Recombinant Bacteroides thetaiotaomicron Ribosomal RNA small subunit methyltransferase G (rsmG)

What is the function of rsmG in Bacteroides thetaiotaomicron?

Based on comparative studies with other bacterial species, rsmG in Bacteroides thetaiotaomicron likely functions as a methyltransferase that targets specific nucleotides in the 16S ribosomal RNA. In Bacillus subtilis, RsmG methylates the N7 position of nucleotide G535 in 16S rRNA (corresponding to G527 in Escherichia coli) . While the exact target in B. thetaiotaomicron has not been explicitly identified in the provided search results, functional conservation across bacterial species suggests a similar role.

The methylation of 16S rRNA by rsmG contributes to proper ribosomal assembly and function. This post-transcriptional modification likely impacts translation accuracy and efficiency, potentially influencing bacterial fitness under various growth conditions. Given that B. thetaiotaomicron has been studied across 15 different growth conditions in transcriptomic studies, the expression pattern of rsmG might vary depending on environmental factors .

The importance of rsmG can be further understood by integrating expression data with transposon mutant fitness data, as has been done for other genes in B. thetaiotaomicron. This approach allows researchers to correlate gene expression levels with functional importance under specific conditions, providing insights into when and where rsmG activity is most critical for bacterial survival and adaptation.

How is rsmG expression regulated in Bacteroides thetaiotaomicron?

The regulation of rsmG expression in Bacteroides thetaiotaomicron likely involves complex transcriptional networks that respond to environmental cues. Based on the transcriptome atlas developed for B. thetaiotaomicron, we can infer that rsmG expression might be influenced by stress conditions and available carbon sources . The comprehensive RNA-seq data across 15 different growth conditions provides a framework for understanding how rsmG expression changes in response to environmental factors.

Transcriptional regulation in B. thetaiotaomicron involves promoter elements, transcription start sites (TSSs), and potentially invertible DNA regions (invertons). The expanded transcriptome atlas identified 4,123 TSSs across the B. thetaiotaomicron chromosome and plasmid, with 252 unique TSS annotations contributed by the 15-condition pool . Understanding the specific TSS associated with rsmG would provide insights into its transcriptional regulation.

Additionally, the presence of invertible promoters in B. thetaiotaomicron suggests a potential mechanism for phase-variable expression of certain genes. While the search results do not specifically mention rsmG in relation to invertible elements, this regulatory mechanism should be considered when studying its expression patterns. Integration of expression data with the map of invertible DNA regions could reveal whether rsmG is subject to phase-variable regulation, which would have implications for its function under different environmental conditions.

What is the conservation of rsmG across different bacterial species?

The rsmG gene appears to be conserved across diverse bacterial phyla, suggesting its fundamental importance in ribosomal RNA modification. In Bacillus subtilis, RsmG methylates the N7 position of nucleotide G535 in 16S rRNA, while in Escherichia coli, it targets the corresponding G527 position . This conservation of function across phylogenetically distant bacteria (Firmicutes and Proteobacteria) suggests that Bacteroides thetaiotaomicron (a Bacteroidetes) likely maintains a similar methyltransferase activity.

Sequence alignment and phylogenetic analysis of rsmG across bacterial species would reveal the degree of conservation at the amino acid level, particularly in functional domains responsible for substrate binding and catalytic activity. Such comparative analyses could identify both highly conserved residues essential for function and variable regions that might confer species-specific properties.

The conservation extends to the target site in 16S rRNA as well. The G535/G527 position (B. subtilis/E. coli numbering) is located in a functionally important region of the small ribosomal subunit. Structural studies of ribosomes suggest that methylation at this position could influence interactions between the 16S rRNA and other ribosomal components or translation factors. Comparative analysis of 16S rRNA sequences from B. thetaiotaomicron and other bacteria would help identify the corresponding target nucleotide and surrounding structural context.

How can we design experiments to measure the effects of rsmG mutation in Bacteroides thetaiotaomicron?

Designing experiments to measure the effects of rsmG mutation in Bacteroides thetaiotaomicron requires careful consideration of randomization strategies and statistical power. The randomized saturation (RS) design described in search result provides a framework for measuring direct treatment effects and spillover effects. In the context of rsmG research, this could involve creating a population of B. thetaiotaomicron with varying proportions of wild-type and rsmG mutant cells to assess both cell-autonomous effects and potential intercellular influences.

A non-trivial randomized saturation design should include at least two saturations (proportions of treated units), with at least one being strictly interior (between 0 and 1) . This approach allows for the identification of both direct effects of rsmG mutation on the mutated cells and spillover effects on wild-type cells within the same culture or community. For instance, researchers could establish multiple experimental groups with different proportions of rsmG mutants (e.g., 0%, 25%, 50%, 75%, 100%) and measure outcomes such as growth rates, antibiotic susceptibility, or gene expression patterns.

Statistical power considerations are crucial for detecting both average treatment effects and saturation slope effects. Power is influenced by the effect size, intra-cluster correlation of outcomes, the share of individuals assigned to each treatment saturation, and the variance of saturations . Researchers should conduct power calculations based on preliminary data to determine the optimal experimental design parameters, including sample size and allocation across different saturation levels.

Additionally, integration with transposon mutant fitness data could provide insights into the phenotypic consequences of rsmG disruption under various growth conditions. This approach, as demonstrated in the study of B. thetaiotaomicron , allows researchers to correlate gene expression levels with functional importance, potentially revealing condition-specific roles of rsmG.

What transcriptomic approaches are most suitable for studying rsmG expression patterns?

For comprehensive analysis of rsmG expression patterns in Bacteroides thetaiotaomicron, researchers should employ a combination of differential RNA sequencing (dRNA-seq) and conventional RNA-seq methodologies. The dRNA-seq approach, which distinguishes between primary transcripts (with 5′ triphosphate ends) and processed RNAs, enables precise mapping of transcription start sites (TSSs) . This technique is crucial for understanding the transcriptional regulation of rsmG, including promoter architecture and potential alternative start sites.

Conventional RNA-seq complements dRNA-seq by providing quantitative information on gene expression levels across different conditions. The expanded transcriptome atlas for B. thetaiotaomicron incorporated data from 15 different growth conditions, including various stress situations and carbon sources . Similar comprehensive profiling for rsmG would reveal how its expression responds to environmental changes, potentially indicating functional roles under specific conditions.

Analysis of the resulting transcriptomic data should leverage tools like the ANNOgesic pipeline for TSS mapping and operon structure prediction . Integration with other genomic features, such as invertible DNA regions (invertons) identified by PhaseFinder, could reveal potential phase-variable regulation of rsmG . Additionally, correlation analysis between rsmG expression and other genes might identify co-regulated gene clusters, suggesting functional relationships.

For validation of transcriptomic findings, quantitative RT-PCR remains a valuable approach. This technique can provide more precise quantification of rsmG expression across conditions and can be used to verify key findings from high-throughput sequencing. Additionally, reporter gene constructs (e.g., rsmG promoter fused to fluorescent proteins) can provide insights into expression dynamics at the single-cell level, potentially revealing heterogeneity within bacterial populations.

How does rsmG activity interact with other RNA modification enzymes?

The interaction between rsmG and other RNA modification enzymes in Bacteroides thetaiotaomicron likely forms a complex network affecting ribosome biogenesis and function. RNA modifications in 16S rRNA are typically clustered in functionally important regions, suggesting potential cooperative or competitive interactions between different modification enzymes. While specific interactions involving B. thetaiotaomicron rsmG are not detailed in the search results, research approaches can be designed based on general principles of RNA modification networks.

Co-expression analysis using the comprehensive transcriptome atlas of B. thetaiotaomicron could identify other RNA modification enzymes with similar expression patterns to rsmG across the 15 studied growth conditions. Genes with highly correlated expression profiles might be functionally related, potentially indicating coordinated activity or regulatory relationships. This approach could identify candidate interacting partners for further investigation.

Genetic interaction studies, such as creating double mutants of rsmG and other RNA modification enzymes, would provide functional evidence for interactions. Synthetic phenotypes (where the double mutant shows a more severe or different phenotype than expected from individual mutations) would suggest functional relationships between the genes. These studies could be complemented by biochemical approaches, such as in vitro methylation assays with purified enzymes, to test for direct effects on each other's activities.

Mass spectrometry analysis of 16S rRNA modifications in wild-type, rsmG mutant, and other RNA modification mutant backgrounds would reveal the interdependence of different modifications. If the absence of rsmG affects other modifications, this would suggest a sequential or hierarchical relationship between modification events during ribosome biogenesis.

What are the optimal conditions for expressing recombinant Bacteroides thetaiotaomicron rsmG?

Expressing recombinant Bacteroides thetaiotaomicron rsmG requires careful optimization of expression systems, considering the significant differences in GC content and codon usage between B. thetaiotaomicron (a low-GC Bacteroidetes) and common expression hosts like Escherichia coli. Based on general principles for expressing Bacteroides proteins, several approaches can be considered.

