Recombinant Enterococcus faecalis Ribonuclease Y (rny), partial

What is the basic function of Ribonuclease Y in Enterococcus faecalis?

Ribonuclease Y (RNase Y) in E. faecalis functions as an endoribonuclease involved in RNA processing and decay, playing a crucial role in the regulation of gene expression. While RNase Y can act on 5ʹ triphosphate RNA ends, it demonstrates preference for 5ʹ monophosphate ends . Unlike some other RNases in E. faecalis (such as RNase J1), RNase Y is not essential for bacterial survival but contributes significantly to general fitness of the organism . The protein participates in the bacterial RNA degradosome complex, a multi-protein machinery responsible for RNA turnover, and interacts with DEAD-box RNA helicases to facilitate RNA processing . Studies have also indicated that RNase Y plays a role in the virulence and stress response of E. faecalis, with its expression being upregulated during host infection scenarios, such as mouse peritonitis .

How conserved is RNase Y across different Enterococcus species?

RNase Y is highly conserved across Enterococcus species, though with some variation in conservation levels compared to other RNA metabolism proteins. Analysis of available genomic data reveals that the gene encoding RNase Y (rny) is present in 97-100% of enterococcal strains . Using the NCBI genome database and tBLASTn tool, researchers have identified genes encoding proteins sharing more than 70% identity with RNase Y in various Enterococci (from a database of 96 complete genomes) . Beyond the Enterococcus genus, RNase Y is also found in related genera such as Carnobacterium, Clostridioides, Tetragenococcus, and Vagococcus, as well as in Listeria monocytogenes, with protein identity levels exceeding 50% . This conservation pattern suggests the fundamental importance of RNase Y in RNA metabolism across Gram-positive bacteria, making it a valuable target for comparative genomic and functional studies.

How does E. faecalis RNase Y differ structurally and functionally from similar enzymes in other bacterial species?

E. faecalis RNase Y shares functional similarities with homologs in other Gram-positive bacteria, particularly Bacillus subtilis, but with distinct structural features that influence its specific activities. While both function as endoribonucleases within their respective degradosome complexes, E. faecalis RNase Y exhibits characteristic domain organization that affects its substrate specificity and protein-protein interactions . Unlike the well-studied RNase E in Gram-negative bacteria, E. faecalis RNase Y belongs to a different structural family but fulfills analogous roles in RNA processing. The E. faecalis enzyme contains membrane-binding domains that localize it to the cell membrane, where it can coordinate with other components of the RNA degradation machinery .

Functionally, E. faecalis RNase Y appears to have a more prominent role in general fitness rather than being essential for viability (as demonstrated by successful creation of deletion mutants), which contrasts with some other bacterial species where RNase Y homologs are essential . This functional difference may reflect the specific adaptation of E. faecalis to its ecological niches and its unique RNA regulatory networks.

What are the optimal conditions for expressing recombinant E. faecalis RNase Y in heterologous systems?

For optimal expression of recombinant E. faecalis RNase Y, researchers should consider both the expression system and purification strategy. Based on structural similarities with other bacterial RNases, expression in E. coli BL21(DE3) with the pET expression system has proven effective for related proteins. The following protocol represents a methodological approach:

• Vector design: Clone the E. faecalis rny gene (partial or full-length) into a pET vector with an N-terminal His-tag for purification. Include a TEV protease cleavage site if tag removal is desired.

• Expression conditions: Grow transformed E. coli at 37°C to OD600 of 0.6-0.8, then induce with 0.5-1.0 mM IPTG. Reduce temperature to 18-25°C post-induction and continue expression for 12-16 hours to maximize proper folding.

• Lysis and purification: Use phosphate buffer (pH 7.4) containing 300mM NaCl, 5-10% glycerol, and protease inhibitors for cell lysis . Purify using Ni-NTA affinity chromatography followed by size exclusion chromatography.

• Stabilizing additives: Include 1mM DTT or 2mM β-mercaptoethanol to maintain reduced cysteines, and consider adding 1-2mM MgCl2 as cofactor for optimal enzyme conformation.

• Storage: Store in buffer containing 20-50% glycerol at -80°C in small aliquots to maintain enzyme activity through multiple freeze-thaw cycles.

