Recombinant Streptomyces coelicolor Oligoribonuclease (orn)

What is the structural homology of S. coelicolor oligoribonuclease compared to other bacterial species?

The S. coelicolor oligoribonuclease (OrnA-c) shares remarkable structural homology with other bacterial oligoribonucleases, particularly within the Streptomyces genus. Sequence analysis reveals that the S. coelicolor OrnA homologue shares 88% amino acid sequence identity with the S. griseus OrnA, indicating strong evolutionary conservation of this enzyme within Streptomyces species . This high degree of identity strongly suggests that the S. coelicolor enzyme maintains the canonical oligoribonuclease activity observed in other species. The conservation extends beyond Streptomyces, with notable homology to oligoribonucleases found in other bacteria like Escherichia coli, though with lower sequence identity percentages.
To characterize structural homology effectively, researchers should employ multiple sequence alignment tools such as Clustal Omega or MUSCLE, followed by phylogenetic tree construction using maximum likelihood methods. Homology modeling using previously resolved oligoribonuclease structures as templates can provide insights into the three-dimensional structure of the S. coelicolor enzyme and help identify conserved catalytic domains.

What is the primary biochemical function of oligoribonuclease in S. coelicolor?

Oligoribonuclease (OrnA) in S. coelicolor functions as a 3′-to-5′ exoribonuclease that specifically degrades short oligoribonucleotides to mononucleotides . This enzyme is critical for completing the final step in RNA degradation pathways, converting small RNA fragments (typically 2-5 nucleotides in length) into individual nucleotides that can be recycled for new RNA synthesis .
The biochemical activity of OrnA can be demonstrated through standard oligoribonuclease assays using synthetic RNA substrates such as ApCpC[5′-32P]pC, which allows monitoring of the release of [32P]CMP as the end product of enzymatic activity . The reaction typically requires divalent metal ions such as Mn2+ or Mg2+ as cofactors and shows optimal activity under defined buffer conditions (commonly Tris-HCl at pH 8.0) . Unlike other exoribonucleases that may halt degradation when reaching short oligonucleotides, OrnA efficiently processes these final fragments, making it essential for complete RNA turnover in the cell.

What are the optimal conditions for heterologous expression of recombinant S. coelicolor oligoribonuclease?

For efficient heterologous expression of recombinant S. coelicolor oligoribonuclease, the ornA gene can be amplified from genomic DNA using high-fidelity polymerase and specific primers designed based on the published sequence . The amplified gene should be cloned into an appropriate expression vector containing an inducible promoter (T7 or similar) and affinity tag for purification.
Expression in E. coli BL21(DE3) or Rosetta strains is recommended due to their reduced protease activity and ability to supply rare codons that may be present in the GC-rich Streptomyces sequence. Optimal induction conditions generally involve growth at 28-30°C rather than 37°C to improve protein solubility, with IPTG concentrations of 0.1-0.5 mM. Because S. coelicolor has a high GC content (approximately 72%), codon optimization may be necessary to achieve efficient expression in E. coli.
Post-induction incubation periods of 4-6 hours or overnight at lower temperatures (16-18°C) can significantly improve yield of soluble protein. Inclusion of additional cofactors such as Mn2+ or Mg2+ in the growth medium may enhance proper folding of the recombinant enzyme.

What purification strategy yields the highest activity of recombinant S. coelicolor oligoribonuclease?

A multi-step purification strategy is recommended to obtain highly active recombinant S. coelicolor oligoribonuclease. Based on protocols adapted from similar enzymes, the following approach yields optimal results:

• Initial capture using affinity chromatography (His-tag or similar) with imidazole gradient elution

• Secondary purification via ion-exchange chromatography (typically Q-Sepharose) exploiting the enzyme's charge properties

• Final polishing step using size exclusion chromatography to remove aggregates and ensure homogeneity
  All buffers should contain reducing agents (typically 1-5 mM DTT or β-mercaptoethanol) to maintain cysteine residues in reduced form, and divalent metal ions (2-5 mM MnCl₂ or MgCl₂) to stabilize the enzyme's active site. Purification should be performed at 4°C to minimize proteolytic degradation.
  The specific activity of purified recombinant oligoribonuclease can be assessed using the standard oligoribonuclease assay with ApCpC[32P]pC substrate as described in previous studies . Enzyme fractions showing the highest specific activity should be pooled, concentrated, and stored with 20-25% glycerol at -80°C to maintain long-term stability.

How can researchers generate and validate an ornA null mutant in S. coelicolor?

Generation of an ornA null mutant in S. coelicolor requires precise genetic manipulation techniques. The following methodology has been successfully employed:

• Design a gene disruption construct containing antibiotic resistance marker (typically kanamycin or apramycin resistance cassette) flanked by homologous regions (500-1000 bp) upstream and downstream of the ornA coding sequence

• Introduce the construct into S. coelicolor by either protoplast transformation or conjugation from E. coli

• Select primary recombinants on appropriate antibiotic-containing media

• Screen for double crossover events by replica plating or PCR analysis

• Confirm gene disruption by Southern blot hybridization using ornA-specific probes
  Validation of the null mutant should include both genetic and functional analyses. PCR amplification across the disrupted region followed by sequencing can confirm the precise location of the antibiotic cassette insertion. RT-PCR or RNA-Seq analysis should verify the absence of ornA transcript. Complementation studies using a plasmid containing the wild-type ornA gene (such as a low-copy-number plasmid like pKU209) should restore the wild-type phenotype, confirming that observed defects are specifically due to the loss of ornA function .

