Recombinant Lactobacillus johnsonii Ribonuclease Z (rnz)

What is Ribonuclease Z (RNase Z) and what is its primary function in Lactobacillus johnsonii?

Ribonuclease Z (RNase Z) in Lactobacillus johnsonii is a metalloenzyme involved in tRNA maturation. It functions primarily as an endonuclease that removes the 3' trailer sequence from precursor tRNAs, which is a critical step in tRNA processing pathways. The enzyme contains a conserved phosphodiesterases (PDE) domain with essential Zn²⁺-binding residues that are crucial for its catalytic activity . RNase Z is part of the β-lactamase superfamily of metal-dependent hydrolases and has been identified as gene rnZ in various Lactobacillus strains. In L. johnsonii specifically, RNase Z plays an important role in adapting to environmental stressors and maintaining proper translation mechanisms through tRNA processing .

How does the structure of Lactobacillus johnsonii RNase Z compare to other bacterial RNase Z enzymes?

Lactobacillus johnsonii RNase Z shares significant structural homology with other bacterial RNase Z enzymes, particularly those from Gram-positive bacteria. Structural analysis reveals that L. johnsonii RNase Z contains the characteristic conserved phosphodiesterases (PDE) domain and critical Zn²⁺-binding residues (His63, His65, Asp67, His68, His145, Asp216, and H274) that are essential for its catalytic function . Homology modeling studies demonstrate structural concordance between RNase Z enzymes across bacterial species, including the long flexible protruding arm that binds to the TψC arm of tRNAs . This structural conservation suggests functional similarity in tRNA processing mechanisms while maintaining species-specific adaptations that may relate to the unique ecological niche of L. johnsonii as a host-adapted commensal bacterium .

What expression systems are most effective for producing recombinant L. johnsonii RNase Z?

For recombinant production of L. johnsonii RNase Z, E. coli-based expression systems remain the most widely utilized due to their versatility, scalability, and established protocols. The pET expression system (particularly pET28a with an N-terminal His-tag) provides efficient expression under IPTG induction while facilitating subsequent purification via nickel affinity chromatography. When expressing L. johnsonii RNase Z, researchers should consider codon optimization for E. coli expression, as Lactobacillus species have different codon usage patterns that can affect heterologous protein expression efficiency. Additionally, expression at lower temperatures (16-20°C) after induction often improves protein solubility and reduces inclusion body formation. For functional studies requiring post-translational modifications or proper folding of this metalloenzyme, consideration should be given to supplementing growth media with zinc and using Lactobacillus-based expression systems that provide the native cellular environment .

How does oxidative stress affect RNase Z activity in Lactobacillus johnsonii?

Oxidative stress significantly impacts RNase Z activity in Lactobacillus species. Research has demonstrated that exposure to hydrogen peroxide (H₂O₂) leads to decreased abundance of RNase Z in bacterial cells . This reduction in RNase Z levels under oxidative conditions impedes tRNA maturation processes, which consequently affects translation elongation and protein synthesis. The molecular mechanism behind this involves the susceptibility of RNase Z to oxidative degradation, particularly at its metal-binding sites which are critical for catalytic function. Studies with S. oligofermentans showed that RNase Z abundance significantly decreased in cells treated with 0.5 mM H₂O₂ . This sensitivity to oxidative stress suggests that RNase Z degradation may be part of a broader bacterial response mechanism that rapidly modulates translation in response to environmental stressors. For L. johnsonii specifically, this response is likely important for its adaptation to the oxidative conditions it encounters in host environments .

What techniques are most effective for assessing the in vivo activity of recombinant L. johnsonii RNase Z?

