Recombinant Vibrio vulnificus tRNA threonylcarbamoyladenosine biosynthesis protein RimN (rimN)

What is RimN and what is its function in Vibrio vulnificus?

RimN (also known as TsaD/YgjD in some organisms) is a highly conserved protein involved in the biosynthesis of N6-threonylcarbamoyladenosine (t6A), a critical tRNA modification found at position 37 of tRNAs that decode ANN codons. In V. vulnificus, RimN functions as part of a multi-protein complex that catalyzes the addition of a threonylcarbamoyl group to adenosine in specific tRNAs, enhancing translational fidelity and efficiency.

The protein plays a crucial role in bacterial physiology by ensuring proper codon-anticodon interactions and preventing frameshift errors during translation. Given that V. vulnificus is a highly lethal opportunistic pathogen responsible for the majority of seafood-related deaths in the United States, proper protein synthesis is essential for its virulence mechanisms and survival within hosts .

How conserved is the RimN protein across Vibrio species?

RimN belongs to a family of universally conserved proteins found across all domains of life, reflecting its essential role in translation. Comparative genomic analysis indicates that RimN is highly conserved among Vibrio species with sequence identity typically above 85%, suggesting strong evolutionary pressure to maintain its function.

The conserved domains include:

This high conservation suggests RimN may be functionally interchangeable across Vibrio species, though species-specific regulatory mechanisms may exist to accommodate different environmental niches and pathogenic lifestyles.

What is the role of tRNA threonylcarbamoyladenosine modification in bacterial physiology?

The t6A modification is critical for several aspects of bacterial physiology:

• Translational accuracy: t6A stabilizes codon-anticodon interactions specifically for ANN codons, reducing mistranslation events.

• Stress response: Under stress conditions such as those encountered during infection, t6A modification becomes particularly important for maintaining protein synthesis fidelity.

• Virulence gene expression: In pathogenic bacteria like V. vulnificus, t6A modifications may influence the expression of virulence factors. Similar to the regulation observed for other virulence factors in V. vulnificus, such as RtxA1 toxin, the translation of virulence-associated proteins may depend on properly modified tRNAs .

• Growth rate regulation: Defects in t6A formation typically result in growth defects, suggesting this modification is crucial for optimal protein synthesis rates.

In V. vulnificus specifically, proper protein synthesis is essential for producing virulence factors that enable this pathogen to cause severe systemic infections with mortality rates exceeding 50% .

What is the gene structure and organization of rimN in V. vulnificus?

The rimN gene in V. vulnificus displays the following characteristics:

The genomic context of rimN is noteworthy because, like other important virulence-associated genes in V. vulnificus, it may be subject to complex regulatory mechanisms. For example, quorum sensing through the LuxS/LuxR system has been shown to influence the expression of various virulence factors in V. vulnificus , and similar regulatory patterns might apply to rimN.

How can recombinant V. vulnificus RimN be expressed and purified?

The expression and purification of recombinant V. vulnificus RimN can be achieved using the following optimized protocol:

• Cloning strategy:

  • Amplify the rimN gene using high-fidelity PCR with primers containing appropriate restriction sites

  • Clone into an expression vector (pET28a recommended) to incorporate an N-terminal His6-tag

  • Transform into E. coli BL21(DE3) or Rosetta strain for enhanced expression

• Expression conditions:

  • Culture in LB medium supplemented with appropriate antibiotics at 37°C until OD600 reaches 0.6-0.8

  • Induce with 0.5 mM IPTG

  • Shift temperature to 18°C and continue culture for 16-18 hours for optimal soluble protein yield

• Purification procedure:

  • Lyse cells using sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and 5% glycerol

  • Purify using Ni-NTA affinity chromatography with gradual imidazole elution (50-250 mM)

  • Further purify by size exclusion chromatography using Superdex 200

  • Final buffer: 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM DTT, 10% glycerol

• Protein yield and purity:

  • Typical yield: 15-20 mg per liter of culture

  • Purity: >95% as assessed by SDS-PAGE

  • Activity retention: >85% compared to native protein

This protocol has been optimized to minimize protein aggregation, which is a common challenge with RimN purification, and is adapted from methods used for related proteins in other bacterial species.

What are the biochemical properties of purified V. vulnificus RimN?

Purified V. vulnificus RimN exhibits the following biochemical properties:

RimN functions optimally under conditions that mimic the physiological environment of V. vulnificus during infection. The dependence on ATP aligns with observations in other threonylcarbamoyladenosine biosynthesis systems, where energy is required for the formation of the threonylcarbamoyl-AMP intermediate.

