Recombinant Burkholderia vietnamiensis Oligoribonuclease (orn)

How conserved is the structure of Oligoribonuclease across bacterial species?

The B. vietnamiensis Oligoribonuclease (UniProt ID: A4JCK6) is a 200-amino acid protein with sequence conservation across bacterial species, particularly in catalytic domains . Studies on E. coli oligoribonuclease have shown that close homologues of the orn gene are found in a wide range of organisms, extending from bacteria to eukaryotes including humans . This extensive conservation suggests strong evolutionary selection pressure to maintain ORN function.

To investigate structural conservation, researchers should employ:

• Multiple sequence alignment using tools like Clustal Omega

• Phylogenetic analysis to determine evolutionary relationships

• Homology modeling based on available crystal structures

• Functional complementation assays testing cross-species activity

The specific sequence of B. vietnamiensis ORN includes several conserved motifs likely involved in catalytic activity and substrate binding, though the degree of conservation with well-studied bacterial ORNs would require direct experimental verification .

What experimental approaches are most effective for determining ORN substrate specificity?

To investigate substrate specificity of B. vietnamiensis ORN, researchers should implement a multi-faceted experimental approach:

• In vitro degradation assays: Using synthetic oligoribonucleotides of different lengths (2-10 nucleotides) with varying sequences and modifications. The commercial recombinant protein (>85% purity by SDS-PAGE) is suitable for such studies .

• Kinetic parameter determination: Measuring Km, kcat, and catalytic efficiency (kcat/Km) for different substrates under standardized conditions (typically Tris-HCl buffer pH 7.5-8.0, 50-100 mM NaCl, 1-5 mM Mg²⁺).

• Competition assays: Using mixtures of oligoribonucleotides to determine preference.

• Product analysis: Employing HPLC, mass spectrometry, or gel electrophoresis to analyze degradation products and determine processivity.

From E. coli studies, we know that ORN specifically degrades oligoribonucleotides of 2-5 residues . Researchers should verify if B. vietnamiensis ORN maintains this specificity or has evolved distinct preferences that might relate to its pathogenic lifestyle.

What are the optimal conditions for expressing and purifying recombinant B. vietnamiensis Oligoribonuclease?

Based on available information for recombinant B. vietnamiensis ORN, researchers should consider the following protocol:

Expression system:

• The commercial recombinant protein is produced in mammalian cells

• Alternative expression systems include E. coli (BL21 strains) with pET vectors

Expression optimization parameters:

• Temperature: 25-30°C often favors proper folding

• Induction conditions: For IPTG-inducible systems, 0.1-0.5 mM IPTG

• Duration: 4-16 hours depending on expression level and solubility

Purification strategy:

• Affinity chromatography (if tagged)

• Ion exchange chromatography

• Size exclusion chromatography for final polishing

Buffer considerations:

• Maintain RNase-free conditions throughout

• Include protease inhibitors in initial lysis steps

• Consider 5-50% glycerol in storage buffer

The purified protein should achieve >85% purity by SDS-PAGE . Researchers should verify enzyme activity after purification using appropriate substrate degradation assays to ensure functional integrity has been maintained throughout the purification process.

How should researchers measure the enzymatic activity of B. vietnamiensis Oligoribonuclease?

To accurately measure the enzymatic activity of B. vietnamiensis ORN, researchers should implement the following methodological approach:

Substrate preparation:

• Synthetic oligoribonucleotides (2-5 nucleotides in length)

• 5'-end labeled with 32P or fluorescent tags for detection

• RNase-free conditions throughout preparation

Standard reaction conditions:

• Buffer: 50 mM Tris-HCl (pH 7.5-8.0)

• Salt: 50-100 mM NaCl or KCl

• Cofactors: 1-5 mM MgCl2 (essential for activity)

• Temperature: 37°C (physiological)

• Enzyme concentration: 10-100 nM

• Substrate concentration: 1-100 μM

Activity measurement techniques:

• Radiolabeled substrate degradation: Separate products by TLC or PAGE, quantify by phosphorimaging

• Fluorescence-based assays: Monitor fluorescence changes upon substrate degradation

• HPLC analysis: Quantify the conversion of oligoribonucleotides to mononucleotides

Data analysis:

• Determine initial reaction velocity from linear portion of progress curves

• Calculate specific activity (nmol substrate cleaved/min/mg enzyme)

• For detailed kinetic analysis, fit data to appropriate enzyme kinetic models

From E. coli studies, we know that tracking enzyme activity over time is crucial to understanding ORN function . Researchers should include appropriate controls such as heat-inactivated enzyme and verify that the assay conditions maintain enzyme stability throughout the measurement period.

