Recombinant Acinetobacter sp. Oligoribonuclease (orn)

How does the Acinetobacter sp. genetic background influence recombinant orn expression and function?

The genetic background of Acinetobacter species significantly impacts recombinant orn expression and function through several mechanisms. Acinetobacter strains, particularly ADP1, offer unique advantages for heterologous protein expression due to their natural competence and relatively simple genetic manipulation requirements. The genomic context affects the expression levels of introduced genes through factors such as codon usage bias, regulatory elements, and cellular metabolism.

Acinetobacter sp. ADP1 has been established as an excellent model organism for genetic studies due to its natural transformation capability, which allows for direct uptake of linear DNA fragments without the need for electroporation or chemical treatments commonly required in other bacterial systems . This characteristic makes it possible to introduce the orn gene with its native or modified regulatory elements directly into the chromosome or expression vectors with high efficiency.

The genetic background also determines the cellular environment in which the recombinant orn will function, including factors such as pH, ion concentrations, and the presence of potential interaction partners or inhibitors. These factors must be considered when designing expression systems or interpreting functional studies of the recombinant enzyme.

What are the key structural and functional differences between Acinetobacter sp. orn and oligoribonucleases from other bacterial species?

While the search results don't directly compare Acinetobacter sp. orn with oligoribonucleases from other species, we can infer potential differences based on general bacterial enzyme evolution and the genomic characteristics of Acinetobacter.

Structurally, oligoribonucleases across bacterial species share a conserved core domain containing the catalytic site, but can differ in auxiliary domains that may influence substrate specificity, protein-protein interactions, or cellular localization. Acinetobacter sp. orn likely maintains the conserved DEDDh catalytic motif characteristic of this enzyme family, which is essential for coordinating metal ions required for catalysis.

Functionally, differences may arise in substrate preference, catalytic efficiency, and regulation mechanisms. Acinetobacter species, known for their adaptability and metabolic versatility, might have evolved specific regulatory mechanisms for orn expression or activity. The relatively high GC content of Acinetobacter genomes (around 39%) compared to some other bacterial species could influence codon usage in the orn gene, potentially affecting translation efficiency when expressed in heterologous systems .

Comparative genomic analyses between Acinetobacter strains have revealed considerable variation in accessory genomes, suggesting that while core functions like orn are likely conserved, their regulation and interaction with other cellular components might differ between strains or species, potentially leading to functional adaptations of the enzyme.

What are the most efficient expression systems for producing recombinant Acinetobacter sp. orn protein?

For efficient expression of recombinant Acinetobacter sp. orn, researchers have multiple systems to consider, each with distinct advantages. Based on the transformation techniques described for Acinetobacter, several approaches can be recommended:

• Acinetobacter sp. ADP1 Homologous Expression: Utilizing the natural competence of Acinetobacter ADP1 provides a straightforward approach for homologous expression. This system allows for chromosomal integration of the orn gene with native or engineered promoters using the simple transformation protocol described in the literature: growing cells to stationary phase in LB medium at 30°C for 12-16 hours, followed by dilution and a 2.5-hour growth period before adding PCR-amplified DNA containing the target gene and appropriate selection markers .

• E. coli Expression Systems: For higher yield production, E. coli expression systems using vectors with IPTG-inducible promoters like the T7 promoter in pET30a can be employed, similar to the approach used for Acinetobacter AmpC expression . This system typically involves:

  • Cloning the Acinetobacter orn gene into the expression vector

  • Transforming E. coli BL21(DE3) or similar strains

  • Inducing expression with IPTG (typically 0.5-1 mM)

  • Culturing at lower temperatures (16-25°C) to enhance proper folding

• Dual Plasmid Systems: For cases where orn activity might be toxic to the host, regulated expression using dual plasmid systems with tight control of expression levels may be necessary.

The choice of expression system should be guided by the specific research requirements, including whether native conformation is critical, the need for post-translational modifications, and the desired yield.

What purification strategy yields the highest purity and activity for recombinant Acinetobacter orn?

A multi-step purification strategy is recommended to achieve high purity and activity for recombinant Acinetobacter orn. Based on general protein purification principles and the specific characteristics of nucleases:

• Affinity Chromatography: The initial purification step typically employs affinity tags such as His6-tag or GST-tag. For a His-tagged orn protein, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with imidazole gradient elution (typically 20-250 mM) provides good initial purification.