Critical considerations include membrane association (which may necessitate detergent use during purification) and potential co-expression with interaction partners to improve solubility and stability. For functional studies, recombinant protein should be validated by testing nuclease activity on model RNA substrates under varying pH and divalent cation concentrations.

What experimental approaches are recommended for studying the in vivo interactions of RNase Y in the E. faecalis RNA degradosome?

To study the in vivo interactions of RNase Y within the E. faecalis RNA degradosome, a multi-faceted approach incorporating several complementary techniques is recommended:

• Gradient profiling by sequencing (Grad-seq): This technique has been successfully employed to comprehensively predict complexes formed by RNA and proteins in E. faecalis . Grad-seq allows the separation of cellular complexes based on their sedimentation behavior in a glycerol gradient, followed by RNA sequencing and mass spectrometry to identify components. This approach can reveal the interactome of RNase Y and its association with different RNAs and proteins in native conditions .

• Co-immunoprecipitation (Co-IP) with RNase Y-specific antibodies: This approach allows for the identification of proteins that directly interact with RNase Y in vivo. Tagged versions of RNase Y (with epitope tags like FLAG or HA) can be used if specific antibodies are unavailable. RNA sequencing of co-precipitated material (RIP-seq) can further identify RNA targets .

• Bacterial two-hybrid assays: These can be used to validate direct protein-protein interactions between RNase Y and other components of the degradosome, such as DEAD-box RNA helicases (CshA, CshB, CshC) that have been implicated in RNA metabolism in E. faecalis .

• Cross-linking and mass spectrometry: This approach can capture transient interactions and provide structural insights into the architecture of the degradosome complex.

• Fluorescence microscopy with fusion proteins: Tagging RNase Y and potential interaction partners with fluorescent proteins can reveal their co-localization patterns within bacterial cells.

For data validation, these approaches should be combined with functional studies using mutant strains lacking specific interaction partners to assess the impact on RNase Y activity and RNA processing patterns. Researchers should particularly focus on interactions with DEAD-box helicases like CshA, which have demonstrated tailing toward RNAP and ribosomal fractions in gradient analyses, suggesting stable interactions with cellular partners .

What controls should be included when designing knockout or depletion experiments for E. faecalis rny?

When designing knockout or depletion experiments for E. faecalis rny, several critical controls should be implemented to ensure reliable and interpretable results:

• Complementation controls: Generate a complementation strain where the rny gene is reintroduced (either on a plasmid or integrated into the chromosome at a neutral site) to verify that observed phenotypes are specifically due to the absence of RNase Y rather than polar effects or secondary mutations .

• Wild-type comparisons: Always include the parental wild-type strain in all experiments as the primary reference point. In studies examining multiple deletion mutants, include each single mutant alongside combination mutants to assess individual contributions .

• Empty vector controls: When using plasmid-based complementation, include strains carrying the empty vector to control for effects of the vector itself or selection markers .

• Conditional expression systems: For depletion experiments, use titratable expression systems and include controls with varying levels of induction to establish dose-dependent relationships between RNase Y expression and observed phenotypes.

• Growth condition controls: Test mutant strains under multiple growth conditions, including standard laboratory conditions and stress conditions (oxidative stress, temperature variation, nutrient limitation) to comprehensively assess the role of RNase Y in different physiological states .

• Molecular verification: Confirm the absence of the rny gene and its product using PCR, RT-qPCR, and Western blotting to verify knockout efficiency. For depletion experiments, quantify residual RNase Y levels.

• Phenotypic stability assessment: Monitor phenotypic stability over multiple generations to identify potential suppressors or compensatory mutations.

Previous research has demonstrated that while RNase Y is not essential in E. faecalis (as deletion mutants could be successfully constructed), it does contribute to general fitness and stress response . Therefore, researchers should be particularly attentive to subtle growth defects and stress response phenotypes when characterizing rny mutants.

How can researchers distinguish between direct and indirect effects of RNase Y activity on gene expression patterns in E. faecalis?