What storage conditions maintain optimal activity of recombinant B. vietnamiensis Oligoribonuclease?

For maintaining optimal activity of recombinant B. vietnamiensis ORN, researchers should follow these evidence-based storage recommendations:

Primary storage recommendations:

• Store at -20°C for routine use

• For extended storage, maintain at -80°C

• Shelf life in liquid form: 6 months at -20°C/-80°C

• Shelf life in lyophilized form: 12 months at -20°C/-80°C

Reconstitution protocol:

• Briefly centrifuge vial before opening

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol to 5-50% final concentration (default: 50%)

• Aliquot into small volumes to minimize freeze-thaw cycles

Working solution management:

• Store working aliquots at 4°C for up to one week

• Avoid repeated freeze-thaw cycles which significantly reduce activity

• For dilute solutions, consider adding carrier protein (BSA, 0.1-1 mg/mL)

Activity preservation factors:

• Maintain RNase-free conditions

• Consider adding reducing agents (1-5 mM DTT) if the protein contains cysteines

• Use sterile, low-binding microcentrifuge tubes for storage

Regular activity testing using standardized assays is recommended to verify enzyme functionality, particularly for samples stored for extended periods. For critical experiments, researchers should prefer freshly thawed aliquots rather than samples that have been stored at 4°C for multiple days.

What is the relationship between oligoribonuclease function and antibiotic resistance in B. vietnamiensis?

Research on B. vietnamiensis reveals a unique susceptibility profile where, unlike other Burkholderia cepacia complex (BCC) species, it is often intrinsically susceptible to aminoglycosides while remaining resistant to cationic antimicrobial peptides . This distinct pattern provides an intriguing context for studying potential relationships between ORN function and antibiotic responses.

Key experimental approaches:

• Expression analysis during antibiotic exposure:

  • RT-qPCR to measure orn mRNA levels following aminoglycoside treatment

  • Proteomic analysis to quantify ORN protein levels under antibiotic stress

  • Activity assays to determine if enzymatic function changes during exposure

• Resistance development studies:

  • Monitor orn expression during acquired resistance development

  • Compare orn sequence and expression between susceptible parent strains and resistant derivatives

  • Investigate the effect of tobramycin or azithromycin pressure on ORN function

• Mechanistic investigation:

  • Analyze RNA degradation patterns in antibiotic-treated cells

  • Determine if altered RNA metabolism contributes to resistance mechanisms

  • Investigate if ORN activity affects expression of other resistance determinants

B. vietnamiensis strains can acquire aminoglycoside resistance during chronic cystic fibrosis infection, a phenomenon that can be induced under tobramycin or azithromycin pressure in vitro . This suggests potential adaptation mechanisms that might involve RNA metabolism pathways where ORN plays a crucial role.

How has oligoribonuclease evolved across bacterial pathogens, and what does this tell us about its role in pathogenesis?

Investigating the evolutionary trajectory of oligoribonuclease across bacterial pathogens requires a comprehensive comparative approach:

Evolutionary analysis methodologies:

• Phylogenetic analysis:

  • Construct phylogenetic trees using ORN sequences from diverse pathogens

  • Compare tree topology with species phylogeny to identify potential horizontal gene transfer

  • Calculate evolutionary rates to identify rapidly evolving regions

• Selection pressure analysis:

  • Calculate Ka/Ks ratios to identify sites under positive or purifying selection

  • Compare selection patterns between environmental and pathogenic isolates

  • Identify lineage-specific selection signatures

• Structural evolution assessment:

  • Map conserved and variable regions onto structural models

  • Identify pathogen-specific structural features

  • Correlate structural conservation with catalytic function

For B. vietnamiensis specifically, comparative analysis should focus on whether its ORN contains unique features that might contribute to its distinct behavior as an opportunistic pathogen in cystic fibrosis patients , potentially relating to its unusual antibiotic susceptibility profile.

How can CRISPR-Cas9 technology be applied to study B. vietnamiensis Oligoribonuclease function?