• Ion Exchange Chromatography: As a second step, ion exchange chromatography can remove contaminants based on charge differences. Since oligoribonucleases typically bind nucleic acids, cation exchange chromatography (e.g., using SP-Sepharose) at pH values below the protein's isoelectric point can effectively separate the target protein from nucleic acid contaminants.

• Size Exclusion Chromatography: As a final polishing step, gel filtration (using Superdex 75 or similar matrices) separates the protein by size, removing aggregates and smaller contaminants while also enabling buffer exchange into a stabilizing storage buffer.

Throughout the purification process, it's critical to:

• Include RNase inhibitors in early purification steps if maintaining enzyme activity is crucial

• Test fractions for nuclease activity using appropriate substrates

• Add stabilizing agents such as glycerol (10-20%) and reducing agents like DTT (1-5 mM) in the final storage buffer

• Avoid freeze-thaw cycles by storing aliquots at -80°C

This multi-step approach typically yields protein with >95% purity suitable for structural and functional studies.

How can researchers verify the structural integrity and activity of purified recombinant Acinetobacter orn?

To comprehensively verify both the structural integrity and enzymatic activity of purified recombinant Acinetobacter orn, researchers should employ multiple complementary approaches:

Structural Integrity Assessment:

• SDS-PAGE and Western Blotting: Evaluate protein purity and molecular weight using SDS-PAGE. Western blotting with antibodies against orn or the affinity tag confirms protein identity.

• Circular Dichroism (CD) Spectroscopy: Analyze secondary structure elements to confirm proper folding. Oligoribonucleases typically display characteristic α-helix and β-sheet distributions that can be detected by CD.

• Dynamic Light Scattering (DLS): Assess protein homogeneity and detect aggregation or oligomerization states.

• Thermal Shift Assay: Determine protein stability and proper folding by measuring melting temperatures.

Activity Verification:

• Oligoribonucleotide Degradation Assay: The standard assay involves incubating purified orn with small RNA oligonucleotides (typically 2-5 nucleotides) and analyzing the products by HPLC or capillary electrophoresis. The conversion of oligoribonucleotides to mononucleotides demonstrates enzymatic activity.

• Fluorescence-Based Assays: Utilize fluorescently labeled RNA substrates that exhibit increased fluorescence upon degradation, allowing real-time monitoring of enzyme activity.

• Enzyme Kinetics Analysis: Determine key parameters including Km, Vmax, and kcat using varying substrate concentrations to characterize the enzyme's catalytic efficiency.

A typical activity assay might include:

• 50 mM Tris-HCl (pH 8.0)

• 5 mM MgCl2 (or MnCl2)

• 1 mM DTT

• 50-100 nM purified orn

• 1-10 μM RNA substrate

• Incubation at 30-37°C for 15-60 minutes

The integrity and activity data should be compared with known oligoribonucleases to establish relative activity and confirm proper folding of the recombinant Acinetobacter orn.

What are the most effective methods for cloning and expressing the orn gene from Acinetobacter species?

Based on the transformation techniques described for Acinetobacter species, particularly ADP1, several effective methods can be employed for cloning and expressing the orn gene:

PCR-Based Direct Transformation Approach:

The most straightforward method leverages the natural competence of Acinetobacter sp. ADP1. This approach involves:

• PCR Amplification of the orn Gene: Design primers that include:

  • 20-25 nucleotides complementary to the orn gene sequences

  • Additional flanking sequences (50-100 bp) homologous to the desired integration site

  • Optional regulatory elements (promoters, ribosome binding sites)

• Direct Transformation: Following the protocol described in the literature:

  • Grow recipient Acinetobacter cells to stationary phase in LB medium (12-16 hours at 30°C)

  • Dilute 20 μl of culture into 300 μl fresh LB

  • Incubate with shaking at 30°C for 2.5 hours

  • Add 100 μl PCR product directly to the culture

  • Incubate for an additional 2 hours at 30°C

  • Plate on selective media

This method eliminates the need for restriction digestion, ligation, and intermediate cloning steps, significantly simplifying the workflow compared to traditional cloning methods.

