Recombinant Protochlamydia amoebophila tRNA (guanine-N (7)-)-methyltransferase (trmB)

What is tRNA (guanine-N(7))-methyltransferase (TrmB) and what is its primary biochemical function?

TrmB is an enzyme that specifically catalyzes the formation of m7G46 (7-methylguanosine) modification in the tRNA variable loop. This post-transcriptional modification plays a critical role in maintaining tRNA structural integrity and translational efficiency. The enzyme functions by transferring a methyl group from S-adenosylmethionine (SAM) to the N7 position of guanosine at position 46 in specific tRNA substrates .

TrmB belongs to the family of SAM-dependent methyltransferases and has the EC designation 2.1.1.33. In Protochlamydia amoebophila strain UWE25, the protein consists of 225 amino acids and is involved in crucial cellular processes related to translation regulation .

What are the recommended protocols for expression and purification of recombinant Protochlamydia amoebophila TrmB?

The recombinant protein can be produced in E. coli expression systems. According to product specifications, the protein achieves >85% purity using SDS-PAGE analysis . For optimal results, researchers should:

• Express the full-length protein (amino acids 1-225) in E. coli

• Purify using affinity chromatography (specific tag determined during manufacturing)

• Store the purified protein with 5-50% glycerol at -20°C/-80°C to maintain stability

• Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Avoid repeated freeze-thaw cycles; working aliquots can be stored at 4°C for up to one week

The expected shelf life is approximately 6 months for liquid preparations stored at -20°C/-80°C and 12 months for lyophilized forms .

How can researchers assess the methyltransferase activity and substrate specificity of TrmB?

Several complementary approaches can be used to evaluate TrmB activity:

• Radiolabel-based methyltransferase assays: Using S-[methyl-14C]-adenosyl methionine (14C-SAM) as methyl donor to detect transfer to tRNA substrates. This method directly measures catalytic activity .

• Electrophoretic mobility shift assays (EMSAs): To evaluate tRNA binding affinity. Researchers can use full-length human tRNAPhe or bacterial tRNAs as substrates to assess binding properties .

• Mass spectrometry: For detailed analysis of tRNA modification profiles before and after TrmB treatment, particularly using comparative NAIL-MS (Nucleic Acid Isotope Labeling coupled with Mass Spectrometry) .

• In vivo complementation assays: ΔtrmB mutant strains show distinct phenotypes that can be complemented by functional TrmB. Successful complementation indicates activity .

When comparing wild-type and mutant enzymes, researchers should assess both tRNA binding capacity and methyltransferase activity, as these can be differentially affected by structural changes .

What role does TrmB play in bacterial oxidative stress responses, and how can this be experimentally validated?

TrmB plays a crucial role in bacterial responses to oxidative stress, particularly in pathogenic bacteria:

In Pseudomonas aeruginosa, TrmB has been shown to mediate translational responses to H2O2. The loss of trmB resulted in strong negative effects on the translation of Phe- and Asp-enriched mRNAs, including catalase genes katA and katB that are critical for detoxifying hydrogen peroxide .

In Acinetobacter baumannii, a ΔtrmB mutant showed extreme sensitivity to oxidative stress, with almost complete killing observed after treatment with 5 mM H2O2, while other tRNA methyltransferase mutants behaved similarly to wild-type bacteria .

Experimental validation approaches:

These experimental approaches can help elucidate the specific mechanisms by which TrmB contributes to oxidative stress responses in different bacterial species .

How does TrmB function contribute to bacterial adaptation to low pH environments?

Research on Acinetobacter baumannii has demonstrated that TrmB plays an important role in adaptation to acidic environments. A ΔtrmB mutant displayed significant growth defects at low pH compared to wild-type bacteria. This finding suggests that TrmB-mediated tRNA modification is critical for maintaining efficient translation under acidic stress conditions .