Distinguishing between direct and indirect effects of RNase Y on gene expression requires a multi-layered experimental approach that combines genomic, transcriptomic, and biochemical techniques:

• RNA-seq with rifampicin treatment: Treat cells with rifampicin to inhibit transcription initiation, then compare RNA decay rates between wild-type and rny mutant strains. RNAs that show differential stability are potential direct targets of RNase Y. This approach allows for genome-wide identification of transcripts whose stability is directly affected by RNase Y activity .

• CLIP-seq or RIP-seq analysis: Perform cross-linking immunoprecipitation followed by sequencing (CLIP-seq) or RNA immunoprecipitation sequencing (RIP-seq) with tagged RNase Y to identify RNAs that physically interact with the enzyme in vivo . This technique has been successfully applied to other RNA-binding proteins in enterococci and provides direct evidence of RNA-protein interactions.

• In vitro RNA cleavage assays: Use purified recombinant RNase Y with synthetic or in vitro transcribed RNA substrates to determine sequence or structural motifs that are directly recognized and cleaved by the enzyme. Compare these results with in vivo data to identify consistent patterns.

• Temporal analysis of transcriptome changes: Perform time-course experiments following RNase Y depletion to distinguish primary effects (occurring rapidly) from secondary effects (occurring after extended depletion).

• Global RNA structure mapping: Combine RNA structure probing techniques (such as SHAPE-seq) with RNase Y activity assays to correlate structural features with cleavage efficiency.

• Comparative analysis with other RNase mutants: Compare transcriptome changes in rny mutants with those in other RNase mutants (such as rnj1, rnj2, rnc) to identify unique versus overlapping effects .

The most compelling evidence for direct effects comes from the convergence of multiple approaches—for example, when a transcript shows altered stability in an rny mutant, physically associates with RNase Y in CLIP-seq experiments, and contains sequences or structures that are cleaved by RNase Y in vitro.

What is the role of RNase Y in E. faecalis stress response and virulence regulation?

RNase Y plays significant roles in both stress response and virulence regulation in E. faecalis, acting as a post-transcriptional regulator that modulates gene expression under changing environmental conditions:

• Stress response regulation: Studies have shown that RNase Y contributes to E. faecalis adaptation to various stressors. While RNases J2 and III have been specifically implicated in cold, oxidative, and bile salt stress responses, RNase Y appears to have a broader role in general fitness and adaptability . The enzyme likely modulates the stability of stress-responsive transcripts, controlling their abundance and thus influencing the cellular response to adverse conditions.

• Virulence factor expression: Experimental evidence indicates that the rny gene is significantly induced (2-4 fold) during mouse peritonitis, suggesting its important role during host infection . This upregulation was confirmed by RT-PCR analysis of bacterial cells recovered after 24 hours of mice peritonitis. The induction of rny alongside other RNA metabolism genes (rnjA, rnjB, rnc, and cshA) during infection indicates that RNA processing and turnover are critical aspects of E. faecalis pathogenicity .

• Host colonization: The elevated expression of RNase Y during infection suggests its contribution to E. faecalis survival or colonization inside the host . The enzyme may regulate the expression of adhesins, immune evasion factors, or metabolic adaptations required for persistence in host tissues.

• Integration with regulatory networks: RNase Y likely operates within a network of regulatory factors, including small RNAs and RNA-binding proteins, to orchestrate complex stress and virulence responses. The Grad-seq analysis revealed potential interactions between RNases, RNA-binding proteins like KhpB, and various RNA species, suggesting coordinate regulation of RNA metabolism during stress and infection .

To fully elucidate the role of RNase Y in these processes, researchers should design experiments comparing wild-type and rny mutant strains in various infection models and stress conditions, combined with transcriptomic and proteomic analyses to identify the specific targets and pathways affected by RNase Y activity.

How does RNase Y interact with small RNAs and other RNA-binding proteins to regulate post-transcriptional gene expression in E. faecalis?

RNase Y interacts with small RNAs (sRNAs) and RNA-binding proteins (RBPs) in E. faecalis to form a sophisticated post-transcriptional regulatory network. These interactions occur through several mechanisms:

• sRNA processing and turnover: RNase Y likely contributes to the maturation and degradation of sRNAs, affecting their abundance and activity. Unlike Gram-negative bacteria that utilize Hfq as their major sRNA-binding protein, E. faecalis lacks Hfq, CsrA, and ProQ homologs . This suggests alternative mechanisms for sRNA-mediated regulation involving other RBPs and RNases, including RNase Y.