The in vivo activity assessment of recombinant L. johnsonii RNase Z requires a multi-faceted approach combining genomic, transcriptomic, and functional analyses. The most effective technique involves complementation studies in RNase Z-deficient bacterial strains, where successful restoration of tRNA processing confirms functional activity. Northern blot analysis targeting precursor and mature tRNAs provides direct evidence of processing activity, with accumulation of 3'-extended pre-tRNAs indicating RNase Z deficiency. RNA-Seq can comprehensively profile the tRNA landscape, while LC-MS/MS proteomics can correlate RNase Z activity with downstream translational effects. For spatial localization, fluorescently tagged RNase Z constructs combined with super-resolution microscopy reveal the enzyme's distribution patterns. Additionally, metabolic labeling with radioactive or stable isotopes can track tRNA processing kinetics in vivo. These techniques should be complemented with appropriate controls and statistical analyses to ensure reliable quantification of enzymatic activity under various physiological conditions .

How do mutations in the zinc-binding residues of L. johnsonii RNase Z affect its catalytic mechanism?

Mutations in the zinc-binding residues of L. johnsonii RNase Z profoundly impact its catalytic functionality through several mechanisms. The conserved residues (His63, His65, Asp67, His68, His145, Asp216, and H274) coordinate Zn²⁺ ions that are essential for the enzyme's hydrolyzing activity on pre-tRNA substrates . Site-directed mutagenesis studies reveal that substitutions at these positions result in measurable decreases in catalytic efficiency (kcat/KM), with complete loss of activity when multiple binding sites are altered simultaneously. Spectroscopic analyses demonstrate that such mutations disrupt metal coordination geometry, affecting transition state stabilization during the phosphodiester bond cleavage. Kinetic studies show that while substrate binding may still occur in some mutants, the hydrolysis reaction rate decreases substantially. Additionally, these mutations can increase susceptibility to oxidative inactivation, as the zinc-binding residues also contribute to the structural stability of the enzyme. The flexible protruding arm that interacts with the TψC arm of tRNAs maintains its conformation through proper zinc coordination, meaning mutations in these residues can also disrupt substrate recognition beyond just catalytic activity .

What is the relationship between RNase Z activity and stress response mechanisms in L. johnsonii?

The relationship between RNase Z activity and stress response mechanisms in L. johnsonii represents a sophisticated regulatory network critical for bacterial adaptation. Under oxidative stress conditions, RNase Z abundance significantly decreases, suggesting that oxidative degradation of this enzyme serves as a rapid response mechanism to modulate translation by affecting tRNA maturation . This creates a regulatory checkpoint where the cell can quickly adjust protein synthesis in response to environmental challenges. Transcriptomic analyses indicate that RNase Z downregulation correlates with the expression of stress-responsive genes, including those involved in oxidative stress defense systems. The enzyme's sensitivity to oxidation appears to function as a sensor mechanism, where its degradation triggers broader stress response pathways. Additionally, evidence suggests that L. johnsonii may utilize differential tRNA processing as a strategy to prioritize the translation of stress-response proteins during adverse conditions. This integration with stress response systems is particularly important for L. johnsonii given its niche as a commensal bacterium that must adapt to various environmental stressors within host environments .

What purification strategy yields the highest activity of recombinant L. johnsonii RNase Z?

The optimal purification strategy for obtaining high-activity recombinant L. johnsonii RNase Z involves a multi-step approach that preserves the enzyme's native conformation and metal coordination. Initial capture using immobilized metal affinity chromatography (IMAC) with Ni²⁺-NTA resin provides efficient isolation of His-tagged RNase Z. Critical parameters include using moderate imidazole concentrations (10-20 mM) in washing buffers to reduce non-specific binding while preventing target protein elution. All buffers should be supplemented with 0.1-0.5 mM ZnCl₂ to maintain metallation of the enzyme and 1-5 mM β-mercaptoethanol or DTT to prevent oxidation of cysteine residues. Following IMAC, size exclusion chromatography using Superdex 75 or 200 columns effectively removes aggregates and improves homogeneity. For applications requiring higher purity, ion exchange chromatography (particularly cation exchange at pH 6.5-7.0) can be employed as an intermediate step. Enzyme activity should be monitored throughout purification using fluorogenic tRNA substrates to track specific activity yield. Typical purification yields approximately 5-10 mg of active enzyme per liter of bacterial culture with >95% purity and specific activity of 100-150 units/mg when using optimized expression conditions in E. coli BL21(DE3) host cells .

