Recombinant Geobacter sulfurreducens Ribosomal protein S12 methylthiotransferase RimO (rimO)

What is the function of RimO in Geobacter sulfurreducens?

RimO in G. sulfurreducens, similar to its homolog in E. coli, functions as a methylthiotransferase that catalyzes the post-translational modification of ribosomal protein S12. Specifically, it methylthiolates residue D88 in S12, a unique modification that may influence ribosomal function . This enzyme belongs to the radical-S-adenosylmethionine protein family and shares significant sequence similarity with MiaB, which methylthiolates tRNA rather than proteins .

To study RimO function experimentally, researchers typically employ gene deletion studies followed by mass spectrometry analysis of S12 proteins. In E. coli models, S12 from wild-type strains shows a mass shift of +46 m/z compared to unmodified protein, while S12 from strains with inactivated rimO genes lacks this modification .

How does G. sulfurreducens RimO compare structurally to homologs in other bacteria?

G. sulfurreducens RimO belongs to a larger family of methylthiotransferases that includes four major subgroups: RimO (protein-modifying), MiaB (tRNA-modifying), YqeV (from B. subtilis), and Mj0867 (from M. jannaschii). Despite their different substrates, these enzymes share remarkable sequence similarity .

The C-terminal TRAM domain, which is responsible for substrate binding and recognition, is particularly well-conserved across these subgroups despite RimO being unique in modifying proteins rather than RNA. This represents an extreme case of resemblance between enzymes that modify different types of macromolecules (proteins versus nucleic acids) .

For structural studies, researchers should consider:

• Employing X-ray crystallography or cryo-EM to resolve substrate binding sites

• Using site-directed mutagenesis to identify key residues involved in substrate recognition

• Conducting comparative analyses with MiaB to understand how similar enzymes evolved different substrate specificities

What are the optimal growth conditions for G. sulfurreducens for recombinant RimO expression?

G. sulfurreducens has unique growth requirements that differ significantly from model organisms like E. coli. For optimal recombinant protein expression:

• Growth medium: Use Geobacter-specific medium rather than minimal media like M9. Studies have shown significant differences in metal content (particularly Fe, Cu, Mn, and Se) between G. sulfurreducens grown in specialized media versus E. coli grown in M9 .

• Electron acceptor options: G. sulfurreducens can grow using:

  • Fumarate as an electron acceptor in anaerobic conditions

  • Fe(III) oxides via direct contact, facilitated by chemotaxis

  • Low levels of oxygen (contrary to earlier assumptions that it was strictly anaerobic)

• Metal supplementation: Ensure adequate iron content in the medium, as G. sulfurreducens has significantly higher iron content than E. coli, which is critical for proper folding of iron-sulfur cluster-containing enzymes like RimO .

Table 1: Comparison of Metal Content in G. sulfurreducens vs. E. coli (μg/g dry weight)

Note: Values represented qualitatively based on research findings

What mechanisms underlie the substrate specificity of RimO compared to other methylthiotransferases?

The substrate specificity of RimO presents a fascinating research question, as it targets proteins while its close homolog MiaB modifies tRNA, despite strong sequence conservation. Two competing hypotheses have been proposed to explain this phenomenon :

• RNA-binding hypothesis: RimO's TRAM domain may bind to an RNA stem-loop proximal to S12 in the assembled ribosome rather than to S12 directly. A candidate for this interaction is the 530 stem-loop, one of the most conserved regions of 16S rRNA. This hypothesis suggests that only a stable S12-rRNA complex (such as the assembled 30S ribosomal subunit) can serve as an efficient substrate for RimO .

• Protein-mimicry hypothesis: Alternatively, RimO's TRAM domain may recognize a portion of S12 that structurally mimics tRNA. This would align with other examples in translation where proteins act as structural mimics of tRNA .

To investigate these hypotheses experimentally:

• Conduct in vitro methylthiolation assays with purified RimO using various substrates: free S12, S12 bound to 16S rRNA fragments, and fully assembled 30S subunits

• Perform mutational analyses of the TRAM domain in both RimO and MiaB, followed by substrate specificity assays

• Use structural techniques (X-ray crystallography, cryo-EM) to visualize RimO-substrate complexes

How does the iron-sulfur cluster assembly in recombinant G. sulfurreducens RimO impact its catalytic activity?

RimO, as a radical-S-adenosylmethionine (radical-SAM) enzyme, requires intact iron-sulfur (Fe-S) clusters for its catalytic activity. G. sulfurreducens presents a unique system for studying this enzyme due to its unusually high cellular iron content compared to other bacteria like E. coli .

When expressing recombinant RimO from G. sulfurreducens, proper Fe-S cluster assembly is critical. Research has shown that:

• Iron limitation inhibits electron transfer in G. sulfurreducens, suggesting Fe-S cluster-dependent enzymes may be particularly sensitive to iron availability .

• Overexpression of Fe-S proteins often results in incomplete cluster assembly, potentially affecting functional studies .

