Recombinant Pseudomonas syringae pv. tomato 23S rRNA (guanosine-2'-O-)-methyltransferase RlmB (rlmB)

What is RlmB and what is its function in bacterial ribosomes?

RlmB is a specialized 2'-O-methyltransferase that catalyzes the methylation of guanosine 2251 in the peptidyltransferase domain of 23S ribosomal RNA. This modification is conserved across bacterial species and contributes to the structural stability and functional integrity of the ribosome. The enzyme transfers a methyl group from S-adenosylmethionine (AdoMet) to the 2'-O position of the ribose moiety in the target guanosine residue . This methylation is critical for proper ribosome assembly and optimal translation efficiency.

What is the structural organization of RlmB methyltransferase?

Based on crystallographic studies, RlmB consists of two main domains connected by a flexible linker:

• N-terminal domain: Shares structural similarity with ribosomal proteins L7 and L30, suggesting its involvement in 23S rRNA recognition

• C-terminal domain: Contains the catalytic center with a divergent methyltransferase fold featuring a unique knotted region

• Linker region: A flexible extended sequence connecting the two domains

The enzyme forms a dimer in solution, which may be functionally significant. The C-terminal domain notably lacks classic AdoMet binding site features found in other methyltransferases. Instead, conserved residues cluster in the knotted region, indicating this area likely contains both the catalytic and AdoMet binding sites .

How does the methyltransferase activity of RlmB affect bacterial physiology?

RlmB-mediated methylation impacts bacterial physiology through several mechanisms:

• Ribosome stability: The methyl group at G2251 stabilizes local RNA structure in the peptidyltransferase center

• Translation efficiency: Proper modification ensures optimal protein synthesis rates

• Stress response: Methylation may protect ribosomes during environmental stress conditions

• Antibiotic resistance: Modified ribosomes can exhibit altered susceptibility to antibiotics targeting the ribosome

The rlmB gene appears to be essential for bacterial viability , underscoring its critical role in cellular function.

How might RlmB function differ between E. coli and Pseudomonas syringae pv. tomato?

While both organisms possess RlmB enzymes that catalyze similar reactions, several potential differences may exist:

These differences could be investigated through comparative biochemical and structural studies to understand pathogen-specific adaptations.

What role might RlmB play in the virulence of Pseudomonas syringae pv. tomato during plant infection?

Although not directly addressed in the search results, several lines of evidence suggest potential roles for RlmB in pathogenesis:

• Ribosomal modifications often enhance bacterial adaptation to stress conditions, which would be advantageous during plant infection

• Optimal translation efficiency is crucial for the expression of virulence factors, including type III secretion system components that are essential for P. syringae pathogenicity

• Plant defense responses create challenging environments for bacterial protein synthesis, potentially requiring fully functional ribosomes

To investigate this relationship, researchers could:

• Create conditional rlmB mutants and assess their virulence in tomato infection models

• Examine ribosome function in wild-type vs. rlmB-deficient strains during exposure to plant defense molecules

• Analyze translation efficiency of key virulence genes in the presence or absence of proper rRNA methylation

How does P. syringae pv. tomato RlmB interact with the bacterial chemotaxis system during infection?

The chemotaxis system is crucial for P. syringae pv. tomato infection, as it guides bacterial entry into plant tissue through perception of plant-derived signals like GABA and l-Pro . The potential interactions between RlmB and the chemotaxis system may include:

• Translational regulation: RlmB-mediated rRNA modification may influence the translation efficiency of chemoreceptor proteins like PsPto-PscC

• Metabolic connections: Both systems respond to environmental cues during infection

• Temporal coordination: Entry into the plant (guided by chemotaxis) and subsequent growth (requiring optimal translation) must be coordinated

Experiments could include:

• Analyzing expression patterns of rlmB relative to chemotaxis genes during infection

• Investigating whether RlmB activity is modulated by the same signals that trigger chemotaxis

• Determining if rlmB mutations affect chemotactic responses toward plant signals

What are the best approaches for generating catalytically inactive RlmB mutants for mechanistic studies?

Creating catalytically inactive mutants is valuable for studying enzyme mechanism without disrupting protein structure. For RlmB, consider:

These mutants would be invaluable for crystallographic studies of enzyme-substrate complexes and for understanding the catalytic mechanism.

What expression systems are most effective for producing recombinant P. syringae pv. tomato RlmB?

Several expression systems can be considered, each with advantages and limitations:

Expression constructs should include:

• Affinity tags (His6, GST) for purification

• Protease cleavage sites for tag removal

• Codon optimization if using heterologous systems

How can the methyltransferase activity of recombinant RlmB be measured in vitro?

