Recombinant Acinetobacter sp. Ribosomal protein S12 methylthiotransferase RimO (rimO)

What is RimO and what is its primary function in bacterial systems?

RimO is a bacterial methylthiotransferase enzyme responsible for the post-translational modification of ribosomal protein S12. Specifically, RimO catalyzes the β-methylthiolation of aspartic acid 88 (D88), a universally conserved residue of S12 within prokaryotes. This modification has been identified in S12 orthologs from several phylogenetically distant bacteria and appears to be unique to bacteria, as there have been no reports of this modification in ribosomal proteins from either eukarya or archaea .

The RimO gene displays striking similarity to the full-length gene sequence of the MiaB enzyme, a bifunctional system characterized in Escherichia coli that is involved in methylthiolation of transfer RNA (tRNA). This structural and functional similarity suggests a conservation of mechanisms across different RNA-modifying enzymes in bacterial systems .

Recent in vitro kinetic studies have demonstrated that recombinant RimO from both E. coli and Thermotoga martima can successfully methylthiolate synthetic peptide substrates mimicking the loop bearing D88, providing biochemical evidence confirming S12 as the probable substrate and indicating that the RimO mechanism is consistent with a MiaB-like sulfur insertion .

Why is the β-methylthiolation of S12 significant in bacterial physiology?

The β-methylthiolation of S12 represents a unique post-translational modification that appears to have evolutionary significance in bacteria. Site-directed mutagenesis studies in Thermus thermophilus revealed that substitutions at the D88 position are lethal, whereas nearby substitutions are tolerated, highlighting the critical nature of this residue. Interestingly, viable mutants with substitutions at nearby positions (P90R and P90W) lacked the D88 modification, presumably because steric hindrance prevented recognition by the modifying enzyme .

Although genetic knockout studies of the rimO gene resulted in viable strains containing only unmodified S12, suggesting the modification itself is not essential for basic survival, the lethality of D88 substitutions indicates that this residue and potentially its modification are likely important under specific environmental or stress conditions that have yet to be fully characterized .

Research suggests this modification may play roles in ribosomal function, translational fidelity, or bacterial adaptation to environmental stresses. Understanding these roles requires careful experimental design that can distinguish between phenotypes resulting from the absence of modification versus those stemming from structural changes to the ribosome.

What experimental approaches are most effective for studying RimO function in Acinetobacter species?

Several complementary experimental approaches have proven effective for investigating RimO function in Acinetobacter and other bacterial species:

• Genetic Manipulation Studies: Gene knockout or knockdown studies of rimO provide fundamental insights into its function. In the case of A. baumannii, genetic knockouts followed by phenotypic characterization, particularly regarding antimicrobial resistance profiles, have been informative .

• Comparative Genomics: Analysis across multiple strains with differing susceptibility profiles helps identify correlations between genetic variations in rimO and phenotypic differences. This approach has been used to study antimicrobial resistance mechanisms in A. baumannii .

• Proteomics and Protein Interaction Studies: Using tagged recombinant proteins as bait (such as SPA-tagged S12) allows identification of interaction partners through affinity purification followed by mass spectrometry. This approach revealed the interaction between RimO, YcaO, and S12 .

• Transcriptomics: RNA-Seq analysis comparing wild-type and rimO-deficient strains reveals transcriptional changes that may point to cellular functions affected by RimO activity. This approach identified overlapping transcriptional phenotypes between RimO and YcaO knockouts, suggesting these proteins share a common function .

• Quantitative Mass Spectrometry: This technique can precisely determine the modification status of S12 under different conditions or in different genetic backgrounds, providing direct evidence of RimO's enzymatic activity in vivo .

For Acinetobacter specifically, an iterative approach combining genomics, transcriptomics, and phenotypic characterization has proven valuable in understanding RimO's role in antimicrobial resistance mechanisms .

How can researchers effectively detect and quantify β-methylthiolation of ribosomal protein S12?

The detection and quantification of β-methylthiolation on ribosomal protein S12 requires specialized analytical techniques:

It's important to note that the study by Anton et al. revealed that soluble S12 was not an efficient substrate for in vitro modification, suggesting that in vivo, S12 is likely modified when it is part of the assembled ribosome rather than in isolation .

