Recombinant Nitrosomonas europaea Ribosomal RNA large subunit methyltransferase E (rlmE)

What is Ribosomal RNA Large Subunit Methyltransferase E (RlmE) and what is its function in Nitrosomonas europaea?

RlmE (previously known as RrmJ or FtsJ) is an S-adenosyl methionine (AdoMet)-dependent methyltransferase that catalyzes the 2'-O-methylation of the ribose at position Um2552 in 23S ribosomal RNA. In N. europaea, as in other bacteria, this modification is crucial for proper ribosome assembly and function.

Unlike many other rRNA modification enzymes whose deletion causes minimal growth defects, RlmE deletion results in a substantial decrease in growth rate (2-4 fold slower than wild-type cells) . This is because:

• RlmE mediates the methylation of Um2552, which is situated adjacent to G2553

• G2553 is an essential base that anchors the 3' CCA terminus of the A-site tRNA in the peptidyl transferase center (PTC)

• The modification affects RNA folding, stabilization, and subsequent ribosome assembly

RlmE functions primarily during the late stages of large ribosomal subunit (LSU) biogenesis, unlike many other PTC region modifications that occur during early or intermediate assembly stages .

How does the RlmE from Nitrosomonas europaea differ from other bacterial RlmE proteins?

N. europaea RlmE shares the core catalytic domains and functions with RlmE from other bacteria, but with some notable differences:

The unique metabolic background of N. europaea as an ammonia-oxidizing bacterium with a limited set of energy sources potentially influences how RlmE functions within its cellular context, particularly under different oxygen availability conditions .

What expression systems are commonly used for producing recombinant N. europaea RlmE?

Several expression systems have been successfully used for recombinant N. europaea proteins, which can be applied to RlmE:

• E. coli expression systems: Most commonly used due to:

  • Rapid growth

  • High protein yields

  • Well-established protocols

  • Compatibility with methyltransferase expression

  Common vectors include pET-based systems with T7 promoters, typically using BL21(DE3) or Rosetta strains to address codon usage differences.

• Homologous expression in N. europaea: While more challenging, this approach preserves native folding and post-translational modifications:

  • Electroporation is the established method for introducing plasmid DNA into N. europaea

  • Promoters from highly expressed N. europaea genes, such as the hao (hydroxylamine oxidoreductase) promoter, have been successfully used to drive expression

  • Selection is typically performed using kanamycin resistance

• Cell-free protein synthesis: Useful for producing potentially toxic proteins:

  • Rapid production

  • Avoids growth-inhibitory effects

  • Allows incorporation of modified amino acids

The choice of expression system should consider the downstream application and whether native folding and activity are critical to your research question.

How should I optimize the purification of recombinant N. europaea RlmE?

Purification of recombinant N. europaea RlmE typically follows these steps, with optimization considerations at each stage:

• Expression optimization:

  • Test multiple temperatures (18-30°C)

  • Vary IPTG concentrations (0.1-1.0 mM)

  • Consider auto-induction media for higher yields

  • Expression time (typically 4-24 hours)

• Cell lysis:

  • Buffer composition: 50 mM Tris-HCl (pH 7.5-8.0), 300-500 mM NaCl, 10% glycerol

  • Include protease inhibitors

  • Add 1-5 mM β-mercaptoethanol or DTT to maintain reduced cysteines

  • Consider adding S-adenosylmethionine (AdoMet) or S-adenosylhomocysteine (AdoHcy) for stability

• Affinity chromatography:

  • His-tagged purification using Ni-NTA is most common

  • Wash with 20-40 mM imidazole to reduce non-specific binding

  • Elute with 250-300 mM imidazole

  • Use gravity flow or FPLC depending on scale

• Secondary purification:

  • Ion exchange chromatography (typically anion exchange)

  • Size exclusion chromatography

  • Remove imidazole via dialysis before enzyme activity assays

• Quality control:

  • SDS-PAGE for purity assessment

  • Western blot confirmation

  • Mass spectrometry for identity confirmation

  • Enzymatic activity assay

For optimal activity, the final storage buffer should contain:

• 20-50 mM Tris-HCl (pH 7.5-8.0)

• 100-200 mM NaCl

• 1-5 mM DTT

• 10-20% glycerol

• Storage at -80°C in small aliquots to avoid freeze-thaw cycles

What methods can be used to accurately assess the enzymatic activity of recombinant N. europaea RlmE?

