Recombinant Chromobacterium violaceum Ribosomal protein S12 methylthiotransferase RimO (rimO)

What is Chromobacterium violaceum and why is its RimO protein of interest?

Chromobacterium violaceum is a gram-negative, facultatively anaerobic bacterium commonly found in soil and water environments in tropical and subtropical regions. It produces a characteristic violet pigment called violacein that has been associated with its virulence properties . The bacterium rarely causes human infection but has a high fatality rate when it does occur .

The RimO methylthiotransferase from C. violaceum is of particular interest because it belongs to an ancient family of enzymes that catalyze the methylthiolation of biological molecules. In the case of RimO, it specifically modifies the ribosomal protein S12 at aspartic acid residue D88 . This posttranslational modification is believed to play a significant role in the functioning of bacterial ribosomes and potentially impacts antibiotic resistance mechanisms, making it an important subject for microbiological and biochemical research.

How does RimO function biochemically?

RimO functions as a radical-S-adenosylmethionine (radical-SAM) enzyme that catalyzes the methylthiolation of aspartic acid residue D88 on ribosomal protein S12 . The enzyme utilizes an iron-sulfur cluster to generate a 5'-deoxyadenosyl radical from S-adenosylmethionine (SAM), which initiates the radical-based reaction mechanism.

What expression systems are commonly used for recombinant RimO production?

Recombinant expression of RimO from Chromobacterium violaceum typically utilizes E. coli-based expression systems due to their efficiency and the extensive toolbox available for genetic manipulation. Common E. coli strains used include BL21(DE3), which lacks certain proteases that might degrade the recombinant protein, and Rosetta strains, which provide tRNAs for rare codons that might be present in the C. violaceum sequence.

Expression vectors such as pET series (particularly pET28a for N-terminal His-tag fusion) are frequently employed, with the gene optimized for E. coli codon usage to enhance expression levels. Induction is typically performed using IPTG at concentrations of 0.1-1.0 mM when cultures reach mid-log phase (OD600 of 0.6-0.8). Since RimO contains iron-sulfur clusters essential for its activity, supplementation with iron (typically ferrous ammonium sulfate) and reduction of growth temperature to 18-25°C after induction often improves the production of properly folded, active enzyme.

For optimal protein folding and iron-sulfur cluster incorporation, expression under microaerobic or anaerobic conditions may be beneficial, as excessive oxygen can interfere with proper cluster assembly.

What are the key challenges in expressing active recombinant RimO from C. violaceum?

Expressing catalytically active recombinant RimO from C. violaceum presents several significant challenges that researchers must address through methodical optimization:

The primary difficulty lies in properly incorporating the iron-sulfur clusters essential for RimO's radical-SAM activity. These clusters are oxygen-sensitive, making conventional aerobic expression systems potentially problematic. Researchers must consider expression under reduced oxygen tension or in specialized anaerobic expression systems. Furthermore, co-expression with iron-sulfur cluster assembly machinery (ISC or SUF system components) may significantly improve the yield of holo-enzyme.

Chromobacterium violaceum's genomic DNA has a high GC content and potentially different codon usage patterns compared to standard expression hosts like E. coli. This necessitates either codon optimization of the rimO gene or use of expression strains supplemented with rare tRNAs.

The violacein pigment produced by C. violaceum can potentially interfere with protein purification and enzymatic assays . When working with genomic DNA or extracting the native enzyme, rigorous purification procedures must be implemented to eliminate pigment contamination.

Additionally, RimO's substrate specificity for ribosomal protein S12 requires either co-expression of the substrate or separate preparation of the S12 protein for in vitro activity assays. The specific interaction between RimO and S12 may involve conformational elements that are challenging to reproduce in recombinant systems.

How can researchers assess the methylthiotransferase activity of recombinant RimO?

Assessment of recombinant RimO methylthiotransferase activity requires a multi-faceted approach that combines biophysical, biochemical, and mass spectrometry techniques:

In vitro enzymatic assays: The primary method involves incubating purified recombinant RimO with its substrate (ribosomal protein S12), S-adenosylmethionine as the methyl donor, a suitable sulfur source (often cysteine or an iron-sulfur cluster protein), and a reducing system (such as dithionite or flavodoxin/flavodoxin reductase/NADPH). Reactions are typically conducted under anaerobic conditions to preserve iron-sulfur cluster integrity.