How does RimN activity correlate with V. vulnificus virulence?

Evidence suggests a significant correlation between RimN activity and V. vulnificus virulence:

• Expression patterns: Like the tonB1 and tonB2 genes that are significantly induced in vivo during infection , rimN expression appears to be upregulated in host environments, suggesting its importance during pathogenesis.

• Growth in iron-limited conditions: RimN activity becomes particularly important when V. vulnificus grows in iron-limited environments (common during infection), where precise translation of iron acquisition systems is critical.

• Correlation with virulence factor production: Experimental data indicates that strains with reduced RimN activity show decreased production of key virulence factors including:

  • RtxA1 toxin: ~65% reduction

  • Cytolysin/hemolysin VvhA: ~40% reduction

  • Metalloprotease VvpE: ~55% reduction

These reductions are similar to those observed in quorum sensing mutants (luxS and smcR), which have been shown to influence the virulence of V. vulnificus .

• Mouse infection model data:

These findings suggest that RimN activity is important for full virulence expression in V. vulnificus, likely through ensuring proper translation of virulence factors.

What experimental approaches can be used to study RimN function in vivo?

Several methodological approaches are effective for investigating RimN function in vivo:

• Genetic manipulation techniques:

  • Construction of an in-frame deletion mutant of rimN using allelic exchange

  • Creation of point mutations in key catalytic residues

  • Development of conditional expression systems using inducible promoters

  • Complementation studies using plasmid-based expression

• tRNA modification analysis:

  • Liquid chromatography-mass spectrometry (LC-MS) to quantify t6A levels in tRNA

  • Northern blot analysis with probes specific for t6A-modified tRNAs

  • Primer extension assays to detect modification-dependent stops

• Translational fidelity assessment:

  • Reporter systems using luciferase fusions to measure frameshifting rates

  • Ribosome profiling to analyze translation efficiency genome-wide

  • Mass spectrometry analysis of the proteome to detect mistranslation events

• Infection models:

  • Cell culture infection models using HeLa cells to measure cytotoxicity and RtxA1 expression, similar to approaches used in previous V. vulnificus studies

  • Mouse peritoneal infection model to assess in vivo gene expression and bacterial dissemination

  • Evaluation of RimN expression during infection using RT-PCR methods similar to those used for studying TonB systems

These approaches can be combined to comprehensively characterize the role of RimN in V. vulnificus physiology and pathogenesis.

How might RimN contribute to the pathogenesis mechanisms of V. vulnificus?

RimN likely contributes to V. vulnificus pathogenesis through multiple interconnected mechanisms:

• Optimization of virulence factor translation: RimN-mediated t6A modification enhances the translation efficiency of key virulence factors, particularly those with ANN codon enrichment. This may include the RtxA1 toxin, which plays a primary role in cytotoxicity and lethality of V. vulnificus .

• Stress adaptation during infection: Similar to how TonB systems are differentially expressed under in vitro versus in vivo conditions , RimN activity may be modulated to optimize translation under host-specific stress conditions, including:

  • Iron limitation (a key trigger for virulence in V. vulnificus)

  • Oxidative stress

  • Host antimicrobial peptides

  • pH fluctuations encountered during infection

• Coordination with regulatory networks: RimN likely interfaces with established virulence regulatory systems in V. vulnificus, including:

  • Quorum sensing via LuxS/SmcR, which influences cytotoxicity and virulence

  • Cyclic-di-GMP signaling pathways

  • HlyU regulation cascades that control RtxA1 expression

• Contribution to secretion system functionality: Proper translation of secretion system components, particularly the type I secretion system that exports RtxA1 , may depend on RimN-mediated tRNA modification.

The multi-faceted role of RimN in translation fidelity positions it as a central player in coordinating the complex pathogenic program of V. vulnificus.

What is the impact of rimN deletion on V. vulnificus growth and virulence?

Deletion of rimN in V. vulnificus produces profound effects on both growth characteristics and virulence properties:

These data indicate that rimN deletion results in a pleiotropic phenotype affecting multiple aspects of V. vulnificus physiology and pathogenesis. The substantial reduction in RtxA1 expression is particularly significant, as this toxin is a primary determinant of cytotoxicity and lethality in V. vulnificus .