The precise mechanism involves post-transcriptional regulation of stress response genes. When bacteria encounter acidic environments, proper tRNA modification by TrmB ensures optimal translation of proteins required for acid stress responses. Without these modifications, translation efficiency is compromised, particularly for certain codon-biased stress response genes .

To investigate this phenomenon, researchers can:

• Compare growth rates of wild-type and ΔtrmB strains in media adjusted to different pH values

• Measure intracellular pH homeostasis capabilities

• Assess expression of acid stress response genes at both transcriptional and translational levels

• Analyze codon usage bias in genes affected by low pH stress

How does TrmB contribute to bacterial virulence in infection models?

TrmB has been identified as a critical factor for bacterial pathogenesis, particularly in Acinetobacter baumannii:

In vitro infection models:
The ΔtrmB mutant of A. baumannii was unable to replicate inside J774A.1 macrophages, while wild-type bacteria doubled in number by 4 hours post-infection. Confocal microscopy revealed fewer bacteria per A. baumannii-containing vacuole (ACV) in the ΔtrmB mutant compared to wild-type and complemented strains. This suggests that while the ΔtrmB mutant could be phagocytosed at similar rates to wild-type bacteria, it failed to replicate within the vacuole, likely due to its inability to respond to stresses imposed by macrophages (oxidative stress and low pH) .

In vivo infection models:
The ΔtrmB mutant showed dramatically attenuated virulence in an acute pneumonia murine model. This substantial reduction in pathogenicity demonstrates that TrmB-mediated stress responses are crucial for successful infection in a mammalian host .

These findings highlight TrmB as a potential therapeutic target for combating multidrug-resistant bacterial infections, particularly in light of the current efforts to use another tRNA methyltransferase, TrmD, as an antimicrobial target .

What is the relationship between TrmB activity and iron acquisition during infection?

A fascinating connection has been established between TrmB activity and iron acquisition systems in Acinetobacter baumannii:

Proteomic analysis revealed that under oxidative stress, wild-type A. baumannii significantly upregulates a siderophore biosynthesis and uptake cluster called acinetobactin. In contrast, the ΔtrmB mutant showed only modest upregulation of these acinetobactin proteins, which impaired its ability to withstand iron deprivation under oxidative stress conditions .

Further analysis using qRT-PCR demonstrated that this regulation occurs at the post-transcriptional level, implying that TrmB-mediated tRNA modifications are critical for efficient translation of iron acquisition proteins .

This relationship is particularly significant because:

• Iron acquisition is essential for bacterial survival during infection

• Host defense mechanisms often restrict iron availability as an antimicrobial strategy

• Iron is required for various cellular processes, including detoxification of reactive oxygen species

These findings suggest that TrmB coordinates bacterial responses to multiple stressors encountered during infection, linking oxidative stress resistance with iron acquisition capabilities through translational regulation .

What substrate recognition mechanisms does TrmB employ, and how do they impact methyltransferase activity?

TrmB recognizes specific tRNA substrates through a combination of sequence and structural elements. In Pseudomonas aeruginosa, TrmB has been shown to recognize and modify 23 tRNA substrates containing a guanosine residue at position 46, including 11 novel tRNA substrates identified through experimental validation .

The substrate recognition involves:

The impact of substrate recognition on methyltransferase activity is significant. Studies in P. aeruginosa demonstrated that loss of TrmB had a strong negative effect specifically on the translation of Phe- and Asp-enriched mRNAs, suggesting that TrmB-mediated m7G modification particularly affects the function of certain tRNA isoacceptors .

How does the m7G46 modification installed by TrmB influence tRNA structure and function?

The m7G46 modification occurs in the variable loop of tRNA and significantly impacts tRNA function through several mechanisms:

These functional consequences highlight the importance of TrmB-mediated m7G46 modification in bacterial physiology and pathogenesis .

How do TrmB functions differ between bacterial species, and what experimental approaches can reveal these differences?