Mass spectrometry analysis: The most definitive method for confirming methylthiotransferase activity is mass spectrometry. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be used to detect the mass shift of +46 Da corresponding to the methylthio (-SCH₃) group addition to aspartate 88 of the S12 protein . This approach allows precise identification and quantification of the modified residue.

Radio-isotope incorporation: Using ³⁵S-labeled cysteine or ¹⁴C-labeled SAM as substrates enables sensitive detection of methylthio transfer through scintillation counting or radiography after SDS-PAGE separation.

UV-visible spectroscopy: RimO contains iron-sulfur clusters that display characteristic absorption spectra. Changes in these spectra during catalysis can provide insights into the redox state transitions of the clusters during the reaction cycle.

Table 1 summarizes typical reaction conditions for assessing recombinant RimO activity:

What structural features distinguish RimO from other methylthiotransferases?

RimO possesses several distinct structural features that differentiate it within the methylthiotransferase family:

The most remarkable aspect of RimO is its extraordinary similarity to MiaB (which methylthiolates tRNA) despite having evolved to modify protein rather than nucleic acid substrates . This represents one of the most extreme known cases of structural conservation between enzymes acting on fundamentally different biomolecules. The radical-SAM domain of RimO contains the characteristic CX₃CX₂C motif that coordinates the [4Fe-4S] cluster essential for generating the 5'-deoxyadenosyl radical from S-adenosylmethionine.

RimO contains a C-terminal TRAM domain (tRNA methyltransferase activator) that in related enzymes like RumA is known to bind RNA substrates . Remarkably, despite the substrate shift from RNA to protein, this domain has not significantly diverged at the sequence level, suggesting a novel adaptation of the RNA-binding architecture to recognize protein substrates.

An additional [4Fe-4S] cluster unique to the methylthiotransferase subfamily is believed to serve as the sulfur donor for the methylthiolation reaction, distinguishing RimO from simple methyltransferases. This auxiliary cluster is coordinated by conserved cysteine residues outside the canonical radical-SAM motif.

Unlike many other radical-SAM enzymes, RimO must recognize a specific three-dimensional protein structure rather than a primary sequence alone, necessitating specialized surface recognition elements that accommodate the folded S12 protein.

How does the C. violaceum RimO compare to homologs from other bacterial species?

The RimO enzyme from Chromobacterium violaceum shares core catalytic mechanisms with homologs from other bacterial species, yet exhibits distinctive characteristics reflecting its evolutionary adaptations:

The C. violaceum bacterium's adaptation to tropical and subtropical environments may have influenced certain properties of its RimO enzyme, potentially including temperature optima and stability parameters that differ from mesophilic counterparts. Additionally, C. violaceum produces the violacein pigment with antimicrobial properties, which may have coevolved with modifications to translational machinery components including RimO-modified S12 .

Functional studies suggest that while the catalytic mechanism is conserved across species, subtle differences may exist in substrate specificity and catalytic efficiency. These differences could be related to the distinct ecological niches occupied by various bacterial species and corresponding adaptations in their translational machinery.

From an evolutionary perspective, C. violaceum RimO belongs to one of four ancient subgroups of methylthiotransferases that also includes MiaB (tRNA modifier), B. subtilis YqeV, and M. jannaschii Mj0867 . This classification highlights the enzyme's place in a larger evolutionary context of specialized RNA and protein modification systems.

What purification strategy is optimal for obtaining high-quality recombinant RimO?

Purification of recombinant RimO from Chromobacterium violaceum requires a specialized strategy that preserves the oxygen-sensitive iron-sulfur clusters while achieving high purity:

Initial clarification: Following cell lysis under anaerobic conditions (preferably in an anaerobic chamber), clarification of the lysate should be performed by centrifugation at 30,000 × g for 30 minutes at 4°C. All subsequent steps should maintain anaerobic conditions or include reducing agents.

Immobilized metal affinity chromatography (IMAC): If the recombinant RimO includes a His-tag, Ni-NTA or TALON resin chromatography represents the primary purification step. Buffers should contain 5-10 mM β-mercaptoethanol or 1-2 mM DTT to maintain reducing conditions. A typical elution gradient would range from 20-500 mM imidazole.