What are the optimal conditions for assaying recombinant L. johnsonii RNase Z activity in vitro?

The optimal conditions for assaying recombinant L. johnsonii RNase Z activity in vitro require careful consideration of buffer composition, metal cofactors, and substrate preparation. The reaction buffer should maintain pH 7.2-7.5 (typically using 50 mM Tris-HCl or HEPES) with 50-100 mM NaCl or KCl for ionic strength. Critical for activity is the presence of divalent metal ions, with 1-2 mM MgCl₂ and 0.1 mM ZnCl₂ providing optimal catalytic efficiency. The addition of 1 mM DTT or β-mercaptoethanol is essential to prevent oxidative inactivation during the assay. Pre-tRNA substrates should be prepared by in vitro transcription followed by refolding through controlled cooling from 65°C to ensure proper secondary structure formation. Substrate concentrations of 0.5-2 μM typically provide conditions for accurate kinetic determinations. Reactions should be conducted at 30-37°C, reflecting the physiological temperature range of L. johnsonii growth. For analyzing reaction products, denaturing PAGE (20% polyacrylamide with 8M urea) provides excellent resolution of processed and unprocessed tRNAs. Quantitative activity measurements can be achieved using 5'-fluorescently labeled substrates with detection by capillary electrophoresis or fluorescence imaging of gels. Standard reaction times of 5-20 minutes with enzyme concentrations of 10-50 nM typically yield linear product formation suitable for initial velocity determinations .

How can gene knockout and complementation strategies be optimized for studying L. johnsonii RNase Z function?

Optimizing gene knockout and complementation strategies for studying L. johnsonii RNase Z function requires tailored approaches given the essential nature of this gene and the unique characteristics of Lactobacillus genetics. For knockout construction, homologous recombination-based methods using the pORI28 system have proven effective in Lactobacillus species. This approach requires designing homology arms of at least 1 kb flanking the rnZ gene and incorporating a selectable marker (typically erythromycin resistance). Since complete deletion may be lethal, conditional knockout strategies using inducible promoters like the nisin-controlled expression system provide better control. For complementation studies, the pSIP vector system with its well-characterized inducible promoters offers tight regulation of expression levels. Complementation constructs should include the native ribosome binding site to ensure proper translation initiation, while incorporating a C-terminal tag rather than N-terminal to minimize interference with the catalytic domain. Integration of the complementation construct at neutral genomic loci like the attB sites prevents position effects on expression. Quantitative verification of knockout and complementation should combine RT-qPCR for transcript levels, western blotting for protein detection, and functional assays measuring pre-tRNA processing efficiency. To distinguish between direct and indirect effects, RNA-Seq analysis comparing the transcriptome profiles of wild-type, knockout, and complemented strains provides comprehensive insights into the regulatory networks affected by RNase Z modulation .

What approaches are effective for studying the interaction between L. johnsonii RNase Z and its tRNA substrates?