TrmB functions show both conservation and species-specific variations across different bacteria:

Observed differences:

Experimental approaches to reveal species-specific differences:

• Comparative genomics and phylogenetics: Analyze TrmB sequence conservation and divergence across species.

• Cross-species complementation assays: Test whether TrmB from one species can functionally complement ΔtrmB mutants of another species.

• Substrate specificity profiling: Compare the range of tRNA substrates modified by TrmB orthologs using mass spectrometry.

• Stress response phenotyping: Systematically compare ΔtrmB mutant phenotypes across species under various stress conditions.

• Structural biology approaches: Compare protein structures to identify species-specific features that might explain functional differences.

These approaches can help elucidate how TrmB functions have evolved to meet the specific physiological needs of different bacterial species .

What insights from pathogenic bacterial TrmB research might apply to understanding Protochlamydia amoebophila TrmB function?

Research on TrmB in pathogenic bacteria like Acinetobacter baumannii and Pseudomonas aeruginosa provides valuable insights that may inform understanding of Protochlamydia amoebophila TrmB:

• Stress response mechanisms: Like pathogenic bacteria, Protochlamydia exists within host cells (amoebae) and likely faces similar stresses including oxidative stress and pH fluctuations. The P. amoebophila TrmB may similarly regulate stress response proteins through tRNA modification .

• Host-pathogen interactions: Protochlamydia is an amoebal endosymbiont, and research has shown that it can induce apoptosis in human immortal HEp-2 cells but not in primary peripheral blood mononuclear cells (PBMCs). This host-specific effect might involve translational regulation mechanisms potentially mediated by TrmB .

• Metabolic adaptation: Studies on Protochlamydia have revealed respiratory activity and D-glucose utilization in elementary bodies (EBs), the infectious stage. TrmB-mediated translational regulation might be important for maintaining metabolic capabilities during different developmental stages .

• Environmental persistence: The ability of Protochlamydia to survive in diverse environments may depend on translational adaptations similar to those observed in pathogenic bacteria, potentially involving TrmB .

• Evolutionary conservation: The conservation of TrmB across diverse bacterial species suggests fundamental importance in bacterial physiology, including in Protochlamydia, despite its unique evolutionary position as an amoebal endosymbiont .

Research methods established for pathogenic bacterial TrmB can be adapted to investigate these aspects in Protochlamydia amoebophila, providing insights into the biological significance of TrmB in this organism's lifecycle and host interactions .

What therapeutic potential does targeting TrmB hold for combating multidrug-resistant bacterial infections?

TrmB represents a promising antimicrobial target for several compelling reasons:

• Essential for virulence: TrmB is critical for bacterial adaptation to host-imposed stresses. In A. baumannii, loss of TrmB dramatically attenuates virulence in murine pneumonia models .

• Conservation across pathogens: TrmB is conserved across many bacterial pathogens, suggesting potential broad-spectrum applications.

• Limited redundancy: Unlike many virulence factors, the specific tRNA modification catalyzed by TrmB cannot be readily compensated by other enzymes.

• Precedent from related targets: Another tRNA methyltransferase, TrmD, is already being explored as an antimicrobial therapeutic target, providing proof-of-concept for this approach .

• Limited off-target effects: The bacterial TrmB has sufficient structural differences from human tRNA methyltransferases to potentially allow selective targeting.

Researchers interested in developing TrmB inhibitors should consider:

• Structure-based drug design targeting the SAM-binding pocket

• High-throughput screening approaches to identify lead compounds

• Rational modification of known methyltransferase inhibitors

• Evaluation of inhibitors against multidrug-resistant clinical isolates

• Assessment of resistance development potential

The development of TrmB inhibitors could provide a novel strategy to combat multidrug-resistant A. baumannii, which represents a major global health threat .

How might CRISPR-Cas systems be utilized to study TrmB function in bacterial species that have been historically difficult to genetically manipulate?