Several complementary techniques can assess RlmB activity:

• Radiometric assays:

  • Incubate enzyme with [³H]-SAM and 23S rRNA substrate

  • Measure incorporation of radioactive methyl groups into RNA

  • Quantify via scintillation counting after RNA precipitation

• Mass spectrometry:

  • React enzyme with SAM and 23S rRNA or synthetic oligonucleotides

  • Digest RNA and analyze by LC-MS/MS

  • Detect mass shift of +14 Da at target nucleotide

• HPLC-based methods:

  • Monitor consumption of SAM or production of SAH

  • Requires sensitive detection methods (fluorescence, UV)

• Biochemical parameters that should be determined:

  • Km for both SAM and RNA substrates

  • kcat and catalytic efficiency

  • pH and temperature optima

  • Divalent metal ion requirements

What techniques are most appropriate for studying the binding of S-adenosylmethionine (AdoMet) to RlmB?

Understanding AdoMet binding is crucial given the unusual binding site in the knotted region :

• Equilibrium binding methods:

  • Isothermal Titration Calorimetry (ITC): Provides complete thermodynamic profile (ΔH, ΔS, Kd)

  • Microscale Thermophoresis: Requires minimal sample, works in solution

  • Fluorescence-based methods: Intrinsic tryptophan fluorescence or fluorescent SAM analogs

• Structural approaches:

  • X-ray co-crystallography with SAM or SAM analogs

  • NMR to map binding interface and detect conformational changes

  • Hydrogen-deuterium exchange mass spectrometry to identify protected regions

• Computational methods:

  • Molecular docking to predict binding mode

  • Molecular dynamics simulations to understand binding dynamics

What is the best methodology for detecting viable Pseudomonas syringae pv. tomato cells during infection studies?

When studying RlmB function in infection contexts, accurate quantification of viable bacteria is essential:

The PMA-qPCR method provides an excellent approach for specific detection:

• Propidium monoazide (PMA) selectively penetrates dead bacterial cells and binds to DNA

• This binding prevents PCR amplification of DNA from dead cells

• Subsequent qPCR specifically amplifies DNA only from viable cells

Optimization parameters include:

• PMA concentration: 10 μmol liter⁻¹ is optimal

• Light exposure time: 10 minutes provides best results

• Target gene: The hrpZ gene offers specificity for P. syringae pv. tomato

This method achieves detection limits of:

• 10² CFU/ml in bacterial suspensions

• 11.86 CFU/g in contaminated tomato tissue

For naturally contaminated samples, this approach can detect bacterial levels ranging from 10² to 10⁴ CFU/g .

How can understanding RlmB function contribute to developing novel antimicrobial strategies?

RlmB presents a promising antimicrobial target for several reasons:

• Essential gene function: The rlmB gene appears essential for bacterial viability

• Unique structure: The knotted catalytic domain offers distinctive binding sites for inhibitor design

• Conservation: Present across bacterial species but with differences from eukaryotic methyltransferases

Potential antimicrobial approaches include:

• Small molecule inhibitors targeting the AdoMet binding site

• Compounds that interfere with RNA substrate recognition

• Peptide-based inhibitors that disrupt dimerization

• CRISPR-based antimicrobials targeting the rlmB gene

These approaches could be particularly valuable against multidrug-resistant P. syringae pv. tomato strains that cause bacterial speck disease in tomato.

How might RlmB function be affected by interactions with plant defense molecules?

During infection, P. syringae pv. tomato encounters numerous plant defense molecules that could impact RlmB function:

• Reactive oxygen species may oxidize critical residues in the enzyme

• Plant antimicrobial peptides could interact with RlmB or its substrates

• Plant-derived amino acids like GABA and l-Pro, which increase during infection , may influence metabolic pathways connected to methylation

Experimental approaches to investigate these interactions include:

• In vitro methyltransferase assays in the presence of plant extracts

• Analysis of rRNA methylation patterns in bacteria isolated from infected versus uninfected plants

• Transcriptional and translational profiling of rlmB expression during infection

What are the emerging technologies that could advance our understanding of RlmB function?

Several cutting-edge technologies could transform RlmB research:

• Cryo-electron microscopy:

  • Visualize RlmB in complex with ribosomes or rRNA substrates

  • Capture conformational states during the catalytic cycle

• Single-molecule techniques:

  • FRET to monitor conformational changes in real-time

  • Optical tweezers to study enzyme-substrate interactions

• RNA structural biology:

  • SHAPE-Seq to probe rRNA structure before and after methylation

  • RNA-protein crosslinking to map interaction interfaces

• Systems biology approaches:

  • Ribosome profiling to assess translation impacts

  • Multi-omics integration to understand RlmB's role in the infection process

These technologies would provide unprecedented insights into the structural basis and biological significance of RlmB-mediated methylation in bacterial pathogenesis.