Recombinant Shewanella baltica Ribonuclease 3 (rnc)

What is Shewanella baltica Ribonuclease 3 (rnc)?

Shewanella baltica Ribonuclease 3 (rnc) is an endoribonuclease that specifically recognizes and cleaves double-stranded RNA structures. It belongs to the RNase III family of enzymes that are widely conserved across bacterial species. The recombinant form is expressed in E. coli expression systems for research purposes, with the full-length protein consisting of 226 amino acids. According to the protein information, it has the Uniprot number A3D1V6 and is sourced from Shewanella baltica strain OS155 / ATCC BAA-1091 . This enzyme plays crucial roles in RNA processing and gene regulation mechanisms, particularly in S. baltica, which is a significant organism in marine environments like the Baltic Sea.

How should recombinant Shewanella baltica Ribonuclease 3 be stored and handled?

Proper storage and handling of recombinant Shewanella baltica Ribonuclease 3 is critical for maintaining its enzymatic activity. The recommended storage protocol includes:

Before opening, it is recommended to briefly centrifuge the vial to bring the contents to the bottom. Importantly, repeated freeze-thaw cycles should be avoided as they can lead to protein degradation and loss of enzymatic activity . For experimental work, researchers should prepare small working aliquots and handle the enzyme in RNase-free conditions to prevent contamination and preserve its activity.

What are the primary functions of Ribonuclease III in bacterial systems?

RNase III serves multiple critical functions in bacterial RNA metabolism and gene regulation:

• rRNA Processing: It plays a central role in the maturation of ribosomal RNA (rRNA) precursors, making it essential for ribosome biogenesis .

• Regulatory RNA Processing: The enzyme processes small non-coding RNAs that are involved in post-transcriptional gene regulation.

• mRNA Stability Control: It regulates the stability of various mRNAs by cleaving double-stranded regions, thereby affecting protein expression levels.

• Cold Adaptation: In Shewanella baltica specifically, RNase III appears to be involved in RNA degradation pathways associated with cold adaptation, which is particularly relevant given S. baltica's ability to thrive at low temperatures .

• Biofilm Formation: Research suggests connections between RNA degradation pathways and biofilm formation in S. baltica, indicating RNase III may influence this process .

The enzyme's activity is highly specific for double-stranded RNA structures rather than specific nucleotide sequences, allowing it to target various structural motifs across different RNA substrates.

How does mutation in the rnc gene affect gene expression patterns?

Mutations in bacterial RNase III genes typically lead to widespread alterations in gene expression patterns. Based on studies of RNase III homologs in other bacteria, rnc gene mutations can result in:

• Approximately 6-18% of genes becoming up-regulated

• Approximately 8-18% of genes becoming down-regulated

These changes reflect both direct effects (where RNase III directly processes the RNA) and indirect effects (downstream consequences of altered RNA processing). In bacteria with multiple RNase III homologs, single mutants often show similar expression patterns that differ notably from double and triple mutants, suggesting both distinct and overlapping functions .

The differential gene expression in RNase III mutants typically affects several key pathways:

These changes in gene expression underline the central role of RNase III in coordinating bacterial physiology and environmental adaptation.

What is the relationship between S. baltica RNase III and food spoilage mechanisms?

Shewanella baltica has been identified as the most important H₂S-producing organism in fish spoilage, particularly in iced marine fish . The connection between S. baltica's RNase III and its food spoilage capabilities can be understood through several mechanisms:

• Cold Temperature Adaptation: S. baltica can grow well at 0°C and dominates the bacterial population in iced fish storage . RNase III likely contributes to gene regulation during cold adaptation, enabling the bacterium to thrive under refrigeration conditions.

• Metabolic Regulation: RNase III may regulate the expression of genes involved in H₂S production and TMAO reduction, which are key metabolic pathways in fish spoilage .

• Biofilm Formation: RNA degradation pathways have been associated with biofilm formation in S. baltica . Biofilms enhance bacterial persistence on food surfaces and resistance to cleaning procedures.

• Stress Response Coordination: RNase III may work in concert with stress regulators like RpoS to coordinate gene expression during environmental stress, potentially enhancing survival under food preservation conditions .

• Competitive Advantage: During ice storage, S. baltica becomes the dominant organism , suggesting that its RNA processing systems may contribute to a competitive advantage over other bacteria in cold storage conditions.

Understanding RNase III's role in S. baltica could potentially lead to novel food preservation strategies targeting RNA processing mechanisms to inhibit spoilage activities.

What are the optimal expression conditions for recombinant Shewanella baltica Ribonuclease 3?

While specific optimal conditions for S. baltica RNase III expression aren't detailed in the search results, based on standard practices for recombinant RNase III proteins and the information provided, researchers should consider:

For purification, the protein should be assessed by SDS-PAGE to verify purity, which should exceed 85% for most research applications . The expression region spans the full 226 amino acids of the native protein, ensuring complete functional domains are present .

What experimental approaches are most effective for studying RNase III activity?

Several complementary experimental approaches can be employed to comprehensively study Shewanella baltica Ribonuclease 3 activity:

• Genetic Approaches:

  • Generation of rnc knockout mutants to study loss-of-function effects

  • Complementation studies to confirm phenotype specificity

  • Site-directed mutagenesis to examine specific functional residues

• Transcriptomic Analysis:

  • RNA-sequencing to identify differentially expressed genes in wildtype versus rnc mutants

  • Comparative analysis of RNA processing patterns

  • Identification of direct RNA targets through techniques like CLIP-seq

• Biochemical Characterization:

  • In vitro cleavage assays using synthetic or in vitro transcribed RNA substrates

  • Gel mobility shift assays to study RNA-protein interactions

  • Structure probing of RNA substrates before and after RNase III treatment

• Physiological Studies:

  • Growth assays under various conditions (temperature, salinity)

  • Analysis of specific phenotypes like H₂S production or biofilm formation

  • Comparative studies of wild-type and mutant strains in fish spoilage models

• Structural Biology Approaches:

  • X-ray crystallography or cryo-EM to determine three-dimensional structure

  • NMR spectroscopy for dynamic studies and RNA interactions

  • Computational modeling of enzyme-substrate interactions

Combining these approaches provides a comprehensive understanding of S. baltica RNase III function in both molecular and ecological contexts.