Ion exchange chromatography: As a secondary step, ion exchange chromatography (typically anion exchange using Q-Sepharose) helps remove contaminants with different charge properties. The buffer system should be maintained at pH 7.5-8.0 where RimO is expected to have negative surface charge.

Size exclusion chromatography: A final polishing step utilizing Superdex 75 or 200 separates any aggregates or degradation products based on molecular size, while also accomplishing buffer exchange to the storage buffer.

Throughout the purification process, it's critical to supplement buffers with sodium dithionite (1-2 mM) or another suitable reducing agent to protect the iron-sulfur clusters. Including 10% glycerol in all buffers helps maintain protein stability.

Spectroscopic assessment (UV-visible absorption at 280 nm and the characteristic Fe-S cluster absorbance at 390-420 nm) should be performed at each purification stage to monitor both protein concentration and iron-sulfur cluster integrity.

How can researchers investigate the substrate specificity of C. violaceum RimO?

Investigating the substrate specificity of C. violaceum RimO requires a systematic approach that combines biochemical assays with structural analysis:

Mutagenesis of the S12 substrate: Creating a panel of S12 variants with mutations at D88 and surrounding residues enables mapping of the recognition elements required for RimO activity. Each variant can be tested in the standard methylthiotransferase assay, with activity quantified by mass spectrometry. Critical residues would show dramatically reduced modification rates.

Chimeric S12 proteins: Constructing chimeras between S12 proteins from C. violaceum and other species that are modified to different extents by C. violaceum RimO helps identify regions critical for recognition. Each chimera can be assayed for methylthiolation efficiency.

Competition assays: Performing enzymatic reactions in the presence of peptide competitors derived from regions of the S12 protein helps define the minimal recognition motif. Peptides that effectively inhibit the reaction likely represent key binding determinants.

Cross-species activity profiling: Testing the activity of C. violaceum RimO against S12 proteins from diverse bacterial species creates a specificity profile that can reveal evolutionary patterns in substrate recognition.

Structural studies: X-ray crystallography or cryo-EM studies of RimO in complex with its S12 substrate (or suitable substrate analogs) provide direct visualization of the binding interface and catalytic site arrangement.

Table 2 presents a systematic approach to investigating substrate recognition elements:

What analytical techniques can detect methylthiolation of ribosomal protein S12 in vivo?

Detecting the methylthiolation of ribosomal protein S12 in vivo requires sophisticated analytical techniques that can identify this specific modification within the complex cellular environment:

Mass spectrometry-based proteomics: The gold standard approach involves isolating ribosomes from C. violaceum cultures, separating the ribosomal proteins by SDS-PAGE or liquid chromatography, and subjecting the S12 band/fraction to tryptic digestion followed by LC-MS/MS analysis. The methylthiolated D88-containing peptide can be identified by its characteristic mass shift of +46 Da . Modern parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM) approaches allow quantitative assessment of the modification stoichiometry.

Antibody-based detection: Development of antibodies specifically recognizing the methylthiolated D88 residue enables techniques such as Western blotting, immunoprecipitation, or immunofluorescence to detect and localize the modified S12 protein.

Ribosome profiling with modification-specific analysis: Combining ribosome profiling techniques with mass spectrometry enables correlation of the methylthiolation status with ribosome positioning on mRNAs, potentially revealing functional consequences of the modification.

CRISPR-based RimO knockout studies: Creating RimO-deficient C. violaceum strains through CRISPR-Cas9 genome editing provides negative controls that lack the methylthiolation, facilitating comparative analyses to confirm modification identification.

Radiolabeling approaches: In vivo incorporation of 35S-labeled methionine or cysteine can trace the incorporation of the methylthio group, with subsequent ribosome isolation and S12 protein analysis revealing the modification.

Table 3 compares the sensitivity and specificity of different analytical approaches:

How does RimO activity influence bacterial antibiotic resistance profiles?

The relationship between RimO activity and bacterial antibiotic resistance presents a complex interplay of ribosomal function modifications and bacterial physiology:

Ribosomal protein S12 methylthiolation by RimO occurs at D88, a residue in close proximity to the decoding center of the bacterial ribosome. This region is critical for translational fidelity and is the target of several aminoglycoside antibiotics. Modification of D88 may alter the conformational dynamics of this region, potentially affecting antibiotic binding or ribosomal response to antibiotic pressure.