What is the functional relationship between RimO and YcaO proteins in the β-methylthiolation process?

The functional relationship between RimO and YcaO represents an important discovery in understanding the mechanism of S12 β-methylthiolation. Proteomic analysis using endogenously expressed tagged S12 protein as bait identified both RimO and YcaO as S12-interacting proteins, suggesting they function together in the modification process .

Transcriptomic analysis of bacterial strains with deleted genes for RimO and YcaO identified an overlapping transcriptional phenotype, providing further evidence that these proteins likely share a common function. Quantitative mass spectrometry additionally indicated that both proteins dramatically impacted the modification status of S12 .

These findings collectively indicate that YcaO, previously a protein of unknown function, is involved in β-methylthiolation of S12, and its absence impairs the ability of RimO to modify S12. This suggests a cooperative mechanism where:

• YcaO may function as a specificity factor that helps RimO recognize S12

• YcaO could act as a scaffold that brings S12 and RimO together

• YcaO might directly participate in the chemical reaction

The discovery of this functional relationship highlights the complexity of post-translational modification systems in bacteria and demonstrates how seemingly unrelated proteins can function together in critical cellular processes.

How does the assembly state of ribosomal protein S12 affect its modification by RimO?

The assembly state of ribosomal protein S12 appears to be a critical factor in its modification by RimO. Proteomic data from studies using tagged S12 as bait provides direct evidence that E. coli-specific β-methylthiolation likely occurs when S12 is assembled as part of a ribosomal subunit, rather than when it exists as an isolated protein .

This finding helps explain the inefficiency observed in in vitro studies where recombinant RimO was used to modify synthetic peptide substrates or soluble S12. The low yield of modified peptide in these studies points to the importance of the proper structural context for efficient RimO activity .

Several factors may contribute to this assembly-dependent modification:

• Structural Conformation: S12 likely adopts a specific conformation within the ribosome that presents the D88 residue in an optimal orientation for RimO recognition and catalysis.

• Cooperative Interactions: Other ribosomal components may provide additional recognition elements or stabilizing interactions that facilitate RimO binding and activity.

• Co-factors or Helper Proteins: The ribosomal environment may include additional factors (like YcaO) that enhance RimO activity through direct or indirect mechanisms.

This assembly-dependent modification mechanism has important implications for experimental design, suggesting that studies seeking to characterize RimO activity or identify inhibitors should utilize intact ribosomes or ribosomal subunits rather than isolated S12 protein.

What evidence connects RimO activity to antimicrobial resistance in Acinetobacter species?

The connection between RimO activity and antimicrobial resistance in Acinetobacter species is an emerging area of research with several lines of evidence suggesting potential links:

• Differential Expression in Resistant Strains: RNA-Seq analysis of paired Acinetobacter baumannii isolates with differing susceptibility profiles has revealed differential expression of rimO and related genes, suggesting a potential role in resistance mechanisms .

• Phenotypic Correlation: Studies comparing phylogenetically-related pairs of A. baumannii isolates with differing susceptibility profiles have identified at least five different potential mechanisms involving gene expression differences, which may include pathways affected by RimO activity .

• Functional Impact on Translation: Since RimO modifies ribosomal protein S12, it potentially affects translational dynamics, which could influence the expression of resistance genes or the interaction of certain antibiotics with their ribosomal targets.

The plasticity of the A. baumannii pan-genome further complicates efforts to establish direct links between genetic elements and resistance phenotypes, highlighting the need for combined approaches that integrate genomics, transcriptomics, and functional studies .

How can bacterial genome-wide association studies (bGWAS) be utilized to investigate RimO's role in drug resistance?

Bacterial genome-wide association studies (bGWAS) represent a powerful approach for investigating RimO's potential role in drug resistance, though they come with specific challenges when applied to highly plastic genomes like those of Acinetobacter baumannii:

In a study of 84 A. baumannii isolates, researchers performed bGWAS looking for associations between all possible 21-mers and resistance phenotypes. This approach generally failed to identify mechanisms that clearly explained resistance phenotypes, highlighting the limitations of genomic approaches alone when dealing with complex resistance mechanisms .