How can researchers purify recombinant S. baltica RNase III for structural studies?

Obtaining high-purity recombinant S. baltica RNase III suitable for structural studies requires a methodical purification strategy:

Expression Optimization:

• Use E. coli expression systems with appropriate fusion tags

• Optimize induction conditions (temperature, IPTG concentration, duration)

• Consider specialized strains if disulfide bonding is critical

Purification Protocol:

• Initial Capture:

  • Affinity chromatography based on fusion tag (e.g., Ni-NTA for His-tagged protein)

  • Include RNase inhibitors during lysis to prevent contamination

  • Use appropriate buffers to maintain protein stability

• Secondary Purification:

  • Size exclusion chromatography to remove aggregates and impurities

  • Ion exchange chromatography for charged contaminant removal

  • Consider tag removal if it might interfere with structural studies

• Quality Assessment:

  • SDS-PAGE to verify purity (target >85% as noted in product specifications)

  • Western blot for identity confirmation

  • Dynamic light scattering to assess homogeneity

Storage Considerations:

• Store at -20°C or -80°C for long-term preservation (with -80°C preferred)

• Add glycerol (5-50%) to prevent freeze damage

• Prepare small working aliquots to avoid repeated freeze-thaw cycles

Following this systematic approach should yield protein of sufficient quality for crystallization trials or other structural biology techniques.

How does S. baltica RNase III function in cold adaptation mechanisms?

Shewanella baltica is notably able to grow well at 0°C and dominates in iced fish storage environments , suggesting specialized cold adaptation mechanisms potentially involving RNase III. While direct experimental evidence is limited in the search results, several mechanistic roles for RNase III in cold adaptation can be proposed:

• RNA Secondary Structure Modulation: At low temperatures, RNA tends to form more stable secondary structures that can impede translation. RNase III may help resolve these structures by cleaving double-stranded regions, potentially enhancing translation efficiency at low temperatures.

• Cold-Responsive Gene Regulation: RNase III likely processes specific mRNAs or regulatory RNAs involved in cold-shock response. This connection is supported by findings that RpoS regulates genes involved in RNA degradation pathways associated with cold adaptation in S. baltica .

• Ribosome Assembly at Low Temperatures: Given RNase III's established role in rRNA processing, it may facilitate specific processing events required for proper ribosome assembly and function under cold conditions.

• Coordination with the RpoS Regulon: RNase III may work in concert with the RpoS regulon to fine-tune gene expression during cold adaptation, as suggested by connections between RpoS, RNA degradation pathways, and cold adaptation in S. baltica .

To directly investigate these hypotheses, researchers could compare wild-type and rnc mutant growth at various low temperatures, perform RNA-seq analysis under cold shock conditions, and identify specific RNA processing events that differ between optimal and low-temperature growth.

How does S. baltica RNase III compare structurally and functionally to other bacterial RNase III enzymes?

While detailed comparative structural studies are not provided in the search results, we can analyze potential similarities and differences between S. baltica RNase III and other bacterial RNase III enzymes:

Functional Comparisons:

• Core Activity: As an RNase III family member, the S. baltica enzyme shares the fundamental function of specifically cleaving double-stranded RNA.

• Environmental Adaptation: Given S. baltica's marine habitat and growth at low temperatures, its RNase III may have evolved specific properties for function under these conditions compared to mesophilic or terrestrial bacterial RNase IIIs.

• Regulatory Networks: The specific regulatory networks and RNA targets in S. baltica may differ from those in other bacteria, reflecting its unique ecological niche and metabolic capabilities.

A comprehensive comparison would require experimental studies including enzyme kinetics, substrate preferences, and structural analysis, which could reveal adaptations specific to S. baltica's lifestyle.

How might substrate specificity of S. baltica RNase III differ from other bacterial RNases?

The substrate specificity of S. baltica RNase III likely reflects both the conserved mechanisms of the RNase III family and adaptations specific to S. baltica's ecological niche:

RNase III Family Characteristics:

• Specifically cleaves double-stranded RNA

• Recognizes structural features rather than specific sequences

• Contains both nuclease and double-stranded RNA binding domains

Potential Unique Features of S. baltica RNase III:

• Cold Adaptation: Given S. baltica's ability to grow at 0°C , its RNase III might have evolved to maintain activity at lower temperatures and recognize RNA structures that form under cold conditions.

• Marine Environment Adaptations: As a marine bacterium, S. baltica's RNase III might function optimally at salt concentrations typical of its natural habitat, potentially differing from enzymes of terrestrial bacteria.

• Comparison with Other RNases:

  • Unlike RNase A or RNase T1, which cleave single-stranded RNA at specific nucleotides

  • Unlike RNase H, which degrades RNA in RNA-DNA hybrids

  • Unlike RNase P, which processes tRNA precursors at specific sites

• Specificity Determinants:

  • The specific amino acid composition of the RNA-binding domains

  • The structure of the catalytic site

  • Potential accessory proteins that might modify substrate recognition

Understanding these specificity determinants could provide insights into S. baltica's unique RNA regulatory mechanisms adapted to marine and cold environments, potentially revealing novel principles of bacterial adaptation.