In Chromobacterium violaceum specifically, the interplay between RimO activity and the bacterium's intrinsic antibiotic resistance profile is particularly noteworthy. C. violaceum exhibits resistance to multiple antibiotics including vancomycin, ampicillin, and linezolid, while remaining susceptible to colistin, oxacillin, gentamicin, norfloxacin, chloramphenicol, and amikacin . This resistance pattern may be partially influenced by ribosomal modifications including those catalyzed by RimO.

Comparative genomic studies of clinical isolates with varying antibiotic resistance profiles can reveal correlations between RimO sequence variants or expression levels and specific resistance patterns. Researchers investigating this relationship should employ targeted gene knockout or knockdown approaches to directly assess how RimO deficiency affects minimum inhibitory concentrations (MICs) for various antibiotic classes.

The sophisticated experimental approach would involve creating RimO deletion mutants in C. violaceum and comparing their antibiotic susceptibility profiles with wild-type strains across a panel of antibiotics. Complementation studies with both native and catalytically inactive RimO variants would confirm the specific contribution of the methylthiotransferase activity to any observed resistance phenotypes.

What experimental controls are essential when characterizing recombinant RimO activity?

Rigorous experimental design for characterizing recombinant RimO activity requires comprehensive controls to ensure reliable and interpretable results:

Negative enzyme controls:

• Heat-inactivated RimO enzyme (95°C for 10 minutes) to confirm that observed activity requires intact protein structure

• Site-directed mutants of the radical-SAM domain's CX₃CX₂C motif to demonstrate the essential nature of the iron-sulfur cluster

• Reactions conducted in the absence of the S12 substrate to confirm substrate dependency

Negative substrate controls:

• S12 protein variant with D88 mutated to alanine or asparagine to verify site-specificity

• Heterologous proteins of similar size/structure to S12 to demonstrate substrate specificity

Cofactor controls:

• Omission of S-adenosylmethionine to confirm its requirement as both methyl donor and radical generator

• Omission of the reducing system to verify the need for electron input

• EDTA treatment to chelate iron and disrupt iron-sulfur clusters, confirming their necessity

Reaction condition controls:

• Aerobic versus anaerobic reaction conditions to verify oxygen sensitivity

• Temperature series to determine optimal reaction temperature and distinguish enzymatic from non-enzymatic modifications

• pH series to establish optimal pH and rule out chemical methylthiolation

Positive controls:

• Recombinant E. coli RimO with established activity as a benchmark

• Pre-validated activity assay with known substrates and products

Table 4 presents critical control experiments and their interpretations:

How can researchers differentiate between the roles of RimO and other posttranslational modifications in bacterial physiology?

Differentiating the specific physiological contributions of RimO-mediated methylthiolation from other posttranslational modifications requires sophisticated experimental approaches that isolate its effects:

Genetic approaches:
Creating precise genetic variants is fundamental to this investigation. Researchers should generate:

• Clean rimO deletion strains of C. violaceum with no polar effects on surrounding genes

• Point mutant strains expressing catalytically inactive RimO (mutations in the radical-SAM domain)

• S12 ribosomal protein variants with D88 replaced by a non-modifiable residue

• Complementation strains expressing wild-type RimO from controlled promoters

• Double/triple mutants lacking rimO and genes for other relevant modifications

Phenotypic profiling:
Systematic characterization of these genetic variants across multiple conditions provides functional insights:

• Growth rate analysis across different temperatures, pH values, and nutrient conditions

• Survival under various stress conditions (oxidative, osmotic, antibiotic challenge)

• Ribosome assembly kinetics and polysome profiling

• Translational fidelity using reporter systems that detect frameshifting or stop codon readthrough

• In vivo translation rate measurements using pulse-labeling techniques

Integrative 'omics approaches:
Multi-omics strategies reveal system-wide impacts:

• Ribosome profiling to identify specific mRNAs with altered translation in RimO-deficient strains

• Comparative proteomics to detect changes in protein expression patterns

• Metabolomics to identify altered metabolic pathways

• Transcriptomics to detect compensatory responses to RimO deficiency

Structural biology:
Direct visualization of molecular consequences:

• Cryo-EM structures of ribosomes from wild-type versus rimO deletion strains

• Hydrogen-deuterium exchange mass spectrometry to detect conformational changes

• Fluorescence techniques to measure ribosomal subunit association kinetics

Table 5 summarizes an experimental matrix for differentiating RimO-specific effects:

What are common pitfalls in recombinant RimO expression and how can they be addressed?