To optimize bGWAS for investigating RimO's role in resistance:

• Control for Population Structure: Given the significant noise associated with population stratification, focused comparisons between phylogenetically-related isolate pairs with differing susceptibility profiles can be more informative than broad population-wide comparisons .

• Integrate Multiple Data Types: Combining genomic data with transcriptomics (RNA-Seq) can reveal differential expression patterns that may not be evident from genomic sequence alone. This approach successfully identified several potential resistance mechanisms in A. baumannii .

• Focus on Specific Pathways: Rather than genome-wide searches, targeted analysis of pathways involving RimO, YcaO, and ribosomal components may yield more meaningful associations with resistance phenotypes.

• Validate Findings Functionally: Computational associations should be followed by experimental validation, such as gene knockouts, complementation studies, or heterologous expression to confirm the functional significance of identified variants or expression changes .

The results from A. baumannii studies suggest that a diagnostic platform based on gene expression rather than genomics alone may be more beneficial for certain surveillance efforts related to antimicrobial resistance .

How should researchers approach contradictory findings in studies of RimO function?

When faced with contradictory findings in RimO research, researchers should consider several methodological approaches:

When reporting seemingly contradictory results, researchers should clearly delineate experimental conditions, genetic backgrounds, and analytical methods to facilitate meaningful comparison across studies.

What strategies can be employed to study the temporal dynamics of ribosomal protein S12 modification?

Investigating the temporal dynamics of S12 β-methylthiolation requires specialized approaches that can track modification status across different growth phases or in response to environmental changes:

• Time-Course Experiments: Sequential sampling of bacterial cultures throughout growth phases, followed by quantitative analysis of S12 modification status, can reveal when β-methylthiolation occurs during the bacterial life cycle.

• Pulse-Chase Methods: Incorporating isotopically labeled amino acids or methyl donors at specific time points, followed by mass spectrometric analysis, can help determine the kinetics of S12 modification and potential turnover of modified proteins.

• Conditional Expression Systems: Using inducible promoters to control rimO or ycaO expression allows researchers to activate modification machinery at specific times and observe the rate and extent of S12 modification.

• Real-time Reporters: Though challenging to develop, fluorescent or luminescent reporters linked to modification-dependent conformational changes could potentially provide real-time monitoring of modification status in living cells.

• Environmental Stress Response: Subjecting bacterial cultures to various stresses (antibiotics, nutrient limitation, oxidative stress) and monitoring changes in S12 modification could reveal conditions that regulate this process.

• Ribosome Assembly Intermediates: Isolation and analysis of ribosome assembly intermediates at different stages could determine precisely when during ribosome biogenesis the S12 modification occurs.

Such temporal studies could help resolve an important question raised in the literature: whether RimO-mediated S12 modification occurs co-translationally during ribosome assembly or post-translationally on mature ribosomes, and whether this timing is affected by environmental conditions .

How do expression levels of rimO and ycaO correlate with S12 modification status?

Based on the findings from transcriptomic and proteomic analyses, there appears to be a strong correlation between rimO and ycaO expression levels and the S12 modification status in bacterial systems:

The transcriptomic analysis revealed that bacterial strains with deleted genes for RimO and YcaO shared an overlapping transcriptional phenotype, suggesting these proteins function in the same pathway. Quantitative mass spectrometry further confirmed that both proteins significantly impact the modification status of S12 .

These findings indicate that both proteins are required for efficient β-methylthiolation of S12, with YcaO likely playing a crucial role in facilitating RimO's enzymatic activity. The absence of either protein results in reduced or absent modification, suggesting a cooperative mechanism rather than redundant pathways .

What is the evolutionary conservation pattern of RimO across bacterial species?

The evolutionary conservation of RimO across bacterial species reflects its important role in ribosomal protein modification:

The RimO protein itself shows striking similarity to the MiaB enzyme involved in tRNA modification, suggesting a common evolutionary origin for these two methylthiotransferases. This relationship provides insight into how specialized modification enzymes may have evolved from more general predecessors .

In Acinetobacter species specifically, genomic analysis indicates RimO is present, but the plastic nature of the A. baumannii pan-genome complicates efforts to establish clear associations between RimO genetic variations and phenotypic differences .