Several complementary methods can be used to assess RlmE methyltransferase activity:

• Primer extension assay:

  • Incubate recombinant RlmE with in vitro transcribed 23S rRNA

  • Use reverse transcriptase to create cDNA from the template

  • Low dNTP concentrations cause reverse transcriptase to pause one nucleotide before methylation sites

  • Analyze products using gel electrophoresis

  • Quantify band intensity compared to controls

• Radiometric methylation assay:

  • Incubate RlmE with substrate RNA and S-[methyl-³H]adenosylmethionine

  • Filter reaction through charged filters to capture RNA

  • Measure incorporated radioactivity by scintillation counting

  • Calculate enzyme kinetics (Km, kcat, kcat/Km)

• HPLC-based assays:

  • Digest modified RNA with nucleases

  • Separate nucleotides using HPLC

  • Detect methylated versus unmethylated nucleotides by their retention times

  • Quantify peak areas for activity assessment

• Mass spectrometry:

  • Analyze methylation by mass difference

  • Can distinguish between different methylation types

  • Provides accurate quantification of modification levels

• In vivo complementation assay:

  • Express recombinant N. europaea RlmE in an E. coli ΔrlmE knockout strain

  • Measure growth rate recovery

  • Analyze ribosome profiles by sucrose gradient centrifugation

  • Quantify 50S, 70S and polysome levels

The table below compares these methods:

How can I assess whether recombinant N. europaea RlmE maintains its native structure and function?

To ensure your recombinant protein maintains native structure and function, employ these techniques:

• Circular dichroism (CD) spectroscopy:

  • Assess secondary structure elements (α-helices, β-sheets)

  • Monitor thermal stability (melting temperature)

  • Compare with known methyltransferase CD profiles

• Thermal shift assays (Thermofluor):

  • Measure protein stability under various conditions

  • Identify optimal buffer compositions

  • Assess the impact of cofactors (SAM/AdoMet)

  • Determine stabilizing ligands

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS):

  • Determine oligomeric state

  • Confirm monodispersity

  • Detect protein aggregation

• Enzymatic assays with various substrates:

  • Test activity on in vitro transcribed 23S rRNA

  • Compare activity with isolated domain V of 23S rRNA

  • Measure activity on ribosomes at different assembly stages

• Structural characterization:

  • X-ray crystallography

  • Cryo-electron microscopy

  • NMR for smaller domains

  • Compare with known methyltransferase structures

The 3D structure prediction (if not experimentally determined) would likely show a catalytic domain with a Rossmann-like fold similar to other methyltransferases, with the characteristic S-adenosylmethionine binding site and catalytic tetrad (K-D-K-E) necessary for methyl transfer activity .

How does oxygen limitation affect the expression and activity of RlmE in Nitrosomonas europaea?

N. europaea is an obligate aerobic chemolithoautotroph that oxidizes ammonia to nitrite, but it can adapt to oxygen-limited conditions by adjusting its metabolism. Research suggests that oxygen limitation affects RlmE and other translation-related factors in the following ways:

• Transcriptional changes:

  • Under oxygen limitation, N. europaea differentially regulates genes involved in energy conservation and ribosome biogenesis

  • Genes encoding ribosomal proteins and translation factors often show altered expression patterns

  • RNA modification enzymes, potentially including RlmE, may be upregulated to maintain ribosome assembly under stress conditions

• Metabolic adaptations:

  • Reduced growth yield during oxygen limitation (0.35 g [dry cell weight] mol⁻¹ NH₃ compared to 0.40 g [dry cell weight] mol⁻¹ NH₃ under ammonia-limited conditions)

  • Altered carbon fixation pathway efficiency

  • Potential accumulation of polyphosphate as energy storage

• Ribosome assembly:

  • Oxygen limitation may affect ribosome assembly kinetics

  • RlmE function becomes particularly critical during stress conditions

  • Proper rRNA modifications help maintain translation fidelity under suboptimal growth conditions

This table summarizes the effects of oxygen limitation on N. europaea growth parameters and potential impacts on RlmE function:

These findings suggest that RlmE activity may be particularly important during oxygen limitation to maintain efficient ribosome assembly and translation .

What is the role of RlmE in antibiotic resistance and how can recombinant N. europaea RlmE be used to study this phenomenon?

RlmE methylates position Um2552 in the 23S rRNA, which is close to the peptidyl transferase center (PTC) of the ribosome. This modification affects ribosome assembly and function, with implications for antibiotic resistance:

What advanced structural and functional studies can be performed to better understand N. europaea RlmE interaction with its RNA substrate?