CRISPR-Cas systems offer powerful approaches for studying TrmB in genetically recalcitrant bacteria:

• Precise gene editing: CRISPR-Cas9 or Cas12a can create targeted mutations or deletions in trmB genes, allowing functional studies even in bacteria that lack established genetic tools.

• CRISPRi for conditional knockdowns: For essential genes like trmD, or potentially trmB in some contexts, CRISPR interference (CRISPRi) using catalytically inactive Cas9 (dCas9) can provide tunable repression without complete gene deletion.

• Base editors for point mutations: CRISPR base editors can introduce specific point mutations to study structure-function relationships in TrmB without requiring homologous recombination.

• Transcriptional activation: CRISPR activation systems (CRISPRa) could be used to upregulate trmB expression to study the effects of enhanced tRNA modification.

• Pooled screenings: CRISPR screens targeting trmB and related genes could identify genetic interactions and pathways connected to tRNA modification systems.

For organisms like Chlamydia, which historically have been challenging to genetically manipulate, CRISPR technologies build upon recent advances in transformation technologies. As noted in search result #8, researchers have already achieved targeted gene disruption in Chlamydia using methods like the TargeTron approach and Himar1-based transposition .

CRISPR-based approaches could be particularly valuable for studying TrmB in organisms like Protochlamydia amoebophila, where genetic tools are less developed compared to model organisms .

What computational approaches can predict tRNA substrates and context-dependent activities of TrmB across different bacterial species?

Advanced computational approaches can enhance our understanding of TrmB substrate specificity and functional context:

• Machine learning prediction models:

  • Train models on known TrmB substrates to predict new substrates

  • Features can include tRNA sequence motifs, secondary structure elements, and tertiary interactions

  • Incorporate evolutionary conservation data across bacterial species

• Molecular dynamics simulations:

  • Model TrmB-tRNA interactions to understand recognition mechanisms

  • Simulate effects of m7G46 modification on tRNA structural dynamics

  • Predict how environmental factors (pH, ionic strength) affect enzyme-substrate interactions

• Systems biology approaches:

  • Integration of transcriptomics, proteomics, and tRNA modification profiling

  • Network analysis to identify genes co-regulated with TrmB under different stress conditions

  • Predict context-dependent roles of TrmB modification in translational regulation

• Comparative genomics:

  • Analyze codon usage patterns across bacterial genomes in relation to predicted TrmB activity

  • Identify bacteria with potentially distinct TrmB functions based on genomic features

  • Study correlation between TrmB sequence divergence and ecological niches

• Structural prediction tools:

  • Use tools like AlphaFold2 to predict species-specific TrmB structures

  • Compare substrate binding pockets across species

  • Model how bacterial adaptations might affect substrate specificity

These computational approaches can guide experimental work by generating testable hypotheses about TrmB function across bacterial species and environmental contexts .

What are the primary technical challenges in expressing and purifying functional recombinant TrmB, and how can they be addressed?

Researchers face several challenges when working with recombinant TrmB proteins:

Challenge 1: Protein solubility

• TrmB may form inclusion bodies when overexpressed in E. coli

• Solution: Optimize expression conditions by using lower IPTG concentrations and lower temperatures (16-18°C). Consider fusion tags such as MBP or SUMO that enhance solubility. Alternative expression hosts like Arctic Express strains containing cold-adapted chaperones may improve folding.

Challenge 2: Maintaining enzymatic activity

• Recombinant TrmB may be structurally intact but enzymatically inactive

• Solution: Ensure proper cofactor (SAM) availability during purification and storage. Include reducing agents to maintain cysteine residues in their reduced state. Test activity immediately after purification and optimize buffer conditions.

Challenge 3: Protein stability during storage

• TrmB may lose activity during storage through aggregation or degradation

• Solution: Store with 5-50% glycerol at -20°C/-80°C as recommended. Avoid repeated freeze-thaw cycles by preparing single-use aliquots. For short-term storage, maintain at 4°C for no more than one week .