What phenotypic changes are observed in S. coelicolor ornA null mutants?

S. coelicolor ornA null mutants exhibit several distinctive phenotypic changes that reflect the importance of oligoribonuclease in growth and development. The primary phenotypes include:

• Reduced growth rate compared to wild-type strains

• Significant defects in morphological differentiation, particularly in aerial mycelium formation

• Conditional defects reminiscent of other developmental mutants, such as bldA mutants

• Delayed but not completely abolished antibiotic production
  These phenotypic changes are conditional, with severity depending on growth conditions. The morphological defects are particularly noteworthy, as they link oligoribonuclease function to the complex developmental program of S. coelicolor. The similarities to bldA mutant phenotypes suggest potential interactions with regulatory pathways controlled by the bldA-encoded tRNA that recognizes the rare UUA codon .
  It's important to note that while ornA disruption causes significant developmental defects, it is not lethal in Streptomyces species, indicating that while the enzyme plays an important role in growth and differentiation, it is not essential for viability .

How does oligoribonuclease expression interact with the developmental program in S. coelicolor?

Oligoribonuclease expression in S. coelicolor shows interesting connections to the organism's developmental program. Transcriptional analysis reveals that the ornA gene is not transcriptionally or translationally coupled to adpA, a key developmental regulator that is itself a target of bldA-mediated regulation . This independence suggests that oligoribonuclease regulation occurs through separate pathways, despite its involvement in developmental processes.
The developmental defects observed in ornA null mutants are reminiscent of those seen in bldA and other developmental mutants , suggesting functional overlap in the pathways affected. This connection may involve RNA processing and turnover mechanisms that influence the expression of key developmental genes. Since bldA encodes a tRNA that recognizes the rare UUA codon used in regulatory genes like adpA, one hypothesis is that oligoribonuclease activity might influence the stability or processing of small RNAs that interact with these regulatory networks.
Researchers investigating this interaction should employ global transcriptomic and proteomic approaches to identify changes in gene expression patterns between wild-type and ornA mutant strains across different developmental stages. ChIP-seq or similar techniques can help identify if any transcription factors directly regulate ornA expression during development.

What is the relationship between oligoribonuclease activity and antibiotic production in S. coelicolor?

The relationship between oligoribonuclease activity and antibiotic production in S. coelicolor appears to be indirect but significant. The ornA null mutant shows delayed but not completely abolished production of antibiotics like actinorhodin . This suggests that oligoribonuclease is not directly involved in antibiotic biosynthesis pathways but may influence them through broader cellular processes.
S. coelicolor produces several distinctive pigmented antibiotics, including the blue-pigmented actinorhodin, red undecylprodigiosin, and yellow coelimycin . These compounds serve as excellent visual markers for studying the effects of genetic manipulations on specialized metabolite production. The production of these compounds is tightly linked to the developmental program and responds to various environmental and nutritional signals.
The mechanistic connection between oligoribonuclease and antibiotic production may involve:

• Influence on the turnover of mRNAs encoding regulatory proteins that control antibiotic biosynthetic gene clusters

• Effects on small RNA processing that modulates expression of biosynthetic or regulatory genes

• Indirect effects through alterations in primary metabolism or stress responses that trigger antibiotic production
  To investigate these connections, researchers should perform time-course analyses of antibiotic production in wild-type versus ornA mutant strains under various growth conditions, coupled with targeted transcriptional analyses of key biosynthetic gene clusters and their regulators.

How do oligoribonucleases from different Streptomyces species compare in structure and function?

Oligoribonucleases from different Streptomyces species show high conservation in both structure and function, reflecting their important cellular roles. A comparative analysis reveals:

What evidence supports the evolutionary conservation of oligoribonuclease across bacterial phyla?

Oligoribonuclease represents a highly conserved enzyme family found across diverse bacterial phyla and extending into eukaryotes. This conservation provides strong evidence for its fundamental importance in cellular RNA metabolism. Key evidence includes:

• Sequence homology: S. coelicolor oligoribonuclease shows significant sequence similarity not only to other Streptomyces enzymes but also to those from phylogenetically distant bacteria like E. coli

• Functional conservation: The 3′-to-5′ exoribonuclease activity targeting small oligoribonucleotides is maintained across species, suggesting a universal requirement for this enzymatic function in RNA degradation pathways

• Structural conservation: Core catalytic domains and metal-binding sites are preserved across bacterial oligoribonucleases, indicating selection pressure to maintain the enzyme's mechanism of action

• Phenotypic impact: While the specific phenotypes of ornA disruption vary between species, the general importance for normal growth and development appears consistent
  This high degree of conservation suggests that oligoribonuclease plays a fundamental role in RNA metabolism that predates the divergence of bacterial phyla. The enzyme's specialization for degrading small oligoribonucleotides represents a critical and non-redundant function in cellular RNA turnover across diverse organisms.