Multiple complementary approaches are effective for characterizing the interaction between L. johnsonii RNase Z and its tRNA substrates. Electrophoretic mobility shift assays (EMSA) provide a straightforward method for determining binding affinities, with gel conditions optimized to preserve enzyme-RNA complexes (typically using 6% native polyacrylamide gels with 5 mM MgCl₂). Surface plasmon resonance (SPR) offers real-time kinetic analysis of binding events, requiring biotinylated tRNA immobilization on streptavidin-coated sensor chips and carefully controlled flow rates of 20-30 μL/min. For structural characterization, chemical probing methods like SHAPE (Selective 2'-hydroxyl acylation analyzed by primer extension) can map the interaction interface by identifying protected nucleotides in the presence of RNase Z. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) provides complementary information from the protein perspective, revealing regions with altered solvent accessibility upon substrate binding. UV-induced crosslinking followed by mass spectrometry can identify direct contact points between the enzyme and tRNA. Computational approaches like molecular docking combined with molecular dynamics simulations predict interaction modes and conformational changes during substrate recognition. Mutation analysis focusing on the flexible protruding arm that interacts with the TψC arm of tRNAs provides functional validation of predicted interaction sites. These approaches together generate a comprehensive map of the molecular recognition events governing substrate specificity and catalytic efficiency .

What are the most significant challenges in expressing and studying L. johnsonii RNase Z in heterologous systems?

Expressing and studying L. johnsonii RNase Z in heterologous systems presents several significant challenges that must be addressed for successful investigation. The primary challenge involves maintaining proper protein folding and metallation, as improper zinc coordination leads to inactive enzyme. This requires careful buffer optimization with zinc supplementation throughout expression and purification. Codon usage differences between Lactobacillus johnsonii and common expression hosts like E. coli can cause translational pausing and protein truncation, necessitating codon optimization of the gene construct. The hydrophobic regions in RNase Z can promote aggregation during high-level expression, requiring lower induction temperatures (16-20°C) and the inclusion of solubility-enhancing fusion partners like SUMO or thioredoxin. The enzyme's sensitivity to oxidation demands strictly reducing conditions during all purification steps, typically maintained using 1-5 mM DTT or β-mercaptoethanol. Additionally, the absence of Lactobacillus-specific chaperones in heterologous hosts may affect proper folding, potentially requiring co-expression of chaperone systems. For functional studies, the specificity for Lactobacillus tRNA substrates means that commercially available tRNA substrates may not accurately reflect native activity, necessitating preparation of species-specific tRNA substrates. Finally, the essential nature of RNase Z means that toxic effects can occur when overexpressed in heterologous systems, requiring tightly controlled inducible expression systems .

How can high-throughput screening be designed to identify inhibitors or activators of L. johnsonii RNase Z?

A comprehensive high-throughput screening platform for identifying modulators of L. johnsonii RNase Z activity should incorporate fluorescence-based assays with real-time monitoring capabilities. The primary assay should utilize a synthetic pre-tRNA substrate labeled with a fluorophore-quencher pair that generates signal upon cleavage, allowing continuous measurement in 384-well format. The assay buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl₂, 0.1 mM ZnCl₂, 0.01% Triton X-100, 1 mM DTT) must maintain enzyme stability while minimizing interference with fluorescence detection. Initial screening concentrations of 10-20 µM for compound libraries with DMSO concentrations below 1% ensure minimal solvent effects. Critical quality control parameters include Z' factor >0.7, signal-to-background ratio >10, and coefficient of variation <10%. Counter-screening against related metalloenzymes helps identify selective inhibitors. For hit validation, dose-response curves should be generated using both the fluorogenic substrate and gel-based assays with native pre-tRNA substrates. Thermal shift assays can distinguish between compounds that directly bind RNase Z versus those affecting substrate recognition. Computational docking should be employed to predict binding modes of confirmed hits, guiding structure-activity relationship studies. Cell-based secondary assays using an L. johnsonii strain with a reporter system linked to tRNA processing provides validation of compound cell permeability and in vivo efficacy .

What experimental design would best evaluate the impact of recombinant L. johnsonii RNase Z on host-microbe interactions?

A comprehensive experimental design to evaluate the impact of recombinant L. johnsonii RNase Z on host-microbe interactions would employ a multi-level approach spanning molecular, cellular, and organism scales. The foundation of this design centers on generating L. johnsonii strains with modified RNase Z expression: a knockout strain complemented with either wild-type RNase Z, catalytically inactive mutant, or overexpression constructs. These strains should be fluorescently labeled for tracking in complex environments and thoroughly characterized for growth kinetics, stress responses, and tRNA processing capacity prior to host interaction studies.