Selection Strategies:

For effective selection of transformants, several antibiotic resistance markers can be used, including:

• Kanamycin resistance (Kan^R)

• Spectinomycin resistance

• Trimethoprim resistance

• Tetracycline resistance

The integration of dual selection markers (e.g., antibiotic resistance coupled with counter-selection genes like sacB) facilitates subsequent genetic manipulations such as marker removal for unmarked gene expression.

For optimal expression, consider using native Acinetobacter promoters or well-characterized heterologous promoters depending on the expression level requirements and experimental goals.

How can researchers create site-directed mutations in Acinetobacter orn to study structure-function relationships?

Creating site-directed mutations in Acinetobacter orn for structure-function studies can be efficiently accomplished using the natural competence of Acinetobacter sp. ADP1 combined with PCR-based mutagenesis techniques. The following comprehensive methodology is based on the transformation techniques described in search result :

Two-Step PCR Mutagenesis and Direct Transformation:

• Design of Mutagenic Primers: Create primers that:

  • Contain the desired mutation in the middle of the primer sequence

  • Have 15-20 nucleotides of perfect complementarity on each side of the mutation

  • Maintain appropriate GC content and melting temperature

• First Round PCR: Generate two PCR fragments that overlap at the mutation site:

  • Fragment 1: Using forward primer (with upstream homology) and reverse mutagenic primer

  • Fragment 2: Using forward mutagenic primer and reverse primer (with downstream homology)

• Overlap Extension PCR: Combine the two fragments from step 2 as templates with the outermost primers to generate a full-length mutated gene.

• Direct Transformation: Transform the PCR product directly into Acinetobacter using the natural transformation protocol:

  • Grow cells to stationary phase (12-16 hours at 30°C)

  • Dilute 20 μl culture into 300 μl fresh LB

  • Incubate for 2.5 hours at 30°C with shaking

  • Add 100 μl PCR product

  • Continue incubation for 2 hours

  • Plate on selective media

• Verification of Mutations: Confirm the presence of the mutation by:

  • Colony PCR and sequencing

  • Restriction fragment length polymorphism (RFLP) analysis if the mutation creates or eliminates a restriction site

  • Functional assays to assess the impact of the mutation

This approach eliminates the need for intermediate cloning steps, significantly reducing the time and resources required for creating mutant libraries. The method can be scaled to create multiple mutations in parallel, facilitating comprehensive structure-function analyses.

For studying catalytic residues, mutations in the conserved DEDDh motif (typically aspartate, glutamate, and histidine residues involved in metal coordination and catalysis) would be primary targets, followed by residues involved in substrate binding and protein stability.

What expression systems allow for controlled induction of potentially toxic orn variants?

When working with potentially toxic orn variants, tightly controlled expression systems are essential to prevent premature expression that could compromise cell viability. Based on genetic manipulation principles applicable to Acinetobacter species, the following controlled expression systems are recommended:

1. IPTG-Inducible T7 Expression System:

This system provides tight regulation through a combination of lac repressor control and the specificity of T7 RNA polymerase:

• Recommended Vectors: pET series vectors (such as pET30a used for AmpC expression in the search results )

• Host Strains:

  • E. coli BL21(DE3)pLysS - contains T7 lysozyme to suppress basal expression

  • E. coli C43(DE3) or C41(DE3) - specialized strains developed for toxic protein expression

• Optimization Parameters:

  • Lower incubation temperature (16-20°C) post-induction

  • Reduced IPTG concentration (0.1-0.5 mM instead of standard 1 mM)

  • Shorter induction times (2-4 hours)

  • Addition of glucose (0.5-1%) to media to suppress basal expression

2. Arabinose-Inducible (PBAD) System:

This system offers titratable expression levels based on arabinose concentration:

• Recommended Vectors: pBAD series vectors

• Key Features:

  • Tighter regulation compared to lac-based systems

  • Dose-dependent induction allowing fine-tuning of expression levels

  • Fast response time for induction and de-induction

  • Compatible with both E. coli and Acinetobacter hosts

3. Tetracycline-Inducible Systems:

These systems work well in Acinetobacter species and provide tight regulation:

• Utilize the tetA(B) regulatory elements mentioned in the resistance islands of Acinetobacter

• Typically employ tetracycline analogs like anhydrotetracycline that bind the repressor but have minimal antibacterial activity

4. Dual-Plasmid Systems for Acinetobacter:

For expression directly in Acinetobacter, consider a dual plasmid approach:

• One plasmid carrying the regulated promoter system

• Second plasmid carrying the orn variant under control of the regulated promoter

• The XerC/XerD-like recombination systems mentioned in result could potentially be adapted for controlled integration of expression cassettes

The ideal system should be evaluated based on:

• Background expression level in uninduced state

• Induction ratio (induced vs. uninduced expression)

• Host compatibility

• Ease of use in the laboratory setting

How does Acinetobacter orn contribute to RNA metabolism and bacterial physiology?

RNA Decay Pathway Completion:

Oligoribonuclease serves as the terminal enzyme in mRNA degradation pathways, hydrolyzing small oligoribonucleotides (2-5 nucleotides) to mononucleotides. This final step is essential because:

• It prevents accumulation of small RNA fragments that could interfere with cellular processes

• It recycles nucleotides for new RNA synthesis, contributing to nucleotide homeostasis

• It completes the degradation initiated by other ribonucleases like RNase II, RNase R, and PNPase

Regulatory RNA Processing:

In Acinetobacter species, which are known for their metabolic versatility and adaptability to different environments, orn likely contributes to:

• Processing and turnover of small regulatory RNAs involved in stress responses

• Maintaining appropriate levels of signaling nucleotides

• Influencing expression of genes associated with virulence and antimicrobial resistance through RNA stability regulation

Connection to Biofilm Formation:

The search results indicate that ribonucleases play a role in Acinetobacter surface colonization. Result mentions that "disruption of A. baumannii ribonuclease T2 family protein (ATCC 17978 locus A1S_3026) severely diminishes the organism's ability to colonize abiotic surfaces." While this specifically refers to a T2 family ribonuclease rather than orn, it suggests that RNA metabolism enzymes contribute to biofilm formation and surface attachment behaviors in Acinetobacter. Orn could potentially influence these processes through:

• Regulation of mRNAs encoding adhesins and biofilm matrix components

• Processing of regulatory RNAs involved in quorum sensing

• Influencing cellular responses to surface contact

Stress Response and Adaptation:

The ability of Acinetobacter species to acquire antimicrobial resistance and adapt to different environments likely involves coordinated RNA metabolism in which orn participates by:

• Facilitating rapid turnover of mRNAs during stress responses

• Contributing to the regulation of horizontally acquired genes, including resistance determinants

• Maintaining RNA quality control during environmental transitions

The essential nature of orn in many bacteria suggests it serves critical housekeeping functions in Acinetobacter physiology that extend beyond simple RNA degradation to influence core aspects of bacterial adaptation and survival.

What are the implications of orn activity for Acinetobacter antimicrobial resistance and pathogenicity?

Oligoribonuclease (orn) activity likely has significant implications for Acinetobacter antimicrobial resistance and pathogenicity through several molecular mechanisms, though these connections must be inferred from broader principles of bacterial RNA metabolism and the specific resistance characteristics of Acinetobacter described in the search results.

Regulation of Resistance Gene Expression:

Acinetobacter species, particularly A. baumannii, possess remarkable ability to acquire resistance determinants through horizontal gene transfer, including OXA-group carbapenemases, AmpC-type β-lactamases, and various efflux pumps . The expression of these resistance genes is regulated at multiple levels, including:

• Transcript Stability: Orn contributes to mRNA turnover pathways that influence the stability and half-life of resistance gene transcripts. Altered orn activity could potentially modify the expression levels of key resistance determinants.

• Small RNA Regulation: Regulatory small RNAs often modulate resistance gene expression. Orn's role in processing small RNA molecules may indirectly affect resistance mechanisms by altering the function of these regulatory RNAs.