To elucidate the precise mechanisms of RlmE-RNA interactions, consider these advanced approaches:

• X-ray crystallography and cryo-EM studies:

  • Crystallize RlmE alone and in complex with SAM and RNA fragments

  • Use cryo-EM to visualize RlmE interactions with full 23S rRNA or pre-50S particles

  • Map the binding interface between RlmE and its substrate

  • Identify key residues involved in substrate recognition and catalysis

• RNA-protein crosslinking studies:

  • UV-induced crosslinking followed by mass spectrometry (MS) analysis

  • Site-specific incorporation of photo-reactive nucleotides in the RNA substrate

  • Identify direct contact points between RlmE and rRNA

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map conformational changes upon substrate binding

  • Identify flexible regions that accommodate RNA

  • Compare with other methyltransferases to identify conserved binding mechanisms

• Single-molecule approaches:

  • Fluorescence resonance energy transfer (FRET) to measure conformational changes during catalysis

  • Single-molecule enzymology to characterize the kinetic mechanism

• Molecular dynamics simulations:

  • Model the interaction between RlmE and its rRNA substrate

  • Predict conformational changes during catalysis

  • Guide mutagenesis studies to validate key interactions

Based on structural studies of related methyltransferases, RlmE likely contains:

• A catalytic domain with a Rossmann-like fold for SAM binding

• A K-D-K-E catalytic tetrad for 2'-O methylation

• An RNA-binding surface with positively charged residues

• Potentially specific recognition elements for the RNA sequence/structure surrounding position U2552

How can recombinant N. europaea RlmE be used as a tool to study bacterial adaptation to environmental stresses?

N. europaea is an environmental bacterium that faces various stresses in its natural habitats. Recombinant RlmE can be used to investigate bacterial adaptation mechanisms in several ways:

• Expression under stress conditions:

  • Generate reporter constructs (e.g., luciferase or GFP fusions) to monitor RlmE expression under different stresses

  • Compare expression levels under ammonia limitation, oxygen limitation, temperature stress, and chemical stressors

  • Correlate RlmE expression with ribosome biogenesis rates

• Ribosome modification dynamics:

  • Analyze how the rate and extent of Um2552 methylation changes under stress conditions

  • Map the complete rRNA modification pattern under different growth conditions

  • Determine whether rRNA modifications serve as a regulatory mechanism during stress response

• In vitro reconstitution experiments:

  • Use purified recombinant RlmE to reconstitute ribosome assembly in vitro

  • Test how factors like pH, temperature, and ionic conditions affect RlmE activity

  • Compare ribosome assembly kinetics with and without RlmE

• Functional role in oxidative stress:

  • N. europaea produces reactive nitrogen species during ammonia oxidation

  • Investigate whether RlmE-mediated rRNA modification helps maintain translation fidelity under oxidative/nitrosative stress

  • Compare with how other bacteria regulate translation during stress

• Competitive fitness assays:

  • Create RlmE knockout or overexpression strains in N. europaea

  • Compete these strains against wild-type under various environmental conditions

  • Quantify the fitness advantage conferred by proper rRNA modification

Studies in E. coli have shown that RlmE knockouts have severe growth defects (2-4 fold decrease in growth rate) , but the phenotype may be even more pronounced in N. europaea due to its more constrained energy metabolism as a chemolithoautotroph.

How does the mechanism of RlmE differ from other rRNA methyltransferases in N. europaea, and what are the implications for ribosome biogenesis?

N. europaea contains several rRNA methyltransferases that modify different positions in ribosomal RNA. Understanding their mechanistic differences provides insights into ribosome biogenesis:

• Comparison of methyltransferase mechanisms:

• RlmE's unique role in ribosome biogenesis:

  • Unlike most modification enzymes that act early, RlmE methylates a late assembly intermediate

  • The Um2552 modification occurs near functionally critical sites in the ribosome

  • Only RlmE knockouts show severe growth defects among methyltransferase mutants

  • RlmE function may couple rRNA modification with large subunit maturation

• Domain organization differences:

  • RlmE contains a characteristic Rossmann-like fold methyltransferase domain

  • Other methyltransferases like RlmKL have additional domains (e.g., THUMP domains)

  • These structural differences reflect their distinct substrate recognition mechanisms

• Research implications:

  • Study the hierarchical nature of rRNA modifications in ribosome assembly

  • Investigate whether certain modifications are prerequisites for others

  • Explore potential interactions between different modification enzymes during assembly

Understanding these differences is crucial for developing a comprehensive model of ribosome biogenesis in bacteria and potentially identifying new antibiotic targets.