Table 1: Experimental Strains for Host-Microbe Interaction Studies

In vitro studies should first assess adhesion to intestinal epithelial cell lines (Caco-2, HT-29) and interaction with immune cells (dendritic cells, macrophages) through co-culture experiments measuring adhesion efficiency, cytokine production profiles, and transcriptional responses in both bacterial and host cells. Ex vivo organ culture systems using intestinal tissue sections would bridge to in vivo studies by allowing evaluation of strain-specific adhesion patterns while maintaining tissue architecture.

For in vivo studies, gnotobiotic mouse models would enable controlled colonization experiments where single strains or defined communities including the modified L. johnsonii strains are introduced. Longitudinal sampling should include:

• Fecal and mucosal samples for bacterial enumeration and localization

• Host tissue for histological examination and immunophenotyping

• Metabolomic analysis of intestinal contents

• Transcriptomic profiling of both host tissues and recovered bacteria

The experimental timeline should include baseline measurements, acute responses (1-7 days post-colonization), and long-term effects (28+ days) to capture the dynamic nature of host-microbe interactions. Multi-omics integration combining bacterial transcriptomics, host response patterns, and metabolic profiles would provide comprehensive insights into how RNase Z activity influences the molecular dialogue between L. johnsonii and its host .

What potential biotechnological applications exist for recombinant L. johnsonii RNase Z?

Recombinant L. johnsonii RNase Z holds significant potential for diverse biotechnological applications across multiple fields. In molecular biology, its precise tRNA processing capability makes it valuable for structural RNA studies and the creation of custom tRNA pools with defined 3' ends. The enzyme's substrate specificity can be exploited for selective RNA degradation in transcriptome manipulation experiments. In synthetic biology, RNase Z could serve as a regulatory component in engineered genetic circuits where specific RNA processing steps control gene expression. For pharmaceutical applications, the essential nature of RNase Z in bacterial physiology positions it as a potential antimicrobial target, with species-specific inhibitors offering narrow-spectrum activity. Conversely, the incorporation of stress-resistant RNase Z variants into probiotic L. johnsonii strains could enhance their survival in manufacturing processes and gastrointestinal transit. In RNA therapeutics, modified RNase Z variants could be developed for targeted RNA editing applications. The enzyme also has potential in industrial RNA processing for manufacturing structured RNAs at scale. Development of these applications will require protein engineering to enhance stability, modify substrate specificity, and optimize catalytic efficiency under various conditions .

How might structural biology approaches advance our understanding of L. johnsonii RNase Z function?

Advanced structural biology approaches would significantly deepen our understanding of L. johnsonii RNase Z function through multi-scale characterization of its structure-function relationships. X-ray crystallography of the enzyme in both apo and substrate-bound states would reveal the precise coordination geometry of the zinc-binding sites and conformational changes upon substrate binding. Cryo-electron microscopy (cryo-EM) could capture dynamic intermediate states during the catalytic cycle, particularly the transition states that are difficult to characterize by other methods. Nuclear magnetic resonance (NMR) spectroscopy would provide insights into solution dynamics, especially the movements of the flexible protruding arm that interacts with tRNA substrates. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) could map conformational changes under different conditions, including oxidative stress that affects enzyme activity. Integrating these structural data with molecular dynamics simulations would generate a comprehensive model of the complete catalytic cycle. Single-molecule techniques like FRET could monitor real-time conformational changes during substrate binding and product release. These structural insights would guide rational engineering efforts to modify substrate specificity, enhance catalytic efficiency, or develop inhibitors targeting specific conformational states of the enzyme. The evolutionary adaptation of RNase Z structure across Lactobacillus species could also be examined to understand how structural variations relate to the diverse ecological niches these bacteria occupy .