• Stress Response Coordination: Antibiotic exposure triggers bacterial stress responses. Orn likely contributes to the coordinated RNA metabolism required for these adaptive responses, potentially influencing:

  • Expression of inducible resistance genes

  • Activation of SOS response pathways

  • Modulation of envelope stress responses

Biofilm Formation and Persistence:

The search results indicate that ribonucleases impact Acinetobacter surface colonization , a critical step in biofilm formation. Biofilms significantly enhance antimicrobial resistance through:

• Physical Barriers: Reduced antibiotic penetration

• Metabolic Adaptations: Slower growth and altered metabolism

• Increased Horizontal Gene Transfer: Enhanced sharing of resistance genes

If orn influences biofilm development through RNA metabolic pathways, it may indirectly contribute to the elevated antimicrobial resistance characteristic of biofilm communities.

Virulence Factor Expression:

Pathogenicity in Acinetobacter involves coordinated expression of virulence factors, many of which are regulated post-transcriptionally. Orn's role in RNA metabolism potentially influences:

• Timing of Virulence Gene Expression: By affecting mRNA turnover rates of virulence genes

• Response to Host Environment: By contributing to adaptation mechanisms during infection

• Quorum Sensing Pathways: By processing regulatory RNAs involved in cell-to-cell communication

While direct experimental evidence linking orn to these processes in Acinetobacter is not provided in the search results, the fundamental role of RNA metabolism in bacterial pathogenesis suggests that orn likely contributes to the remarkable adaptability and pathogenic potential of Acinetobacter species.

How can recombinant Acinetobacter orn be used as a tool in RNA biology research?

Recombinant Acinetobacter orn represents a valuable enzymatic tool for RNA biology research that can be applied across multiple experimental contexts. Its specific catalytic properties make it particularly useful for several applications:

1. RNA Sample Preparation and Quality Control:

Recombinant orn can be employed to:

• Remove small RNA contaminants from RNA preparations

• Prepare RNA samples for high-resolution techniques like RNA-seq by eliminating small oligoribonucleotide fragments

• Serve as a quality control tool for checking the purity of synthetic RNA preparations

2. Studying RNA Degradation Pathways:

As the terminal enzyme in RNA decay pathways, purified orn enables:

• Reconstitution of complete RNA degradation pathways in vitro

• Analysis of degradation intermediates by preventing their complete breakdown to mononucleotides when used in controlled amounts

• Investigation of the coordination between different ribonucleases in the RNA decay process

3. Analytical Applications:

The specific activity of orn against small oligoribonucleotides makes it useful for:

• Analyzing the composition of small RNA pools in different cellular states

• Developing assays to detect specific small RNA species by selective degradation of others

• Structure-function studies through comparison with other bacterial oligoribonucleases

4. Tool for Genetic Studies:

The recombinant enzyme can be employed in genetic research approaches such as:

• Complementation studies in orn-deficient strains to investigate the physiological importance of this enzyme

• Creation of conditional knockdown systems to study the effects of reduced orn activity

• Structure-function analyses through expression of mutant variants

5. Development of RNA-Based Technologies:

In biotechnology applications, recombinant orn can serve as:

• A processing enzyme for RNA-based therapeutic development

• A tool for controlling RNA stability in cell-free protein synthesis systems

• A component in nucleic acid detection and amplification methodologies

For optimal use in these applications, researchers should consider the following parameters:

• Buffer conditions (typically containing divalent cations like Mg²⁺ or Mn²⁺)

• Temperature optimum (likely 30-37°C based on Acinetobacter's growth temperature)

• pH requirements (typically 7.5-8.5 for most ribonucleases)

• Storage conditions to maintain activity (usually -20°C to -80°C in buffer containing glycerol)

The unique properties of Acinetobacter orn, including its potential thermal stability and specific activity profile, may offer advantages over oligoribonucleases from other bacterial species in certain experimental contexts.

How can researchers use genetic approaches to study orn essentiality and conditional phenotypes in Acinetobacter?

Investigating the essentiality of orn and its conditional phenotypes in Acinetobacter requires sophisticated genetic approaches that take advantage of the organism's natural competence and tractable genetics. Based on the transformation and genetic manipulation techniques described in the search results, the following methodological approaches are recommended:

1. Conditional Expression Systems:

To study an essential gene like orn, researchers can employ regulatable promoter systems:

• Tetracycline-Responsive Expression: Utilizing the tetA(B)-tetR(B) regulatory elements mentioned in the resistance islands of Acinetobacter , researchers can create a construct where orn expression is controlled by tetracycline concentration.

