Phylogenetic analysis of RimN sequences from diverse piezophilic and non-piezophilic bacteria

• Identification of selective pressure signatures in the rimN gene sequence using dN/dS analysis

• Structural modeling to identify conserved domains specific to piezophilic variants

• Correlation of sequence variations with depth and pressure adaptation across species

While specific data on RimN conservation in piezophiles is limited, comparative genomic studies of Photobacterium strains have revealed high genomic diversity within the genus . The genus Photobacterium contains both piezophilic and non-piezophilic members, making it an excellent model for studying the evolution of pressure adaptation. Analysis should examine whether rimN was acquired through horizontal gene transfer, similar to other genes in P. profundum that show evidence of HGT .

What insights can comparing RimN from P. profundum with homologs in other extremophiles provide about adaptation to extreme environments?

Comparing RimN from P. profundum with homologs in other extremophiles can reveal convergent or divergent evolution strategies for maintaining translation fidelity under extreme conditions. Methodological approaches should include:

• Comparative biochemical characterization of RimN from:

  • Piezophiles (P. profundum)

  • Psychrophiles (e.g., polar bacteria)

  • Thermophiles (e.g., Thermus thermophilus)

  • Halophiles (e.g., Halobacterium species)

• Analysis of amino acid composition and structural features correlated with specific environmental adaptations

• Functional complementation assays where rimN genes from different extremophiles are expressed in P. profundum rimN mutants, followed by growth assessment under varying pressure conditions

This research would reveal whether adaptations in tRNA modification systems represent a common strategy for extremophiles or if different extreme environments select for distinct adaptive mechanisms in these pathways .

How does the ribosomal occupancy profile change in P. profundum when RimN function is compromised?

Ribosomal occupancy profiles in P. profundum with compromised RimN function would likely show significant changes, particularly at ANN codons. Similar to observations in Candida albicans where deletion of the tRNA modification enzyme Hma1 altered ribosome occupancy at 37°C , P. profundum would likely show pressure-dependent translation effects.

Methodological approach:

• Generate conditional rimN mutants in P. profundum

• Perform ribosome profiling (Ribo-seq) under varying pressure conditions

• Analyze codon-specific translation pauses, particularly at ANN codons

• Correlate with quantitative proteomics to identify most affected proteins

Expected outcomes would include increased ribosomal pausing at ANN codons, particularly under high pressure conditions, and potential accumulation of mistranslated proteins leading to stress response activation. This would be similar to the proteotoxic stress response observed in t6A-deficient strains , but potentially magnified under pressure stress.

What is the relationship between tRNA modifications and CRISPR-Cas systems in P. profundum?

The relationship between tRNA modifications and CRISPR-Cas systems in P. profundum represents an unexplored frontier. Photobacterium strains contain diverse CRISPR-Cas architectures, with P. profundum SS9 containing two clusters similar to those in Y. pestis and E. coli, encoded in the chromosome and plasmid respectively .

A methodological investigation would include:

• Transcriptomic analysis comparing expression of rimN and CRISPR-Cas components under varying pressure conditions

• Functional analysis of CRISPR-Cas activity in rimN-deficient strains

• Assessment of tRNA modification profiles in CRISPR-Cas mutants

• Testing whether stress responses from compromised tRNA modification affect CRISPR-Cas expression or activity

This research could reveal novel connections between translational fidelity mechanisms and bacterial immune systems, potentially identifying pressure-dependent regulatory networks that coordinate these systems in deep-sea environments .

What are the critical considerations for designing knockout or knockdown experiments targeting RimN in P. profundum?

Designing genetic manipulation experiments targeting RimN in P. profundum requires careful consideration of several factors:

• Essentiality assessment: Based on studies in other bacteria, t6A pathway genes are essential in most prokaryotes . Therefore, conditional knockout systems may be necessary.

• Recommended approach:

  • Use gene disruption methods similar to those employed for recD mutations , where internal portions of the target gene are amplified by PCR and cloned into suitable vectors

  • Consider temperature-sensitive or pressure-sensitive conditional systems

  • Employ inducible antisense RNA approaches for knockdown experiments

• Controls and validation:

  • Confirm mutants by PCR, sequencing, and RT-PCR

  • Verify changes in t6A modification levels using LC-MS analysis of tRNA

  • Include complementation with wild-type rimN to confirm phenotype specificity

• Special considerations:

  • Growth under different pressure conditions may affect transformation efficiency

  • Use pressure-resistant antibiotics for selection

  • Consider the multi-chromosomal nature of P. profundum (two circular chromosomes)

What analytical techniques are most appropriate for characterizing tRNA modifications in P. profundum cultured under different pressure conditions?

Analysis of tRNA modifications in P. profundum under different pressure conditions requires specialized approaches:

• tRNA isolation protocol:

  • Total RNA isolation using acidic phenol-TRIzol extraction

  • tRNA purification by gel extraction from denaturing 8 M urea 8% polyacrylamide gels

  • Maintain samples on ice during processing to prevent hydrolysis of labile modifications

• Comprehensive modification analysis:

  • Reversed-phase nano liquid chromatography mass spectrometry of ribonucleosides after complete tRNA hydrolysis

  • Targeted analysis of t6A using selective reaction monitoring mass spectrometry

  • Northern blot analysis with modification-specific probes

• Quantitative comparison methodology:

  • Include internal standards for accurate quantification

  • Prepare multiple technical replicates for each biological sample

  • Normalize to total tRNA amount and unmodified nucleosides

• Pressure effect considerations:

  • Compare samples from cultures grown at atmospheric pressure (0.1 MPa) versus high pressure (10-28 MPa)

  • Analyze samples from different growth phases to detect temporal changes

  • Consider temperature interactions, as P. profundum shows different temperature optima at different pressures