• Co-regulatory activities with DEAD-box RNA helicases: RNase Y functionally interacts with DEAD-box RNA helicases (CshA, CshB, CshC), which are key players in RNA metabolism . These helicases can remodel RNA structures, potentially facilitating RNase Y access to cleavage sites. Interactions between RNase Y and these helicases have been observed in experiments, suggesting coordinated activity in RNA processing .

• Degradosome formation: Evidence suggests that E. faecalis possesses a degradosome structure similar to that in Bacillus subtilis, involving interactions between RNases and DEAD-box helicases . Within this complex, RNase Y likely cooperates with other enzymes to regulate RNA turnover. The Grad-seq analysis has revealed potential components of this machinery and their sedimentation profiles, providing insights into complex formation .

• Interactions with emerging RBPs: The Grad-seq analysis identified potential RBPs such as KhpA and KhpB, whose sedimentation profiles correlate with sRNA clusters . These proteins showed strong tailing toward the RNAP and 30S fractions, suggesting their involvement in RNA metabolism. RNase Y may interact with these proteins to regulate target transcripts.

• Regulatory RNA-protein networks: The analysis of RNA-protein complexes in E. faecalis identified established complexes such as the 6S RNA-RNA polymerase complex . Similar regulatory networks likely exist involving RNase Y, sRNAs, and target mRNAs.

To study these interactions, researchers should employ techniques such as RIP-seq to identify RNA targets of specific proteins (as demonstrated with KhpB ), combined with genetic studies examining the effects of deleting multiple components of these regulatory networks. Understanding these interactions will provide insights into the unique mechanisms of post-transcriptional regulation in E. faecalis, which lacks many of the classic regulatory proteins found in other bacteria.

What are the common challenges in purifying active recombinant RNase Y and how can these be overcome?

Purifying active recombinant RNase Y from E. faecalis presents several technical challenges that researchers should anticipate and address:

• Membrane association: RNase Y contains membrane-binding domains that can cause aggregation and reduced solubility during purification. To overcome this:

  • Consider expressing truncated versions lacking the membrane-binding domain for structural studies

  • Use mild detergents (0.1-1% Triton X-100 or n-dodecyl β-D-maltoside) in extraction buffers

  • Perform extraction at lower temperatures (4-16°C) to minimize aggregation

• Enzyme instability: RNases are generally prone to denaturation and loss of activity during purification. Strategies to maintain stability include:

  • Adding RNase inhibitors during initial extraction steps to prevent uncontrolled RNA degradation

  • Including stabilizing agents (10% glycerol, 1mM DTT) in all buffers

  • Incorporating divalent cations (1-2mM MgCl2 or MnCl2) that serve as cofactors

• Contaminating RNase activity: Bacterial expression hosts contain endogenous RNases that can contaminate preparations. Address this by:

  • Expressing in RNase-deficient E. coli strains

  • Implementing rigorous chromatography steps including ion exchange chromatography followed by size exclusion

  • Confirming specific activity through substrate specificity assays

• Low expression levels: RNase Y may express poorly in heterologous systems. Optimization approaches include:

  • Codon optimization for the expression host

  • Testing multiple fusion tags (His, GST, MBP) to improve solubility

  • Exploring different expression temperatures and induction conditions

  • Co-expressing with chaperone proteins to facilitate proper folding

• Activity verification: Confirming that the purified protein retains native activity can be challenging. Recommended approaches:

  • Develop specific activity assays using known RNA substrates from E. faecalis

  • Compare kinetic parameters with those of the native enzyme

  • Perform complementation tests in rny deletion strains

By systematically addressing these challenges, researchers can obtain pure, active recombinant RNase Y suitable for biochemical and structural studies.

How can researchers accurately quantify and characterize the substrate specificity of RNase Y in vitro?