CRISPR-Cas9 technology offers sophisticated approaches to investigate ORN function in B. vietnamiensis, especially considering the likely essential nature of this gene:

Strategic CRISPR applications:

• Inducible CRISPRi systems:

  • Design sgRNAs targeting the orn promoter or non-catalytic regions

  • Use dCas9-repressor fusions (e.g., dCas9-KRAB) for titratable knockdown

  • Create expression systems regulated by inducers like rhamnose or tetracycline

  • Monitor phenotypic changes under various repression levels

• CRISPR-based tagging approaches:

  • Generate C- or N-terminal fluorescent protein fusions to study localization

  • Create epitope-tagged versions for co-immunoprecipitation studies

  • Implement proximity labeling approaches (APEX, BioID) to identify interacting partners

• Base editing applications:

  • Introduce point mutations in catalytic residues without double-strand breaks

  • Create libraries of orn variants with specific amino acid substitutions

  • Engineer temperature-sensitive alleles for conditional studies

• Genetic interaction screening:

  • Perform CRISPRi screens to identify synthetic interactions with orn

  • Discover genes that become essential when orn is partially repressed

  • Map genetic networks involving RNA metabolism pathways

Given that complete deletion of orn is likely lethal based on E. coli studies , these conditional approaches provide crucial methodological flexibility. Researchers must develop appropriate genetic safeguards, such as complementation constructs, when manipulating this essential gene to maintain bacterial viability during experiments.

What structural biology approaches would provide insights into B. vietnamiensis Oligoribonuclease catalytic mechanism?

Advanced structural biology techniques can reveal the molecular basis of B. vietnamiensis ORN function, similar to approaches used in studying other enzymes like ornithine decarboxylase :

Comprehensive structural biology strategy:

• X-ray crystallography:

  • Crystallize ORN in multiple states (apo, substrate-bound, product-bound)

  • Collect high-resolution diffraction data at synchrotron sources

  • Solve structures by molecular replacement or experimental phasing

  • Refine structures using programs like PHENIX

• Cryo-electron microscopy:

  • Employ single-particle analysis for high-resolution structure determination

  • Visualize conformational states during catalytic cycle

  • Analyze potential oligomeric assemblies or complexes with other proteins

• Solution NMR studies:

  • Investigate dynamics of substrate binding and product release

  • Map interaction surfaces through chemical shift perturbation

  • Study conformational changes during catalysis

• Computational structure analysis:

  • Perform molecular dynamics simulations to model catalytic mechanisms

  • Use quantum mechanics/molecular mechanics (QM/MM) for reaction pathway modeling

  • Implement virtual screening to identify potential inhibitors

Expected structural insights:

These structural studies would provide atomic-level insights into ORN function and potentially reveal unique features of the B. vietnamiensis enzyme that could be exploited for selective inhibition .

How can researchers investigate the interaction of Oligoribonuclease with the RNA degradosome in B. vietnamiensis?

Understanding how ORN interacts with the RNA degradosome machinery requires a multi-faceted approach to protein-protein and protein-RNA interactions:

Interaction mapping methodology:

• Protein-protein interaction analysis:

  • Co-immunoprecipitation using tagged ORN

  • Bacterial two-hybrid assays to screen for direct interactions

  • Crosslinking mass spectrometry (XL-MS) to identify interaction interfaces

  • Surface plasmon resonance to determine binding kinetics

• In vivo interaction visualization:

  • Fluorescence resonance energy transfer (FRET) with fluorescently tagged components

  • Bimolecular fluorescence complementation to confirm direct interactions

  • Super-resolution microscopy to track co-localization during RNA degradation

  • Proximity labeling (BioID, APEX) to identify the ORN interactome

• Functional interaction studies:

  • RNA decay kinetics in strains with various degradosome component mutations

  • Reconstitution of degradosome activities in vitro with purified components

  • RNA substrate competition assays between ORN and other exoribonucleases

Studies in E. coli have shown that ORN completes the final step in RNA degradation by processing small oligoribonucleotides (2-5 nucleotides) to mononucleotides . In most bacteria, earlier steps in RNA decay involve the degradosome complex containing endoribonucleases, exoribonucleases, a helicase, and often metabolic enzymes.