Researchers working with recombinant RimO from C. violaceum frequently encounter several challenges that can be systematically addressed:

Problem: Low expression yield

• Potential causes: Codon bias, protein toxicity, mRNA secondary structures, improper induction conditions

• Solutions: Optimize codon usage for the expression host; use tight expression control with lower inducer concentrations; test multiple expression strains; consider fusion partners that enhance solubility (e.g., MBP, SUMO); optimize induction temperature (typically lowering to 16-18°C improves yield for complex proteins)

Problem: Formation of inclusion bodies

• Potential causes: Rapid overexpression, improper folding, iron-sulfur cluster assembly failure

• Solutions: Co-express with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE); co-express with iron-sulfur cluster assembly proteins (IscS, IscU, IscA); use auto-induction media for gradual protein expression; optimize cell lysis conditions to minimize protein aggregation

Problem: Inactive enzyme despite successful purification

• Potential causes: Oxygen exposure damaging iron-sulfur clusters, incomplete cluster assembly, improper protein folding

• Solutions: Maintain strictly anaerobic conditions during purification; supplement purification buffers with iron and sulfide for cluster reconstitution; include small molecules that stabilize iron-sulfur clusters (e.g., 5% glycerol, 2 mM DTT)

Problem: Protein degradation during purification

• Potential causes: Protease contamination, intrinsic instability, oxidative damage

• Solutions: Add protease inhibitors to all buffers; include reducing agents (DTT, β-mercaptoethanol) to prevent oxidative aggregation; optimize buffer composition (pH, salt concentration, additives); perform all steps at 4°C; consider rapid purification approaches

Problem: Poor substrate binding

• Potential causes: Improper folding of the TRAM domain, interference from purification tags, non-native buffer conditions

• Solutions: Test alternative tag positions or cleavable tags; optimize buffer conditions to better mimic physiological environment; verify proper folding using circular dichroism spectroscopy

Table 6 presents a troubleshooting guide for common RimO expression issues:

How can researchers optimize in vitro methylthiotransferase assays for RimO?

Optimization of in vitro methylthiotransferase assays for RimO requires careful consideration of multiple parameters to achieve maximum enzyme activity and reliable detection:

Reaction buffer optimization:
The buffer composition significantly impacts RimO activity. Researchers should systematically test:

• Buffer type: HEPES, Tris, and phosphate buffers in the pH range of 7.0-8.5

• Ionic strength: NaCl or KCl concentrations from 50-300 mM

• Divalent cations: MgCl₂ and/or MnCl₂ at 1-10 mM concentrations

• Reducing agents: DTT, β-mercaptoethanol, or sodium dithionite at various concentrations

• Stabilizing agents: Glycerol (5-20%) or bovine serum albumin (0.1-1 mg/ml)

Substrate presentation:
The S12 substrate configuration affects recognition and modification efficiency:

• Test both full-length S12 and minimal peptide substrates containing the D88 residue

• Compare free S12 versus S12 incorporated into ribosomal subunits

• Evaluate the impact of S12 protein tags on modification efficiency

• Determine optimal substrate concentration through Michaelis-Menten kinetic analysis

Cofactor optimization:
As a radical-SAM enzyme, RimO requires multiple cofactors:

• SAM concentration: Typically 0.5-2 mM, with potential inhibition at higher concentrations

• Electron donation system: Compare chemical reductants (dithionite) versus biological systems (flavodoxin/flavodoxin reductase/NADPH)

• Iron-sulfur cluster integrity: Test addition of Fe²⁺/Fe³⁺ and sulfide during the reaction

• Sulfur source: Compare cysteine, sulfide, or specialized sulfur carrier proteins

Reaction conditions:
Environmental parameters critically affect iron-sulfur enzyme activity:

• Temperature range: Test 25-37°C for optimal activity versus stability

• Incubation time: Establish time course to determine linear range of activity

• Oxygen exclusion: Compare various anaerobic techniques (enzymatic oxygen scavenging systems, glove box, vacuum/gas cycling)

• Light exposure: Minimize due to potential photosensitivity of iron-sulfur clusters

Table 7 presents a systematic optimization matrix for RimO activity assays: