Recombinant Vibrio vulnificus Ribosomal RNA large subunit methyltransferase I (rlmI)

What is Vibrio vulnificus and why is it significant for rRNA methyltransferase research?

Vibrio vulnificus is an estuarine bacterium capable of causing severe and rapidly disseminating septicemia in susceptible hosts, as well as serious wound infections. While septicemia primarily affects immunocompromised individuals or those with conditions like cirrhosis or hemochromatosis, healthy individuals can become infected through wounds . The bacterium expresses numerous virulence determinants, making it an important model for studying how post-transcriptional modifications of rRNA, including methylation by enzymes like rlmI, might influence pathogenicity.

Research methodology for establishing this relationship typically involves:

• Molecular characterization of clinical and environmental isolates

• Comparative genomic analysis across Vibrio species

• Functional studies using knockout mutants

• Virulence assessment using in vitro and in vivo models

How do specialized ribosomes contribute to bacterial adaptation in Vibrio vulnificus?

Specialized ribosomes bearing heterogeneous rRNAs appear to play a conserved role in translational regulation among Vibrio species. Studies have demonstrated that V. vulnificus possesses distinct rRNA operons (such as rrnI) that contribute to specialized ribosomes (I-ribosomes) with unique functions . These I-ribosomes preferentially associate with specific mRNAs, such as those encoding heat shock proteins like HspA.

The methodology to investigate this phenomenon includes:

• Cloning and expressing different rRNA operons in V. vulnificus strains

• Co-immunoprecipitation studies to identify ribosome-nascent peptide associations

• RT-PCR quantification of specific rRNAs in different cellular fractions

• Functional assays to assess the impact on stress tolerance

For example, when rrnI is overexpressed in V. vulnificus MO6-24/O strains, there is increased bacterial survival under heat shock conditions, suggesting that these specialized ribosomes selectively translate stress-response mRNAs .

What experimental approaches are used to study rRNA modifications in Vibrio species?

The investigation of rRNA modifications in Vibrio species employs several complementary experimental approaches:

• Genetic manipulation techniques:

  • Random transposon mutagenesis for screening phenotypes

  • Allelic exchange for targeted gene inactivation

  • Complementation studies using broad-host-range vectors

• Structural analysis methods:

  • Secondary structure prediction of rRNAs using M-fold program

  • Phylogenetic analysis of rRNA operons across different strains

  • Sequence alignments to identify conserved modification sites

• Functional characterization:

  • Heat shock susceptibility assays

  • CFU measurements under varying conditions

  • Western blot analysis to determine protein expression levels

  • Co-immunoprecipitation to study RNA-protein interactions

These approaches have revealed that specific rRNA operons, which may be targets for methyltransferases, significantly impact bacterial survival during stress conditions .

How do virulence factor secretion systems interact with ribosomal components in Vibrio vulnificus?

Vibrio vulnificus employs various secretion systems to export virulence factors, with potential interactions between these systems and specialized ribosomes. For example:

• The type II secretion pathway exports multiple extracellular degradative enzymes, including cytolysin/hemolysin .

• The RTX toxin secretion system requires an ATP binding protein (RtxE) for proper function .

• Expression of secretion system components may be regulated at the translational level by specialized ribosomes.

Research has shown that mutations in secretion components significantly reduce cytotoxicity, adherence to host cells, and virulence in mouse models . For instance, an RtxE mutant exhibited a 10⁴ to 10⁵-fold increase in LD₅₀ compared to the wild type in mouse infection models . These studies suggest possible coordination between specialized translation machinery (potentially involving methyltransferases) and virulence factor production.

What is the relationship between rRNA methyltransferases and specialized ribosome formation?

RNA methyltransferases, including those targeting the large ribosomal subunit like rlmI, are believed to contribute to ribosome heterogeneity through post-transcriptional modifications. While the search results don't directly address rlmI, they do demonstrate that heterogeneous rRNAs play important roles in translational regulation in Vibrio species.