• Implementation Protocol:

  • Generate a PCR construct containing the orn gene under control of the tet promoter with appropriate homology arms

  • Transform this construct into Acinetobacter using the natural transformation protocol

  • Select transformants on appropriate media

  • Verify replacement of the native orn promoter with the regulatable system

  • Study phenotypes under various inducer concentrations

2. Depletion Strain Construction:

A complementation-based approach for studying essential genes:

• Methodology:

  • Introduce a second copy of orn controlled by an inducible promoter

  • Delete or disrupt the chromosomal copy using selection/counter-selection cassettes (such as KanR/sacB described in result )

  • Maintain the strain with inducer present

  • Study the phenotypic consequences of orn depletion by removing the inducer

• Experimental Controls:

  • Include wild-type strains in all analyses

  • Monitor growth rates, cell morphology, and RNA profiles during depletion

  • Complement with wild-type orn to confirm phenotype specificity

3. Domain Mapping through Truncation and Chimeric Proteins:

To understand functional domains of orn:

• Generate a series of truncated orn variants through PCR with primers that amplify specific portions of the gene

• Create chimeric proteins by fusing domains from oligoribonucleases of different species

• Express these constructs using the transformation techniques described for Acinetobacter ADP1

• Assess functionality through complementation experiments

4. Site-Directed Mutagenesis for Structure-Function Analysis:

• Target residues predicted to be involved in catalysis (DEDDh motif)

• Create an allelic replacement construct containing the mutated orn gene

• Transform using natural competence protocols

• Select for marker integration

• Use counter-selection to isolate unmarked mutants

• Characterize phenotypes associated with specific amino acid substitutions

5. Synthetic Lethality Screening:

• Construct a strain with reduced orn activity (using the techniques above)

• Create a random transposon mutant library in this background using EZ-Tn5

• Screen for synthetic growth defects

• Identify genetic interactions through sequencing of insertion sites

These approaches take advantage of the natural transformation capability of Acinetobacter sp. ADP1 and the selection systems described in the search results, providing powerful tools for dissecting the essential functions of orn and its role in Acinetobacter physiology.

What are the current challenges in studying Acinetobacter orn and how can they be addressed?

Research on Acinetobacter oligoribonuclease (orn) faces several technical and biological challenges. Based on the broader context of Acinetobacter genetics and protein expression research, these challenges and their potential solutions can be systematically addressed:

Gene Essentiality Constraints

Challenge: As orn is likely essential for viability in Acinetobacter (based on its essentiality in related bacteria), direct knockout studies are problematic.

Solutions:

• Implement conditional expression systems using the tetracycline-regulatable promoters mentioned in the resistance islands of Acinetobacter

• Apply CRISPR interference (CRISPRi) for partial knockdown rather than complete knockout

• Utilize temperature-sensitive mutants by introducing mutations that compromise protein stability at elevated temperatures

• Employ the dual selection/counter-selection cassettes (KanR/sacB) described in the literature to facilitate precise genetic manipulations

Protein Solubility and Activity Issues

Challenge: Ribonucleases often form inclusion bodies when overexpressed or lose activity during purification due to aggregation or misfolding.

Solutions:

• Express orn as a fusion with solubility-enhancing tags (MBP, SUMO, Trx)

• Optimize expression conditions using lower temperatures (16-20°C) and reduced inducer concentrations

• Implement on-column refolding protocols during purification

• Co-express with chaperone proteins to enhance folding

• Screen multiple Acinetobacter species to identify naturally more soluble orn variants

Substrate Specificity Determination

Challenge: Distinguishing orn activity from other ribonucleases and precisely defining its substrate range is technically challenging.

Solutions:

• Design fluorescently labeled oligoribonucleotide substrates of defined length

• Develop high-performance liquid chromatography (HPLC) methods to separate and quantify reaction products

• Utilize synthetic RNA substrates with modified ends to prevent degradation by other nucleases

• Apply RNA sequencing approaches to identify natural substrates in vivo

Functional Redundancy

Challenge: Potential redundancy with other ribonucleases may mask phenotypes in genetic studies.