How can recombinant S. coelicolor oligoribonuclease be used in in vitro RNA degradation studies?

Recombinant S. coelicolor oligoribonuclease provides a valuable tool for in vitro RNA degradation studies, particularly for investigating the final stages of RNA degradation. The following methodological approach is recommended:

• Substrate preparation: Synthesize or in vitro transcribe RNA oligonucleotides of varying lengths (2-10 nucleotides) with appropriate end-labeling (5′-32P or fluorescent labels)

• Reaction conditions: Standard assays employ 100 mM Tris-HCl (pH 8.0), 5 mM MnCl₂, and substrate concentrations in the low nanomolar range

• Enzyme titration: Use various amounts of purified recombinant enzyme to determine concentration-dependent activity profiles

• Product analysis: Monitor the release of labeled mononucleotides using techniques such as paper chromatography, thin-layer chromatography, or high-resolution gel electrophoresis

• Kinetic analysis: Determine essential enzymatic parameters (Km, kcat, substrate preferences) by analyzing reaction progress over time with varying substrate concentrations
  This approach can be extended to study the effects of potential inhibitors, the influence of different metal cofactors on activity, or the impact of pH and ionic conditions on enzyme function. The data obtained can provide insights into the mechanistic details of oligoribonuclease function and its substrate specificity.

What techniques are most effective for studying the in vivo role of oligoribonuclease in RNA metabolism within S. coelicolor?

Investigating the in vivo role of oligoribonuclease in S. coelicolor RNA metabolism requires sophisticated approaches that can track RNA processing and degradation in living cells. The following techniques have proven most effective:

• RNA-Seq with size selection: Compare short RNA profiles (1-10 nt) between wild-type and ornA mutant strains to identify accumulating oligoribonucleotides

• Metabolic labeling: Use pulse-chase experiments with radioactive nucleosides to track RNA degradation kinetics in vivo, comparing wild-type and ornA mutant strains

• CLIP-Seq (Crosslinking and Immunoprecipitation followed by sequencing): Identify RNA species that directly interact with tagged oligoribonuclease in vivo

• Ribosome profiling: Assess the impact of ornA deletion on translation efficiency and accuracy, which may be affected by altered RNA turnover

• Conditional expression systems: Employ tunable promoters to control oligoribonuclease levels and observe the immediate effects on RNA metabolism
  When implementing these techniques, researchers should consider the developmental stage of S. coelicolor cultures, as the impact of oligoribonuclease may vary during vegetative growth versus aerial mycelium formation and sporulation. Time-course experiments across developmental stages can provide a comprehensive picture of how oligoribonuclease function changes throughout the S. coelicolor life cycle.

How might targeting oligoribonuclease function affect antibiotic production in industrial Streptomyces strains?

Based on the connection between RNA metabolism and secondary metabolite production, modulating oligoribonuclease function in industrial Streptomyces strains presents an interesting approach for optimizing antibiotic yields. The strategic considerations include:

• Conditional modulation: Rather than complete deletion, which causes growth defects , tunable expression systems could allow precise control of oligoribonuclease levels at specific developmental stages

• Targeted timing: Since the delay in antibiotic production in ornA mutants appears to be primarily due to growth effects , synchronized cultures with stage-specific oligoribonuclease modulation might optimize both growth and production phases

• Strain-specific effects: The impact of oligoribonuclease modulation likely varies between Streptomyces species and even between strains of the same species producing different antibiotics
  The observed connection between oligoribonuclease and the distinctive pigmented antibiotics of S. coelicolor (blue actinorhodin, red undecylprodigiosin, and yellow coelimycin) suggests that similar relationships may exist for other commercially valuable compounds produced by various Streptomyces species.
  Researchers exploring this approach should implement carefully controlled expression systems and monitor both growth parameters and antibiotic production profiles using quantitative analytical methods such as HPLC or LC-MS/MS.

What role might oligoribonuclease play in stress responses and adaptation in Streptomyces coelicolor?

Oligoribonuclease likely plays a significant role in stress responses and adaptation in S. coelicolor through its function in RNA turnover. While direct experimental evidence is limited, several connections can be inferred:

• Transcriptional control: The regulation of oligoribonuclease expression through specific promoters suggests responsiveness to environmental conditions

• RNA quality control: By completing RNA degradation, oligoribonuclease may prevent the accumulation of potentially interfering short RNAs during stress conditions

• Resource recycling: The conversion of oligoribonucleotides to mononucleotides by oligoribonuclease enables the recycling of nucleotides for new RNA synthesis , which may be particularly important under nutrient limitation

• Developmental program integration: The conditional defects in differentiation observed in ornA mutants suggest that oligoribonuclease activity is integrated with developmental responses to environmental cues To investigate these connections, researchers should examine oligoribonuclease expression and activity under various stress conditions (nutrient limitation, pH stress, osmotic stress, antibiotic exposure) and compare the stress response profiles of wild-type and ornA mutant strains using transcriptomic and metabolomic approaches.