Methodological approaches to investigate this relationship include:

• Comparative genomic analysis:

  • Identifying methyltransferase genes across Vibrio species

  • Analyzing conservation patterns and genomic context

• Biochemical characterization:

  • Expressing recombinant methyltransferases

  • In vitro methylation assays with purified rRNA substrates

  • Mass spectrometry to map modification sites

• Functional studies:

  • Generating methyltransferase knockout strains

  • Ribosome profiling to assess translation patterns

  • Assessing impacts on stress responses and virulence

The analysis of specialized ribosomes has revealed that I-ribosomes from V. vulnificus CMCP6 preferentially translate specific mRNA targets, contributing to bacterial survival under stress conditions .

How do environmental conditions influence expression of rRNA operons and methyltransferases?

The expression of rRNA operons and potentially their modification by methyltransferases appears to be regulated by environmental conditions, particularly host cell contact. For example:

• The rtxBDE genes in V. vulnificus are transcribed as one transcriptional unit under the control of a single promoter, P_rtxBDE .

• Activity of this promoter is induced specifically by exposure to INT-407 intestinal epithelial cells .

• Induction requires direct contact between the bacteria and host cells, as exposure to cell culture medium alone does not elicit the same response .

This host-responsive regulation suggests complex environmental sensing mechanisms that may extend to rRNA operons and modification enzymes. Research methodologies to investigate these phenomena include:

• Primer extension analysis to quantify transcription from specific promoters

• RNA isolation from bacteria exposed to different environmental conditions

• Comparative transcriptomics in the presence/absence of host cells

• Promoter-reporter fusion constructs to visualize gene expression

What computational approaches can identify potential methyltransferase domains and targets?

Advanced computational methods are essential for identifying methyltransferase domains and their potential targets in Vibrio species:

• Sequence-based approaches:

  • BLAST searches against known methyltransferase databases

  • Multiple sequence alignments to identify conserved catalytic motifs

  • Phylogenetic analysis to classify enzyme families

• Structural prediction:

  • Homology modeling based on characterized methyltransferases

  • Molecular docking to predict rRNA binding sites

  • Molecular dynamics simulations to assess interaction stability

• Target identification:

  • Consensus sequence analysis for modification sites

  • Secondary structure prediction of rRNA using tools like M-fold

  • Comparative analysis of rRNA operons within and between species

The search results demonstrate the application of some of these approaches, such as neighbor-joining phylogenetic trees for 16S and 23S rRNA sequences showing the positions of rRNA operons within Vibrio genomes .

How do mutations in rRNA methyltransferases affect bacterial physiology and virulence?

While the search results don't directly address mutations in rRNA methyltransferases, they do provide insights into how alterations in related systems affect Vibrio vulnificus physiology and virulence:

• Physiological impacts:

  • Disruption of type IV leader peptidase/N-methyltransferase (PilD) prevents pili expression

  • Mutants lacking functional secretion systems show growth defects under certain conditions

  • Heat shock susceptibility increases in strains lacking specialized ribosomes

• Virulence effects:

  • PilD mutants exhibit decreased cytotoxicity, reduced adherence to host cells, and attenuated virulence in mouse models

  • RtxE mutants show 10⁴-10⁵ times higher LD₅₀ values compared to wild-type strains

  • Specialized ribosomes contribute to bacterial survival in host environments

Methodological approaches to study these effects include:

• Construction of isogenic mutants through allelic exchange

• Complementation studies to confirm phenotype specificity

• Mouse infection models with LD₅₀ determination

• In vitro cytotoxicity assays using human cell lines

What is the role of specialized ribosomes in translational regulation during stress response?

Research indicates that specialized ribosomes bearing heterogeneous rRNAs play a critical role in translational regulation during stress responses in Vibrio vulnificus:

• Preferential translation:

  • I-ribosomes preferentially associate with HspA nascent peptides in V. vulnificus

  • This association was demonstrated through co-immunoprecipitation studies

• Stress tolerance:

  • Overexpression of rrnI in V. vulnificus MO6-24/O leads to increased bacterial survival during heat shock

  • Wild-type strains show higher survival rates at 45°C compared to strains lacking specialized ribosomes

• Conservation across species:

  • Phylogenetic analysis reveals conservation of specialized rRNA operons across Vibrio species

  • This conservation suggests fundamental importance in bacterial adaptation

The table below summarizes experimental findings regarding specialized ribosome function in V. vulnificus:

This experimental data demonstrates that specialized ribosomes, potentially modified by methyltransferases, significantly impact bacterial adaptation to environmental stresses .