For B. vietnamiensis specifically, researchers should investigate whether pathogen-specific factors affect degradosome assembly or function, particularly in the context of infection environments. The unique antibiotic susceptibility profile of B. vietnamiensis raises questions about whether RNA metabolism machinery might be organized differently in this organism compared to other Burkholderia species.

What is the potential of B. vietnamiensis Oligoribonuclease as an antimicrobial target?

Given that oligoribonuclease is essential for bacterial viability as demonstrated in E. coli , it represents a promising antimicrobial target, particularly for pathogens like B. vietnamiensis that can cause severe disease in cystic fibrosis patients :

Target validation approach:

• Essentiality confirmation:

  • Develop conditional knockdown strains using inducible promoters

  • Employ CRISPRi for titratable repression

  • Determine the minimum expression threshold required for viability

  • Assess growth kinetics and morphological changes upon depletion

• Inhibitor discovery pipeline:

  • Structure-based virtual screening using the solved crystal structure

  • High-throughput biochemical assays screening compound libraries

  • Fragment-based drug discovery approaches

  • Rational design based on substrate analogs

• Selectivity assessment:

  • Comparative analysis with human oligoribonuclease homologs

  • Identification of bacterial-specific structural features

  • Counter-screening against human enzymes

  • Cytotoxicity testing in mammalian cell cultures

Therapeutic potential advantages:

The development of ORN inhibitors could be particularly valuable for B. vietnamiensis infections in cystic fibrosis patients, where strains can acquire resistance to current antibiotics during chronic infection .

How can researchers engineer B. vietnamiensis Oligoribonuclease for enhanced catalytic properties?

Protein engineering approaches can potentially enhance the catalytic properties of B. vietnamiensis ORN for both research and biotechnological applications:

Engineering methodologies:

• Rational design approaches:

  • Site-directed mutagenesis of catalytic residues based on structural data

  • Introduction of stability-enhancing mutations

  • Engineering of substrate specificity through binding pocket modifications

  • Creation of fusion proteins with additional functional domains

• Directed evolution strategies:

  • Error-prone PCR to generate variant libraries

  • DNA shuffling with homologous enzymes

  • Selection systems based on essential ORN function

  • Screening for variants with enhanced stability or activity

• Computational design methods:

  • In silico prediction of stabilizing mutations

  • Molecular dynamics simulations to identify catalytic bottlenecks

  • Enzyme redesign using Rosetta or similar platforms

  • Machine learning approaches incorporating experimental data

Properties for enhancement:

Engineered variants should be thoroughly characterized through activity assays, stability measurements, and structural analysis. The recombinant B. vietnamiensis ORN available commercially (>85% purity) provides a starting point for such engineering efforts.

What RNA-based biotechnology applications could utilize B. vietnamiensis Oligoribonuclease?

B. vietnamiensis Oligoribonuclease offers unique capabilities for RNA processing that can be harnessed in various biotechnology applications:

Biotechnological applications:

• RNA sample preparation:

  • Selective removal of small RNA fragments from RNA preparations

  • Cleanup of RNA sequencing libraries

  • Removal of RNA primer remnants in DNA sequencing applications

  • Processing of RNA prior to mass spectrometry analysis

• Diagnostics development:

  • RNA degradation-based detection systems

  • Removal of background short RNAs in diagnostic samples

  • Component in isothermal nucleic acid amplification techniques

  • Sample preparation for pathogen detection assays

• RNA therapeutics manufacturing:

  • Quality control of synthetic RNA

  • Removal of abortive transcripts from in vitro transcription

  • Processing of RNA for therapeutic applications

  • Selective degradation of undesired RNA species

• Synthetic biology tools:

  • Component in RNA-based circuit design

  • Tunable RNA degradation modules

  • Processing of structured RNA elements

  • Control of RNA half-life in engineered systems

Implementation considerations:

The recombinant B. vietnamiensis ORN product specifications indicate stability for 6 months in liquid form at -20°C/-80°C and 12 months in lyophilized form . For biotechnology applications, researchers should optimize buffer conditions, potentially including glycerol (5-50%) as a stabilizing agent . Immobilization strategies could further enhance stability and enable reusable formats for commercial applications.

Given that B. vietnamiensis ORN specifically degrades small oligoribonucleotides (based on homology to E. coli ORN which processes 2-5 nucleotide fragments) , this narrow substrate specificity provides unique advantages for applications requiring selective RNA processing.