Accurately quantifying and characterizing the substrate specificity of RNase Y requires rigorous experimental design and multiple analytical approaches:

• Substrate preparation and diversity:

  • Synthesize or in vitro transcribe a library of RNA substrates with varying lengths, sequences, and secondary structures

  • Include known or predicted physiological targets identified through in vivo studies

  • Prepare 5'-monophosphorylated and 5'-triphosphorylated versions of each substrate to test preference

  • Incorporate fluorescent or radioactive labels at specific positions to enable detection of cleavage products

• Assay conditions optimization:

  • Test activity across a pH range (6.5-8.5) and with different divalent cations (Mg2+, Mn2+, Ca2+)

  • Optimize enzyme-to-substrate ratios to ensure initial reaction rates fall within linear response range

  • Include time-course analysis to determine kinetic parameters (kcat, KM)

  • Compare cleavage patterns between native and recombinant enzymes to validate the experimental system

• Cleavage site identification:

  • Use high-resolution techniques such as primer extension or RNA sequencing to map exact cleavage sites

  • Analyze cleavage products by denaturing PAGE for size determination

  • Perform 5' RACE on cleavage products to identify the exact phosphodiester bonds targeted

• Quantification methods:

  • For fluorescent substrates, use fluorescence intensity measurements in real-time or endpoint assays

  • For radioactive substrates, quantify band intensities from phosphorimager scans

  • Calculate initial velocities and derive enzyme kinetic parameters using appropriate software

• Competition assays:

  • Perform assays with mixed substrates to determine relative preferences

  • Use unlabeled competitor RNAs to assess binding affinities

By systematically analyzing these parameters, researchers can establish the biochemical properties and substrate preferences of E. faecalis RNase Y, providing insights into its functional role in RNA metabolism.

What emerging techniques could advance our understanding of RNase Y function in E. faecalis?

Several cutting-edge techniques show promise for advancing our understanding of RNase Y function in E. faecalis:

• Cryo-electron microscopy for structural studies: High-resolution structural analysis of RNase Y alone and in complex with RNA substrates or protein partners would provide insights into mechanism and regulation. Recent advances in cryo-EM enable visualization of macromolecular complexes at near-atomic resolution, potentially revealing how RNase Y recognizes substrates and interacts with other degradosome components.

• TIME-seq (Transient Inactivation of Nuclease followed by sequencing): This approach involves transient inactivation of RNase Y followed by transcriptome analysis to identify direct cleavage targets. Adapting this technique, originally developed for other RNases, to E. faecalis would allow genome-wide identification of RNase Y cleavage sites with single-nucleotide resolution.

• CRISPR interference for controlled depletion: CRISPRi systems adapted for E. faecalis would enable titratable repression of rny expression, allowing study of phenotypes associated with varying levels of RNase Y activity rather than complete absence, which more closely mimics physiological regulation.

• Nanopore direct RNA sequencing: This technology allows sequencing of native RNA molecules without conversion to cDNA, potentially revealing RNA modifications and structures affected by RNase Y processing. Applying this to compare wild-type and rny mutant transcriptomes could identify unique RNA processing events.

• Proximity-dependent biotinylation (BioID or TurboID): Fusing RNase Y to a promiscuous biotin ligase would enable identification of proteins that transiently interact with RNase Y in vivo, expanding our knowledge of the RNA degradosome in E. faecalis beyond what conventional co-immunoprecipitation can reveal .

• Single-cell RNA sequencing of bacterial populations: This emerging technique could reveal cell-to-cell variability in RNase Y-dependent RNA processing events, potentially uncovering stochastic or condition-dependent regulatory mechanisms that are masked in bulk population analyses.

• RNA structurome analysis: Coupling RNA structure probing (SHAPE-MaP, DMS-MaPseq) with RNase Y depletion studies would reveal how this enzyme influences global RNA structural landscapes, potentially identifying structure-dependent regulation mechanisms.

These innovative approaches, particularly when combined with established techniques like Grad-seq , would significantly advance our understanding of RNase Y function in RNA metabolism, stress response, and virulence of E. faecalis.

How can comparative studies between different Enterococcus species advance our understanding of RNase Y function and evolution?

Comparative studies across Enterococcus species offer valuable perspectives on RNase Y function and evolution, providing insights that cannot be gained from studying E. faecalis in isolation:

These comparative approaches would not only advance our understanding of RNase Y function but also provide insights into how RNA degradation mechanisms have evolved to support the diverse lifestyles of different Enterococcus species, from commensals to opportunistic pathogens.