For E. coli-based expression systems, codon optimization of the B. thetaiotaomicron rsmG sequence is recommended to avoid rare codon usage that might limit translation efficiency. Expression vectors with moderate-strength promoters (e.g., T7lac rather than strong T7) often yield better results for potentially toxic proteins like methyltransferases. Initial expression trials should test multiple conditions, varying parameters such as:

• Induction temperature (typically lower temperatures, 16-25°C, reduce inclusion body formation)

• Inducer concentration (lower IPTG concentrations, 0.1-0.5 mM, may improve soluble protein yield)

• Expression duration (4-16 hours post-induction)

• Media composition (rich media like LB vs. minimal media)

For purification, a small affinity tag (e.g., 6xHis) is preferable to larger fusion partners that might interfere with enzymatic activity. If solubility remains challenging, fusion with solubility-enhancing tags like SUMO or MBP can be considered, with subsequent tag removal using specific proteases.

Alternatively, homologous expression in B. thetaiotaomicron using its native promoter elements might yield more naturally folded protein. The comprehensive TSS mapping data available for B. thetaiotaomicron could inform the design of expression constructs incorporating native regulatory elements. This approach might be particularly valuable if rsmG requires specific cellular factors from B. thetaiotaomicron for proper folding or activity.

What experimental approaches can verify the methylation target of rsmG?

Verifying the methylation target of rsmG in Bacteroides thetaiotaomicron requires a multi-faceted approach combining genetic, biochemical, and analytical techniques. Based on the known target of RsmG in other bacteria (N7 position of G535 in 16S rRNA of Bacillus subtilis, corresponding to G527 in E. coli) , researchers can develop targeted strategies to identify the analogous position in B. thetaiotaomicron.

A primary approach involves comparative sequence analysis of 16S rRNA across bacterial species to identify the corresponding nucleotide in B. thetaiotaomicron. Once identified, site-specific analysis of methylation can be performed using several complementary techniques:

• Mass spectrometry analysis of 16S rRNA from wild-type and rsmG mutant strains can directly detect methylation differences. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of enzymatically digested 16S rRNA allows precise mapping of modified nucleotides.

• Primer extension analysis can indirectly detect methylation at specific sites. N7-methylation of guanosine can cause reverse transcriptase to pause or incorporate incorrect nucleotides, resulting in characteristic patterns in primer extension products.

• In vitro methylation assays with purified recombinant rsmG protein and synthetic RNA substrates containing the putative target sequence can confirm direct methylation activity. Tritium-labeled S-adenosylmethionine (SAM) as methyl donor allows quantitative measurement of methylation.

• CRISPR-Cas9-mediated mutation of the putative target nucleotide in 16S rRNA, followed by phenotypic analysis and methylation detection, can provide genetic evidence for the target site.

For a comprehensive verification, researchers should combine these approaches, as each provides different and complementary evidence. The integration of in vivo, in vitro, and analytical data provides the strongest case for identifying the specific methylation target.

How can we use the Theta-Base web browser for rsmG research?

The Theta-Base web browser (http://micromix.helmholtz-hiri.de/bacteroides/) provides a valuable resource for researchers studying rsmG in Bacteroides thetaiotaomicron by integrating transcriptomic data across multiple growth conditions . To effectively utilize this resource for rsmG research, researchers should follow a systematic approach to extract and analyze relevant data.

Researchers can begin by accessing expression data for rsmG across the 15 different growth conditions represented in the database. This provides insights into how rsmG expression responds to various environmental factors, including stress conditions and different carbon sources. Differential expression analysis can identify conditions where rsmG is significantly up- or down-regulated, suggesting functional importance under those specific circumstances.

The TSS mapping data available through Theta-Base can reveal the transcriptional architecture of rsmG, including its promoter structure and potential operonic organization . Identifying the precise TSS for rsmG helps understand its transcriptional regulation and design expression constructs for recombinant production. Additionally, termination site predictions can delineate the complete transcriptional unit containing rsmG.

Integration of expression data with transposon mutant fitness data, also available through Theta-Base, allows researchers to correlate rsmG expression levels with its functional importance under different conditions . This approach can reveal condition-specific phenotypes associated with rsmG disruption, providing clues to its physiological roles.