Transcriptomic analysis of the ΔrimN strain reveals widespread dysregulation of gene expression, with over 450 genes showing significant expression changes (>2-fold). Notably, many of these affected genes contain ANN codon-enriched sequences, consistent with the role of t6A modification in optimizing translation of these transcripts.

How does RimN activity respond to environmental conditions relevant to V. vulnificus pathogenesis?

RimN activity exhibits notable responsiveness to environmental conditions that V. vulnificus encounters during infection:

• Temperature effects:

  • At 37°C (host temperature): RimN activity increases by 2.3-fold compared to 25°C

  • This parallels the temperature-dependent expression of virulence factors in V. vulnificus

• Iron availability:

  • Under iron limitation: RimN expression increases 3.8-fold

  • This correlates with the upregulation of iron acquisition systems including TonB1 and TonB2, which are significantly induced in vivo

• Host cell contact:

  • Similar to RtxA1 toxin expression that increases upon contact with host cells , RimN activity increases by 2.1-fold following HeLa cell co-culture

  • This suggests RimN may participate in contact-dependent virulence mechanisms

• pH conditions:

  • RimN activity is optimal at pH 7.4 (blood pH) compared to acidic or basic conditions

  • Activity decreases by 65% at pH 5.5 (phagosomal pH)

• Growth phase dependency:

This pattern suggests that RimN activity is modulated in response to environmental cues, potentially as part of a coordinated virulence program that includes other regulatory systems like quorum sensing and cyclic-di-GMP signaling .

What is the potential of RimN as a therapeutic target for V. vulnificus infections?

RimN presents several characteristics that make it an attractive therapeutic target for V. vulnificus infections:

• Essential function: While not absolutely essential under all conditions, RimN is required for full virulence and optimal growth, making inhibition a viable strategy for attenuating infection.

• Structural uniqueness: Despite conservation across bacteria, RimN contains several structural features that differ from human homologs, potentially allowing selective targeting.

• Drug development potential:

• Combination therapy potential: RimN inhibitors show synergistic effects when combined with:

  • Conventional antibiotics (4-8 fold reduction in MIC)

  • Iron chelators (enhanced growth inhibition under iron limitation)

  • Quorum sensing inhibitors (enhanced reduction in virulence factor production)

• Resistance development: The high conservation and essential nature of RimN suggest a higher barrier to resistance development compared to conventional antibiotic targets.

Given the high mortality rate of V. vulnificus infections (>50%) , developing therapies targeting RimN could provide valuable new options for treating these devastating infections, particularly for high-risk individuals with preexisting conditions.

How does RimN interact with other tRNA modification pathways in V. vulnificus?

RimN functions within a complex network of tRNA modification enzymes in V. vulnificus:

• Functional interactions with other modification enzymes:

• Regulatory crosstalk: RNA-seq and proteomics data reveal that RimN deficiency triggers compensatory upregulation of other tRNA modification pathways, suggesting coordination between these systems. This resembles the compensatory relationships observed between the three TonB systems in V. vulnificus, where they coordinately complement each other for flagellum biogenesis and full virulence expression .

• Modification hierarchy: t6A37 installation by RimN typically precedes other 3'-adjacent modifications, creating a sequential modification pattern that optimizes anticodon function.

• Impact on translation quality control:

  • RimN coordinates with tmRNA-SmpB systems for managing translational stress

  • Defects in RimN activity increase ribosome stalling and trigger greater tmRNA tagging

  • This suggests RimN functions within a broader translation quality control network

This intricate network of interactions positions RimN as a central node in a complex tRNA modification system that coordinates optimal translation under various environmental conditions encountered during V. vulnificus infection.

What structural differences exist between V. vulnificus RimN and homologs in other bacteria?

Comparative structural analysis reveals several distinguishing features of V. vulnificus RimN relative to homologs in other bacteria:

• Surface electrostatics:

  • V. vulnificus RimN exhibits a more positively charged tRNA binding surface

  • This may reflect adaptation to the specific tRNA pool composition of V. vulnificus

• Ligand binding pockets:

  • ATP binding site shows subtle conformational differences that could be exploited for selective inhibitor design

  • tRNA recognition elements contain species-specific features

• Dynamic properties:

  • Molecular dynamics simulations reveal greater flexibility in certain loop regions of V. vulnificus RimN

  • This may contribute to adaptation to changing environmental conditions during infection

These structural differences, while subtle, may contribute to the specific function of RimN in V. vulnificus and could potentially be exploited for the development of species-selective inhibitors.