Challenge 4: Establishing accurate activity assays

• Confirming TrmB activity requires specialized assays

• Solution: Implement multiple complementary assays, including radiometric methyltransferase assays with 14C-SAM and EMSA for binding studies. Consider mass spectrometry for direct detection of m7G46 in tRNA substrates .

Challenge 5: Species-specific optimization

• Expression conditions optimal for one species' TrmB may not work for another

• Solution: Tailor expression and purification protocols for each species' TrmB based on characteristics like pI, hydrophobicity, and predicted structure. Consider testing multiple constructs with different boundaries.

Addressing these challenges will increase the likelihood of obtaining functionally active recombinant TrmB for biochemical and structural studies .

How can researchers effectively measure and interpret changes in tRNA modification profiles in complex bacterial systems?

Analyzing tRNA modifications in complex bacterial systems presents unique challenges that require sophisticated approaches:

Comprehensive analytical methods:

• Liquid chromatography-mass spectrometry (LC-MS):

  • Provides quantitative analysis of multiple tRNA modifications simultaneously

  • Can detect changes in m7G46 and other modifications

  • Enables comparison between wild-type and ΔtrmB mutant strains

• NAIL-MS (Nucleic Acid Isotope Labeling coupled with Mass Spectrometry):

  • Allows differentiation between newly synthesized and pre-existing tRNAs

  • Provides temporal resolution of modification events

  • Can reveal dynamic changes during stress responses

• Next-generation sequencing approaches:

  • Techniques like ARM-seq (AlkB-facilitated RNA methylation sequencing) can map m7G positions transcriptome-wide

  • tRNA-seq with specialized libraries can reveal modification-induced reverse transcription stops

Interpretation challenges and solutions:

Data integration strategies:

• Correlate tRNA modification profiles with translational efficiency using ribosome profiling

• Integrate with transcriptomics and proteomics data to identify genes most affected by TrmB activity

• Apply mathematical modeling to understand how modification levels impact translation dynamics

• Compare results across different growth conditions and genetic backgrounds

These approaches allow researchers to move beyond merely detecting modifications to understanding their functional significance in bacterial physiology and pathogenesis .

What potential roles might TrmB play in bacterial biofilm formation and persistence?

Although direct evidence linking TrmB to biofilm formation is limited in the provided search results, several lines of reasoning suggest this could be an important area for future investigation:

Researchers investigating this connection should consider:

• Comparing biofilm formation between wild-type and ΔtrmB mutants

• Examining tRNA modification profiles in biofilm versus planktonic cells

• Analyzing expression of TrmB during different stages of biofilm development

• Evaluating the impact of TrmB inhibition on established biofilms

• Investigating potential interactions between TrmB and known biofilm regulatory pathways

How might TrmB function be integrated into bacterial regulatory networks responding to multiple simultaneous stressors?

TrmB appears to function as a hub in bacterial stress response networks, integrating responses to multiple stressors:

• Multi-stress integration: Research in A. baumannii reveals that TrmB mediates responses to oxidative stress, low pH, and iron limitation simultaneously . This suggests TrmB may serve as a node integrating multiple stress signals.

• Hierarchical stress responses: The regulatory relationship between TrmB and stress-responsive proteins like catalases in P. aeruginosa suggests TrmB may occupy a specific position in stress response hierarchies.

• Post-transcriptional regulation: TrmB-dependent regulation of acinetobactin in A. baumannii was shown to be post-transcriptional , indicating that TrmB provides an additional layer of regulation beyond transcriptional responses.

• Translational reprogramming: By modifying specific tRNAs, TrmB could facilitate global translational reprogramming when bacteria face multiple stressors.

• Adaptation timing: TrmB may influence the kinetics of stress responses, potentially allowing bacteria to prioritize certain adaptive mechanisms when facing multiple challenges.