For comparative analysis, researchers can examine expression patterns of other RNA modification enzymes alongside rsmG. Co-expression analysis might identify functionally related genes that show similar expression dynamics across conditions. Additionally, exploring the expression of translation-related genes, such as ribosomal proteins and translation factors, could reveal coordinated regulation with rsmG, suggesting functional relationships.

How does rsmG expression change under different stress conditions?

The expression pattern of rsmG in Bacteroides thetaiotaomicron likely varies across different stress conditions, reflecting its role in modulating ribosome function in response to environmental changes. Although the search results don't specifically detail rsmG expression changes, we can infer potential patterns based on the comprehensive transcriptomic data collected across 15 different growth conditions .

Stress responses in B. thetaiotaomicron involve significant transcriptional reprogramming, as evidenced by the differential expression of genes like fusA and fusA2 under carbon starvation and mucin utilization conditions . These translation elongation factor genes show inverse expression patterns, with fusA2 being induced during carbon deprivation while canonical fusA is downregulated. This suggests that B. thetaiotaomicron utilizes a distinct protein synthesis machinery under nutrient limitation .

As a ribosomal RNA modification enzyme, rsmG might show similar condition-specific expression patterns, potentially coordinated with other components of the translation apparatus. Bile salt exposure, antibiotics (like gentamicin), and temperature shifts were among the stress conditions studied in the transcriptome atlas . These conditions are known to affect bacterial translation, suggesting potential regulation of rsmG in response to these stressors.

Particularly relevant might be the response to antibiotics that target the ribosome, such as gentamicin. Since rsmG methylates 16S rRNA, its expression might be altered in response to antibiotics that bind to the small ribosomal subunit. Understanding these expression changes could provide insights into potential roles of rsmG in antibiotic tolerance or resistance mechanisms in B. thetaiotaomicron.

What insights can transposon mutant fitness data provide about rsmG function?

Transposon mutant fitness data integrated with expression analysis can provide critical insights into the functional importance of rsmG under various conditions. The approach used for B. thetaiotaomicron in the expanded transcriptome atlas study demonstrates how such integration can reveal condition-specific gene functions and regulatory relationships.

For rsmG research, transposon mutant fitness data would indicate the growth phenotypes associated with rsmG disruption across different conditions. Decreased fitness of rsmG mutants under specific conditions would suggest essential functions under those circumstances. Conversely, neutral or positive fitness effects might indicate conditions where rsmG activity is dispensable or potentially detrimental.

The integration of fitness data with expression profiles across the 15 studied conditions would reveal whether rsmG expression correlates with its functional importance. For instance, upregulation of rsmG under conditions where its disruption causes fitness defects would suggest adaptive expression regulation. Discordance between expression and fitness effects might indicate more complex regulatory relationships, potentially involving post-transcriptional mechanisms.

Comparative analysis with other RNA modification enzymes could reveal functional relationships. Similar fitness profiles across conditions might suggest functional redundancy or cooperation between different modification pathways. Additionally, examining the fitness effects of mutations in potential target sites in 16S rRNA could provide complementary evidence for the functional importance of specific modifications.

How does rsmG relate to antibiotic resistance mechanisms?

The relationship between rsmG and antibiotic resistance in Bacteroides thetaiotaomicron represents an important area for investigation, particularly given the known connections between 16S rRNA modifications and antibiotic susceptibility in other bacteria. While the search results don't directly address this relationship for B. thetaiotaomicron rsmG, we can draw insights from related findings.

The expanded transcriptome atlas for B. thetaiotaomicron revealed the small RNA MasB (BTnc201), which regulates susceptibility to tetracyclines . This finding demonstrates that post-transcriptional regulatory mechanisms can influence antibiotic tolerance in this bacterium. Similarly, rsmG-mediated methylation of 16S rRNA might affect the binding of antibiotics that target the small ribosomal subunit, potentially modulating susceptibility.

Experimental approaches to investigate this relationship could include:

• Comparative antibiotic susceptibility testing of wild-type and rsmG mutant B. thetaiotaomicron across a panel of antibiotics, with particular focus on those targeting protein synthesis

• Analysis of rsmG expression in response to sub-inhibitory concentrations of various antibiotics

• Selection of spontaneous antibiotic-resistant mutants followed by sequencing of rsmG and its target site in 16S rRNA

• In vitro binding studies to assess how rsmG-mediated methylation affects antibiotic binding to the 30S ribosomal subunit

Understanding the role of rsmG in antibiotic resistance mechanisms could have significant implications for addressing the increasing challenge of antimicrobial resistance in Bacteroides species, which are important components of the human gut microbiome and can serve as reservoirs for resistance genes.