• G. sulfurreducens has an extensive network of cytochromes and Fe-S proteins that may utilize specialized assembly machinery different from model organisms .

To ensure proper Fe-S cluster assembly in recombinant RimO:

• Co-express Fe-S cluster assembly proteins specific to G. sulfurreducens

• Supplement expression media with sufficient iron (10-50 μM ferric citrate)

• Conduct expression under microaerobic or strictly anaerobic conditions to prevent cluster oxidation

• Verify cluster integrity using UV-Vis spectroscopy and EPR analysis prior to activity assays

What are the challenges in distinguishing the methylation and thiolation activities of recombinant RimO?

RimO catalyzes a complex reaction involving both methylation and thiolation of its target. Distinguishing these activities experimentally remains challenging but is essential for understanding the reaction mechanism. Current research suggests several approaches :

• Isolate intermediates: Design trapping experiments to capture reaction intermediates using rapid quenching methods combined with mass spectrometry analysis.

• Identify domain-specific functions: The N-terminal domain of RimO has been postulated to potentially involve cobalamin in the methylation reaction, while the central radical-SAM domain likely facilitates the radical-based thiolation .

• Use specific inhibitors: Selective inhibition of either methylation or thiolation activity can help delineate the reaction sequence.

Table 2: Experimental Approaches to Distinguish RimO Activities

What expression systems are optimal for producing active recombinant G. sulfurreducens RimO?

Expression of active G. sulfurreducens RimO requires careful consideration of host systems, vectors, and conditions to ensure proper folding and Fe-S cluster incorporation:

• Host selection:

  • E. coli strains engineered for Fe-S proteins (e.g., SufFeScient™) provide higher yields of active protein

  • Native G. sulfurreducens expression may offer proper post-translational modifications but with lower yields

  • Cell-free systems supplemented with Fe-S cluster assembly machinery offer a promising alternative

• Vector design:

  • Include a C-terminal His-tag for purification, as N-terminal tags may interfere with Fe-S cluster coordination

  • Consider inducible promoters with moderate expression levels to prevent inclusion body formation

  • Bicistronic designs co-expressing Fe-S assembly proteins can improve yield of active enzyme

• Expression conditions:

  • Grow cultures in Geobacter-specific medium enriched with trace metals

  • Maintain microaerobic conditions (1-2% O2) as G. sulfurreducens can utilize low oxygen levels

  • Supplement with iron sources (ferric citrate) during induction

When testing complementation of rimO mutants, researchers have observed that even with plasmid-based expression, only partial restoration of S12 modification occurs , suggesting that expression level and timing are critical factors.

How can researchers optimize the purification of recombinant RimO while maintaining its catalytic activity?

Purification of RimO presents significant challenges due to its oxygen sensitivity and complex Fe-S cluster requirements. A methodological approach includes:

• Anaerobic techniques:

  • Perform all purification steps in an anaerobic chamber with O₂ < 1 ppm

  • Use oxygen-scrubbed buffers containing reducing agents (2-5 mM DTT or β-mercaptoethanol)

  • Consider vacuum/gas cycling of collection tubes and buffer reservoirs

• Column chromatography sequence:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA for His-tagged RimO

  • Ion exchange chromatography (typically Q-Sepharose) for removing contaminating proteins

  • Size exclusion chromatography for final polishing and buffer exchange

• Fe-S cluster reconstitution:

  • In vitro reconstitution with Fe³⁺, sulfide, and reducing agents may be necessary if clusters are lost during purification

  • Monitor UV-Vis absorbance at 280 nm (protein) and 400-420 nm (Fe-S clusters) to assess cluster integrity

Throughout purification, samples should be analyzed by SDS-PAGE and activity assays to track protein purity and functional integrity. Typical yields of 2-5 mg per liter of culture can be expected, with specific activity measurements providing the critical assessment of functional protein recovery.

What assays are most effective for measuring RimO activity in vitro?

Assessing RimO activity requires methods that can detect both the enzymatic reaction and the modified product. The following approaches have proven effective:

• Mass spectrometry-based assays:

  • Use MALDI-TOF MS to detect the characteristic +46 m/z shift in modified S12

  • Employ LC-MS/MS for detailed characterization of the methylthiolated aspartate residue

  • Develop multiple reaction monitoring (MRM) methods for quantitative analysis

• Substrate preparation:

  • Isolate native S12 from ribosomes using established protocols

  • Express recombinant S12 with proper folding

  • Consider using synthetic peptides containing the target sequence for high-throughput screening

• Reaction conditions:

  • Include SAM (200-500 μM) as methyl donor

  • Provide reducing system (5 mM DTT, flavodoxin/flavodoxin reductase/NADPH)

  • Supply sulfur source (typically Na₂S or cysteine)

  • Maintain anaerobic conditions throughout the assay

The reaction kinetics should be monitored over time (typically 30-120 minutes) with regular sampling to determine initial rates. Controls should include reactions without SAM, without sulfur source, with heat-inactivated enzyme, and with known RimO inhibitors.