Experimental approaches to investigate these network interactions include:

• Network analysis of transcriptomic and proteomic data from ΔtrmB mutants under various stress conditions

• Identification of genetic interactions between trmB and other stress response genes

• Time-resolved studies of tRNA modification dynamics during exposure to multiple stressors

• Mathematical modeling of TrmB's contribution to bacterial stress response networks

• Synthetic biology approaches to rewire TrmB-dependent regulatory circuits

Understanding these integrated networks could provide insights into bacterial adaptation mechanisms and identify potential vulnerabilities for therapeutic targeting .

What are the most critical unresolved questions regarding TrmB function that could significantly advance our understanding of bacterial physiology and pathogenesis?

Despite significant progress in understanding TrmB function, several critical questions remain:

• Structural basis of substrate recognition: How does TrmB specifically recognize its tRNA substrates? Determining the co-crystal structure of TrmB with its tRNA substrates would significantly advance our understanding of recognition mechanisms.

• Regulatory control of TrmB: How is TrmB itself regulated under different stress conditions? Understanding the transcriptional, translational, and post-translational regulation of TrmB could reveal how bacteria modulate tRNA modification in response to environmental cues.

• Species-specific functions: Why does TrmB deletion produce different phenotypes across bacterial species? Comparative studies examining the specific tRNA substrates and downstream effects of TrmB across diverse bacteria could elucidate evolutionary adaptations.

• Interaction with other tRNA modifying enzymes: Does TrmB function within a larger network of tRNA modification enzymes? Investigating potential interactions or coordination between TrmB and other modification enzymes could reveal higher-order regulation of translation.

• Host-pathogen interactions: How does TrmB-mediated tRNA modification influence bacterial interactions with host immune cells beyond what's currently known? Deeper investigation of TrmB's role in evasion of host defenses could uncover new aspects of pathogenesis.

• Potential role in antimicrobial resistance: Does TrmB contribute to antimicrobial resistance mechanisms? Although initial studies suggested limited involvement in antibiotic resistance , more comprehensive investigations might reveal connections to adaptive resistance.

• Small molecule inhibition: Can effective small molecule inhibitors of TrmB be developed, and what would be their impact on bacterial virulence and survival? Developing such inhibitors could provide valuable tools for both research and potential therapeutic applications.

Addressing these questions will require multidisciplinary approaches combining structural biology, biochemistry, genetics, systems biology, and infection models .

How might emerging technologies advance our understanding of TrmB biology and its potential as a therapeutic target?

Emerging technologies are poised to revolutionize TrmB research in several ways:

• Cryo-electron microscopy: High-resolution structures of TrmB-tRNA complexes can reveal atomic details of substrate recognition and catalysis, guiding structure-based drug design approaches.

• Single-cell translatomics: Technologies that profile translation at the single-cell level can reveal how TrmB-mediated tRNA modifications affect protein synthesis in heterogeneous bacterial populations, particularly within infection contexts.

• Nanopore direct RNA sequencing: This technology allows direct detection of tRNA modifications without requiring reverse transcription, potentially enabling comprehensive profiling of the full spectrum of tRNA modifications regulated by TrmB.

• CRISPR-based screening platforms: High-throughput CRISPR screens can identify genetic interactions with TrmB, revealing potential combination targets for therapeutic intervention.

• Microfluidic approaches: Devices allowing precise control of bacterial microenvironments can help understand how TrmB functions under the complex, dynamic conditions of host tissues.

• AI-driven drug discovery: Machine learning approaches can accelerate the identification of TrmB inhibitors by predicting binding modes and optimizing lead compounds.

• Synthetic biology tools: Engineered bacteria with modified or regulated TrmB activity can serve as powerful tools to dissect TrmB functions in vivo.

• In situ structural biology: Techniques like cryo-electron tomography might eventually allow visualization of TrmB-tRNA interactions within the native cellular context.