Solutions:

• Construct double or triple mutants with reduced activity in multiple RNases

• Apply RNA-seq to comprehensively analyze changes in the transcriptome and small RNA profile

• Use comparative genomic approaches across Acinetobacter strains to identify co-evolving RNases

• Implement ribosome profiling to detect translation effects that might be masked at the RNA level

Structural Characterization

Challenge: Obtaining structural information about Acinetobacter orn has been difficult.

Solutions:

• Screen multiple Acinetobacter species and growth conditions for protein crystallization

• Apply cryo-electron microscopy as an alternative to crystallography

• Use hydrogen-deuterium exchange mass spectrometry to probe protein dynamics

• Implement molecular modeling approaches based on structures from related organisms

In vivo Relevance

Challenge: Connecting biochemical activities to physiological roles remains difficult.

Solutions:

• Develop reporter systems to monitor RNA decay in vivo

• Apply metabolomics to detect changes in nucleotide pools

• Implement RNA immunoprecipitation approaches to identify RNA binding partners

• Develop animal infection models to study the role of orn in Acinetobacter pathogenesis

Addressing these challenges requires interdisciplinary approaches combining the genetic tools described for Acinetobacter with advanced biochemical and biophysical methods.

How does orn activity integrate with other ribonucleases in Acinetobacter RNA metabolism pathways?

Understanding the integration of oligoribonuclease (orn) with other ribonucleases in Acinetobacter RNA metabolism requires examining the complex network of enzymes involved in RNA processing and degradation. While the search results don't directly address this network in Acinetobacter, we can construct a model based on general bacterial RNA decay mechanisms and the specific genomic features of Acinetobacter species.

Hierarchical RNA Degradation Pathway in Acinetobacter:

RNA decay in bacteria typically follows a hierarchical process involving multiple ribonucleases with distinct specificities and functions:

• Initiation of mRNA Decay:

  • RNase E (endonuclease) likely initiates decay by cleaving within mRNA transcripts

  • RppH (pyrophosphohydrolase) removes 5' pyrophosphate to facilitate decay initiation

  • These initial steps generate RNA fragments with accessible 3' and 5' ends

• Processive Degradation:

  • 3'→5' exoribonucleases (RNase II, RNase R, PNPase) process longer fragments

  • These enzymes stall when reaching short oligoribonucleotides (2-5 nucleotides)

  • The genomic analysis of Acinetobacter strains suggests the presence of these conserved RNases

• Terminal Degradation by orn:

  • Orn specifically processes the short oligoribonucleotides that other exoribonucleases cannot degrade

  • This step completes the RNA degradation pathway and recycles nucleotides

  • Orn's activity is essential as accumulation of oligoribonucleotides is toxic to cells

Integration with Specialized RNA Processing Pathways:

Beyond the main decay pathway, orn likely integrates with specialized RNA processing systems:

• tRNA Processing and Quality Control:

  • RNase P and RNase Z process tRNA precursors

  • Orn may degrade oligoribonucleotides generated during tRNA quality control

  • This integration ensures proper tRNA pool maintenance

• rRNA Maturation:

  • Various RNases (including RNase III) process rRNA precursors

  • Orn potentially degrades oligoribonucleotide byproducts of this processing

• sRNA Regulation:

  • Small regulatory RNAs influence gene expression in response to environmental cues

  • The presence of ribonuclease T2 family protein in A. baumannii and its role in surface colonization suggests complex RNA regulatory networks

  • Orn likely contributes to sRNA turnover and regulation

Spatial and Temporal Coordination:

The integration of these ribonucleases requires spatial and temporal coordination:

• Degradosome Formation:

  • In E. coli, RNase E forms a multienzyme complex (degradosome) with PNPase, RNA helicase, and enolase

  • While not directly confirmed in Acinetobacter, similar complexes likely exist based on genomic conservation

  • Orn presumably acts downstream of these complexes rather than as a component

• Compartmentalization:

  • RNA degradation may be spatially organized within the bacterial cell

  • Membrane association of some RNases may create degradation microenvironments

  • Orn's cellular localization would influence its access to substrates generated by other RNases

The essential nature of orn in this integrated network is underscored by the fact that its absence cannot be compensated by other RNases, highlighting its unique and critical role in completing RNA degradation pathways in Acinetobacter species.