How should researchers interpret conflicting data on iron content in G. sulfurreducens?

The literature contains conflicting reports regarding iron content in G. sulfurreducens, presenting a challenge for researchers working with iron-dependent proteins like RimO. To navigate these contradictions, consider the following analytical approach:

Table 3: Factors Affecting Iron Content Measurements in G. sulfurreducens

What bioinformatic approaches can identify potential RimO substrates in G. sulfurreducens?

Identifying potential RimO substrates beyond S12 requires sophisticated bioinformatic approaches:

• Sequence-based analysis:

  • Search for proteins containing the consensus sequence surrounding the methylthiolated aspartate in S12

  • Employ position-specific scoring matrices (PSSMs) derived from known RimO substrates

  • Use machine learning algorithms trained on known methylthiolation sites

• Structural prediction:

  • Identify proteins with structural motifs similar to the methylthiolation site in S12

  • Focus on exposed aspartate residues in loop regions similar to the S12 loop containing D88

  • Consider proteins that interact with RNA, given the potential RNA-binding preference of RimO

• Integration with experimental data:

  • Analyze proteomics data for mass shifts consistent with methylthiolation (+46 Da)

  • Cross-reference with G. sulfurreducens expression data under conditions where RimO is highly expressed

  • Perform RimO pull-down experiments followed by mass spectrometry to identify interacting proteins

Given that RimO has not significantly diverged from other methylthiotransferase subgroups at the sequence level , researchers should consider the possibility that RimO in G. sulfurreducens might have broader substrate specificity than observed in E. coli.

How can researchers correlate RimO activity with G. sulfurreducens' electron transfer capabilities?

G. sulfurreducens is known for its remarkable electron transfer capabilities, allowing it to "breathe" metals and generate electrical current in microbial fuel cells . Understanding if and how RimO activity influences these capabilities requires systematic analysis:

• Physiological characterization of rimO knockouts:

  • Compare growth rates on various electron acceptors (fumarate, Fe(III) oxides, electrodes)

  • Measure current production in bioelectrochemical systems

  • Assess ability to respond to changes in electron acceptor availability

• Ribosomal function analysis:

  • Evaluate translation rates and fidelity in rimO mutants

  • Test antibiotic sensitivity profiles (particularly aminoglycosides that target the ribosome)

  • Analyze expression of key electron transfer proteins via proteomics

• Integrated systems approach:

  • Combine transcriptomics, proteomics, and metabolomics to build a systems-level understanding

  • Develop computational models incorporating RimO activity into cellular electron flow networks

  • Use adaptive laboratory evolution to identify compensatory mechanisms in rimO mutants

Given that mutations in residues surrounding the RimO modification site in S12 have been shown to affect translational accuracy and ribosome function , researchers should investigate whether RimO activity influences the expression of the extensive cytochrome network required for G. sulfurreducens' electron transfer capabilities.

What are the potential applications of understanding RimO function in G. sulfurreducens biotechnology?

Understanding RimO function in G. sulfurreducens opens several avenues for biotechnological applications:

• Enhanced bioelectricity production:

  • If RimO influences expression of electron transfer proteins, engineered RimO variants could potentially enhance power output in microbial fuel cells

  • Optimize S12 modification to improve translation of key cytochromes and electron transfer proteins

• Bioremediation applications:

  • G. sulfurreducens is being explored for bioremediation of metal-contaminated environments

  • RimO engineering could potentially improve metal reduction capabilities and survival under environmental stress conditions

• Synthetic biology tools:

  • RimO's unique post-translational modification mechanism could be harnessed as a novel tool for protein engineering

  • Development of orthogonal translation systems with modified S12 proteins for specialized protein production

The connection between RimO activity and G. sulfurreducens' unique metabolism represents an underexplored area with significant potential for biotechnological innovation.

How might structural studies of RimO advance our understanding of radical-SAM enzymes?

Structural characterization of G. sulfurreducens RimO would significantly advance our understanding of radical-SAM enzymes, particularly those that perform complex methylthiolation reactions:

• Mechanistic insights:

  • Resolve the precise arrangement of SAM, substrate, and Fe-S clusters during catalysis

  • Determine how the enzyme coordinates both methylation and thiolation activities

  • Identify structural adaptations that allow protein substrate recognition versus RNA

• Evolutionary perspectives:

  • Compare RimO structure with MiaB to understand how closely related enzymes evolved different substrate specificities

  • Investigate conservation of catalytic residues across the four methylthiotransferase subgroups

  • Explore the evolutionary trajectory of radical-SAM enzymes in electron transfer-specialized bacteria

• Practical applications:

  • Structure-guided engineering of RimO for altered substrate specificity

  • Rational design of inhibitors for antimicrobial applications

  • Development of improved heterologous expression systems based on structural requirements

As noted in previous research, comparing RimO to MiaB "should prove an interesting study of the evolution of substrate specificity of modifying enzymes" , with potential implications beyond G. sulfurreducens biology.